Recent advances in microbial transglutaminase biosynthesis and its application in the food industry

Trends in Food Science & Technology 110 (2021) 458–469

Available online 19 February 2021 0924-2244/© 2021 Elsevier Ltd. All rights reserved.

Recent advances in microbial transglutaminase biosynthesis and its application in the food industry

Mehdi Akbari a, Seyed Hadi Razavi a,**, Marek Kieliszek b,* a Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, College of Agriculture and Natural Resource, University of

Tehran, Karaj, Iran b Department of Food Biotechnology and Microbiology, Institute of Food Sciences, Warsaw University of Life Sciences—SGGW, Nowoursynowska 159 C, 02-776,

Warsaw, Poland A R T I C L E I N F O Keywords:

Microbial transglutaminase Biosynthesis Genetic manipulation

Recombinant A B S T R A C T Background: Microbial transglutaminase (MTGase) has been widely used to modify the functional properties of proteins in food systems. In the last 30 years since the discovery of MTGase, many efforts have been made on new strain isolation, culture media optimization, and fermentation procedure optimization to obtain MTGase with higher activity. Additionally, over the last decade, many studies have switched the focus from conventional optimization to genetic engineering in order to develop a highly efficient MTGase expression system with desired properties such as thermostability, activity, and yield by using genetic manipulation of strains such as Escherichia coli, Bacillus subtilis, and Pichia pastoris.

Scope and approach: In this review, we describe not only the recent advances and limitations related to MTGase biosynthesis but also the potential of MTGase for application in the food industry for some food products, including meat products, cheese, yogurt, and bread. Promoter engineering, gene codon optimization, signal peptide fusion, constitutive expression, random and rotational mutagenesis, etc. have been applied to enhance the recombinant expression system of MTGase. After three decades of research, the expression of recombinant

MTGase has been significantly improved from the formation of inclusion body and enzyme with very low activity to the soluble form with high activity.

Key findings and conclusions: Recombinant MTGase technology could also resolve problems related to post- translational modification in MTGase biosynthesis, resulting in facilitating downstream processing. In the future, it has been predicted that the scope of research will expand to work on heterologous expression by combination of genetic engineering tools. Further research is also needed to evaluate the biosynthesis of re­ combinant MTGase on a larger scale.

1. Introduction Protein-glutamine γ-glutamyltransferase, i.e., transglutaminase (TGase; EC 2.3.2.13), belongs to the transferase family that is widely distributed in nature. Animals, plants, and microorganisms are the source of this enzyme (Kieliszek & Misiewicz, 2014; Singh & Kumar,

2019). TGase catalyzes the acyl transfer reaction between γ-carbox­ amide of glutaminyl residues as acyl donors and primary amines as acyl acceptors (Kieliszek & Bła˙zejak, 2017; Santhi, Kalaikannan, Malairaj, &

Arun Prabhu, 2017) (Fig. 1 a). When the ε-amino groups of lysine resi­ dues in proteins act as acyl acceptors, the transamidation reaction oc­ curs. In this case, the transfer of acyl onto a lysine residue results in the formation of both intermolecular and intramolecular covalent cross-links of ε-(γ-glutamyl)lysine (Gln-Lys), and the protein is enriched with the essential amino acids (Giosafatto, Al-Asmar, & Mariniello,

2018) (Fig. 1 b). Furthermore, this enzyme catalyzes the deamidation or esterification reaction of γ-carboxamide of glutaminyl residues when primary amines are absent. Under this condition, water or alcohol molecules become the acyl acceptors with their hydroxyl groups (Mar­ iniello, Di Pierro, Giosafatto, Sorrentino, & Porta, 2008) (Fig. 1 c, d).

These reactions catalyzed by TGase can be used to modify the functional properties of food proteins, such as solubility, water holding capacity (WHC), emulsifying capacity, foaming, viscosity, elasticity, and gelation (Martins et al., 2014; Wang, Yu, Wang, & Xie, 2018). It has been

* Corresponding author.

** Corresponding author.

E-mail addresses: srazavi@ut.ac.ir (S.H. Razavi), marek-kieliszek@wp.pl, marek_kieliszek@sggw.edu.pl (M. Kieliszek).

Contents lists available at ScienceDirect Trends in Food Science & Technology journal homepage: www.elsevier.com/locate/tifs https://doi.org/10.1016/j.tifs.2021.02.036

Received 16 June 2020; Received in revised form 25 January 2021; Accepted 14 February 2021

Trends in Food Science & Technology 110 (2021) 458–469

459 suggested that TGase can modify many proteins such as milk caseins, whey proteins, soybean globulins, wheat gluten, and meat myosins (Martins et al., 2014).

Research on the application of TGase in food products started with the isolation of the enzyme from animal tissues. Until the end of the 18th century, extraction of the enzyme from animal tissues (especially Guinea pig liver) was the only commercial approach. The rare source and the complicated separation and purification procedure led to an extremely high cost of the enzyme (Zhu & Tramper, 2018). Moreover, animal

TGase is calcium (Ca2+)-dependent, and this ion resulted in protein destabilization in some food proteins such as caseins and soy proteins.

Nevertheless, tissue TGase has been rarely used in food products on an industrial scale (Cui, Du, Zhang, Liu, & Chen, 2007).

Natural TGase is also found in plant tissues such as soybean, maize, tobacco, and orchard apple (Giosafatto et al., 2018). Compared to ani­ mal TGase, a specific feature of plant TGase is sensitivity to light. It is difficult to obtain pure TGase from plants because photosynthesis and photoprotection processes can affect plant TGase expression. Moreover, plant TGase is Ca2+-dependent similar to animal TGase (Aloisi Cai,

Serafini-Fracassini, & Duca, 2016). Moreover, none of these enzymes have been commercialized, which have led to intense research to find a convenient commercial source.

TGase derived from microorganisms was first isolated from the cul­ ture medium of Streptoverticillium mobaraense, also known as Strepto­ myces mobaraensis, and characterized by Ando et al. (1989). Since then, new strains have been selected for higher enzyme activity, and enzyme production has been optimized through different strategies by conven­ tional fermentation (de Souza, Rodrigues, & Ayub, 2009; Eshra, El-Iraki,

& Bakr, 2015; Sorde & Ananthanarayan, 2019; Xavier, Ramana, &

Sharma, 2017).

Microbial transglutaminase (MTGase) is a Ca2+-independent enzyme and is stable over a wide range of pH and temperatures, which is in sharp contrast to tissue TGase (Kieliszek &

Misiewicz, 2014). However, for some uses of MTGase, it is favorable to perform the cross-linking reaction at a higher temperature with high activity. In recent years, genetic engineering has enabled a wider and more practical application of MTGase with improvement in its proper­ ties such as activity and stability. The purpose of this review is to report the recent advances and limitations related to TGase biosynthesis and highlight the influence of MTGase on the physicochemical and sensory properties of some food products.

2. Enzymatic properties of MTGase The MTGase isolated from S. mobaraensis is initially expressed as a zymogen (pro-MTGase) containing a signal peptide, a 45-amino-acid pro-region (pro-peptide), and a 331-amino-acid mature enzyme domain, which is then converted into active MTGase by proteolytic processing for the deletion of pro-peptide (Yokoyama et al., 2010).

Pro-peptide is essential for efficient secretion, correct folding, and sup­ pression of the enzymatic activity of MTGase (Lin, Hsieh, Lai, Chao, &

Chu, 2008). The active center of MTGase contains Cys 64, His 274, and

Asp 255 residues. The thiol group of cysteine, which attacks the side-chain of glutamine residue of the substrate, is essential for the enzyme activity and is covered by an α-helix, which is separated during activation (Kieliszek & Misiewicz, 2014).

The optimum reaction temperatures of MTGase are 45–55 ◦C,

Fig. 1. The reactions catalyzed by transglutaminase included: (a) acyltransfer reaction; (b) cross-linking reaction between Gln and Lys residues of proteins; (c) deamidation with water; (d) deamidation with alcohol.

M. Akbari et al.

Trends in Food Science & Technology 110 (2021) 458–469

460 depending on the species. For example, S. mobaraensis MTGase has an optimum temperature of 55 ◦C, while the optimum temperature of

MTGase produced by Streptomyces cinnamoneum and Streptomyces gri­ seocarneum is 45 ◦C. The optimum pH of MTGase produced by different microorganisms ranges between 5.0 and 7.0, while this enzyme is stable over a wide range of pH values from 4.0 to 9.0 (Romeih & Walker,

2017). In contrast to many other TGases, the microbial isoform shows low substrate specificity; therefore, it can react with different types of food proteins either in the absence or presence of a reducing agent (Gundersen, Keillor, & Pelletier, 2014). TGases biosynthesized by mi­ croorganisms have a low molecular weight (approximately 38 kDa) as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography (Duarte, Matte, Bizarro, & Ayub,

2020).

The colorimetric hydroxamate procedure (hydroxamate assay) is the most common method for estimating MTGase activity. In this method, the broth is centrifuged after fermentation, and cell-free supernatant is collected for measuring enzyme activity. Fifty microliters of the cell-free supernatant is incubated at 37 ◦C for 10 min. The substrate solution is added to the supernatant, and the reaction mixture is incubated at 37 ◦C for 60 min. The reaction is terminated by the addition of ferric chloride- trichloracetic acid reagent. The resultant color is measured at 525 nm by using a spectrophotometer. One unit of MTGase activity is defined as the amount of enzyme needed for the formation of 1 μmole of hydroxamate from hydroxylamine and carbobenzoxy-L-glutamylglycine per minute at

37 ◦C and pH 6.0 (Martins et al., 2014).

3. MTGase biosynthesis 3.1. Microorganisms producing transglutaminase

TGase is synthesized by many species of bacteria, fungi, and Acti­ nomycetes. Many studies have been conducted to find microbial sources capable of producing MTGase (Table 1). Among all the strains investi­ gated, Streptomyces sp. CBMAI 1617 (SB6) (Ceresino et al., 2018) and

Actinomycetes strains (Eshra et al., 2015) were found to have the highest (~6 U/mL) and the lowest (~0.04 U/mL) enzyme activities, respectively. Generally, the strains of Streptomyces, Bacillus, Enterobacter,

Providencia, and Actinomycetes are known as the main sources for

MTGase biosynthesis.

3.2. Culture media optimization Apart from isolation of new strains for producing MTGase, selection of the most appropriate media composition is extremely important. This fact demonstrates that special attention should be given to design fermentation systems in terms of novel culture media for helping bac­ teria to produce enzyme with high activity. The culture media compo­ sition in the fermentation procedure for MTGase biosynthesis from

Streptomyces sp. is almost the same in most studies. Glucose and soluble starch are the common carbon sources and peptone and yeast extract are the common nitrogen sources used in culture media for MTGase biosynthesis. Necessary minerals and trace elements are phosphate, magnesium, potassium, iron, copper, zinc, and vitamins. Nonionic sur­ factant and antifoam can be added if required. It seems that salt sup­ plements are effective in enhancing MTGase biosynthesis probably due to their role in faster conversion of pro-MTGase to mature enzyme domain by increasing the total protease production (Fatima, Tiwari, &

Khare, 2019).

Fortifying culture media with some ingredients such as amino acids can increase MTGase biosynthesis. Zhu, Rinzema, Tramper, and Bol (1996) designed a new medium based on the stoichiometric analysis of

MTGase biosynthesis by S. mobaraense; this medium contained starch 20 g/L, peptone 20 g/L, MgSO4 2 g/L, KH2PO4 2 g/L, K2HPO4 2 g/L, yeast extract 2 g/L, arginine 0.30 g/L, aspartate 0.649 g/L, asparagine 0.155 g/L, cysteine 0.023 g/L, glycine 0.145 g/L, histidine 0.083 g/L, isoleu­ cine 0.063 g/L, methionine 0.092 g/L, and polypropylene glycol (foam suppressor) 0.5 g/L. The authors indicated that amino acids played an important function in the biosynthesis of MTGase because the addition of amino acids to the unmodified medium significantly increased

MTGase biosynthesis.

Recently, Ceresino et al. (2018) cultured Streptomyces sp. CBMAI

1617 (SB6) as a novel source for MTGase biosynthesis. They indicated that the fermentation medium with optimal concentrations of the components provided a yield of 6.074 ± 0.019 U/mL of MTGase, which was the highest MTGase yield obtained by fermentation to date. Ac­ cording to their results, glucose, casein peptone, and KH2PO4.7H2O had the most positive and significant effect on MTGase biosynthesis.

3.3. Fermentation process optimization Similar to other enzymes, the formation of MTGase is dependent on the environmental conditions used in the fermentation process, such as temperature, pH, dissolved oxygen, and shear rate. Therefore, these parameters are required to be optimized to improve MTGase activity. To improve the yield of MTGase produced by Streptomyces, additional studies have been conducted on environmental control strategies.

However, conflicting results have been reported by recent researchers in this field. For example, Zhang et al. (2012) cultured S. mobaraensis at

30 ◦C and a stable pH value of 7.0 with an agitation of 180 rpm, while

Jin et al. (2016) cultured S. mobaraensis at 30 ◦C for 24 h with a pH of 7.4 (shaking at 200 rpm). In another study, Turker, Domurcuk, Tokatli,

Isleroglu, and Koc (2016) showed that the best conditions to achieve the highest enzyme activity are pH 6.0 and 30 ◦C for 14 days. They used two culture media based on glucose-starch and soy. It seems that the opti­ mum temperature and pH to obtain the enzyme is around 30 ◦C and 7.0, respectively, and the fermentation time is normally 72–96 h, depending on the culture conditions and determined by the highest MTGase ac­ tivity that can be achieved.

3.4. Use of agricultural waste in culture media As mentioned above, peptone and yeast extract are general media components for the growth of Streptomyces species. These components are expensive and are not economically feasible. Therefore, in the

MTGase biosynthesis process, culture medium highly influences the final cost of the enzyme and can cover up to approximately 30% of the

Table 1 MTGase production by wild-type strains.

Strains Procedure Enzyme activity (U/mL) References

Streptomyces mobaraensis CECT 3230 Low-cost culture media optimization

2.95 Guerra-Rodríguez and V´azquez (2014) Actinomycetes strains

Strain isolation and identification 0.04 Eshra et al. (2015)

Streptomyces mobaraensis TX Purification and characterization of a high-salt-resistant

MTGase 1.75 Jin et al. (2016) Streptomyces sp.

D1 Strain isolation and culture media optimization

4.1 Xavier et al. (2017) Streptomyces sp.

CBMAI 1617 (SB6) Production optimization, enzyme characterization

6.07 Ceresino et al. (2018) Bacillus nakamurai B4 Isolation, screening, and optimization of bacterial strains

1.71 Sorde and Ananthanarayan (2019) Bacillus subtilis C2

Isolation, screening, and optimization of bacterial strains

1.61 Sorde and Ananthanarayan (2019) M. Akbari et al.

Trends in Food Science & Technology 110 (2021) 458–469

461 production cost (Portilla-Rivera, T´ellez-Luis, de Le´on, & V´azquez, 2009).

On the other hand, industrial production of MTGase requires culture media made of cheaper raw materials due to economic reasons. For this purpose, various attempts have been made to find alternative cheaper media based on agricultural wastes for MTGase biosynthesis.

Guerra-Rodríguez and V´azquez (2014) evaluated the biosynthesis of

MTGase by S. mobaraensis in a minimal nutritional medium based on noncommercial potatoes. According to their results, the best medium was gelified nonhydrolyzed potato that enabled to obtain up to 2.72

U/mL of the enzyme at 96 h of culture. The authors concluded that the milk–potato–glycerol medium was feasible for the biosynthesis of

MTGase, achieving a high MTG activity in a simple natural medium. In another study, T´ellez-Luis, Gonz´alez-Cabriales, Ramírez, and V´azquez (2004) evaluated the biosynthesis of MTGase by Streptoverticillium ladakanum NRRL–3191 on the culture media containing a mixture of hydrolysate of sorghum straw and xylose 20 g/L and obtained an activity level of 0.348 U/mL after 72 h of culture. They also showed that by using media containing commercial xylose 20 g/L, MTGase activity of up to

0.282 U/mL was obtained at 96 h of culture, and they demonstrated that hydrolysates of sorghum straw were suitable media components for

MTGase biosynthesis by S. ladakanum.

Portilla-Rivera et al. (2009) evaluated the biosynthesis of MTGase by

S. ladakanum NRRL–3191 on media prepared from sugar cane molasses and glycerol. They reported that the highest MTGase activity (0.460

U/mL) was obtained in the medium containing a mixture of molasses and glycerol, while in the medium containing sugar cane molasses alone and glycerol alone, the activity was 0.240 and 0.250 U/mL, respectively.

These results showed that sugar cane molasses combined with glycerol was a suitable medium for MTGase biosynthesis.

Glodowsky, Ruberto, Martorell, Mac Cormack, and Levin (2020) conducted research on the biosynthesis of transglutaminase isolated from the antarctic strain of Penicillium chrysogenum. The microorganisms were grown in a medium containing soybean husk waste, a waste product from soybean processing for the extraction of meal and oil. The authors found that the maximum enzymatic activity of psychrophilic

TGase (7.81 mU) was at pH 8.0 and 30 ◦C. The molecular weight of

TGase as determined by SDS-PAGE analysis was approximately 67 kDa.

The obtained purified TGase was used as an additive to modify the rheology of a cold-curing gelatin gel.

4. Genetic engineering approaches in MTGase biosynthesis

To date, commercial MTGase has mainly been produced as an extracellular protein by conventional cultivation of wild-type S. mobaraensis strain. Under this condition, the obtained enzyme with a yield of 100–150 mg/L fermenter volume is activated by cellular pro­ teases. This system has some drawbacks, including complex fermenta­ tion procedure and problems related to post-translational modification, for example, presence of proteases that can hydrolyze the target proteins (Marx, Hertel, & Pietzsch, 2007; Wang et al., 2018). Moreover, for some uses of MTGase, it is favorable to conduct the cross-linking reaction at a higher temperature with higher activity. Therefore, over the last decade, various attempts have been made to improve the properties of MTGase, such as activity, thermostability, and yield, by genetic engineering.

Several expression systems, including but not limited to Escherichia coli (Juettner et al., 2018; Rickert et al., 2016), Streptomyces lividans (Liu,

Wang, Du, & Chen, 2016; Noda, Miyazaki, Tanaka, Ogino, & Kondo,

2012), Corynebacterium glutamicum (Umakoshi et al., 2011), Bacillus subtilis (Mu, Lu, Qiao, et al., 2018), Yarrowia lipolytica (Liu et al., 2015), and Pichia pastoris (Yang & Zhang, 2019; ¨Ozçelik, Ers¨oz, & ˙Inan, 2019), have been established (Table 2).

It should be noted that the use of genetic engineering in the pro­ duction of MTGase has many advantages and disadvantages. The dis­ advantages include mainly creating mutants on a large scale and laboriousness. Other disadvantages of genetic engineering include the creation of genetically modified foods that can cause disease or allergies.

There are many arguments for and against genetic engineering, and it is difficult to clearly define which ones prevail because science is still developing and the consequences of new technologies are not yet fully known. Among the positive aspects of the application of genetic engi­ neering, one can mention the possibility of producing new food, obtaining microbial metabolites in a very short time. Overall, genetic engineering is a very promising new technology. It gives the opportunity and hope for a better life in the future, but for its safe use, appropriate legal standards should be developed and the necessary long-term research carried out.

4.1. Recombinant MTGase expression in E. coli: from inclusion body to overexpression

E. coli is widely used as a host strain to produce recombinant proteins due to its rapid growth and high yield. However, in most attempts that investigated the feasibility of expressing MTGase in E. coli, the levels of expression were low due to the formation of intracellular inclusion body of MTGase instead of enzyme secretion into the culture medium (Mu, Lu,

Qiao, et al., 2018). The biosynthesis of recombinant MTGase as an in­ clusion body is not cost-effective because it requires a cell disruption step to recover and purify the enzyme. Moreover, refolding of insoluble

Table 2 Improvement of MTGase properties by genetic engineering.

Origin strain Host strain Procedure Activity/yield/ specific activity

References Streptomyces hygroscopicus WHS03- 13 Yarrowia lipolytica

Po1h Promoter engineering, fusing signal peptide, and mutation of the Asn-linked glycosylation sites

7.8 U/mL Liu et al. (2016) Streptomyces hygroscopicus WHS03- 13

Streptomyces lividans TK24 The deletion of the negative element of the promoter and gene codon optimization

5.73 U/mL Liu et al. (2016) Streptomyces mobaraensis

Escherichia coli TOP10 and BL21 Alanine-scan of pro-peptide and insertion of the 3C protease cleavage site

30–75 mg/L Juettner et al. (2018) Streptomyces mobaraensis

Escherichia coli BL21 Using constitutive expression system without extracellular protease addition

1 U/mL Javitt et al. (2017) Streptomyces mobaraensis

Bacillus subtilis Using constitutive promoter of PhpaII

29.6 U/mg Mu, Lu, Qiao, et al. (2018) Streptomyces mobaraensis

Pichia pastoris The control of constitutive GAP promoter

37 U/mL ¨Ozçelik et al. (2019) Streptomyces fradiae

Pichia pastoris GS115 Heterologous expression 0.7 U/mL (Yang and Zhang (2019))

Streptomyces mobaraensis Escherichia coli Using a chimeric protein of tobacco etch virus proteas with mutations in the native pro-peptide

22.7 U/mL Sato et al. (2020) Streptomyces mobaraensis

Bacillus subtilis WB600 Replacing native pro-peptide with that of S. hygroscopicus, adding a self- cleaving intein between pro-peptide and mature enzyme domain

16.1 U/mg Fu et al. (2020) M. Akbari et al.

Trends in Food Science & Technology 110 (2021) 458–469

462 MTGase would be too time-consuming and is not economically feasible for the application in the industry.

For the first time, Liu et al. (2015) succeeded in the efficient secretion of pro-MTGase from S. hygroscopicus in E. coli BL21 by fusing the PelB signal peptide to the N-terminal of pro-MTGase. In that study, after the addition of dispase to the culture supernatant, pro-MTGase was con­ verted into an active form, with an enzymatic activity of 4.5 U/mL.

However, when Marx et al. (Marx et al., 2007) used the strategy of signal peptide fusion for cloning pro-MTGase from S. mobaraensis (DSM40847) carrying a histidine tag (pro-MTG-His6) and expressing in E. coli, it resulted in the intracellular production of MTGase in soluble form. This is due to a high difference in the amino acid sequence between the pro-peptide of MTGases from S. mobaraensis and S. hygroscopicus.

Pro-MTGase from S. hygroscopicus may have a secretion-component pro-peptide that is different from that of pro-MTGase from

S. mobaraensis.

In a remarkable study, Liu et al. (2011) successfully developed a novel method for the direct production of soluble and active MTGase from S. hygroscopicus in E. coli by a polycistronic sequence expressing the pro-peptide and mature MTGase domain as a separate polypeptide in the order of pro-peptide and MTGase under a single T7 promoter. The specific activity of the produced recombinant enzyme after purification by nickel affinity chromatography was 22 U/mg. By using this method, recombinant active MTGase was produced without downstream pro­ teolytic cleavage processing. This approach was validated by Javitt,

Ben-Barak-Zelas, Jerabek-Willemsen, and Fishman (2017) who used a constitutive expression system of active MTGase in E. coli without pro­ tease addition. They concluded that recombinant MTGase had catalytic properties similar to those of the wild-type MTGase. For both strategies, the overall yield of active MTGase was dependent on the expression level of two genes that were expressed separately. More recently, Sato,

Minamihata, Ariyoshi, Taniguchi, and Kamiya (2020) reported another approach to obtain soluble and active MTGase by constructing a chimeric protein of tobacco etch virus protease and pro-MTGase as a single polypeptide. The author reported that when pro-peptide was mutated, active MTGase showed 5-fold higher specific activity than the one from native pro-peptide due to self-cleaving of the mutated pro-peptide.

Duarte, Bars´e et al. (2020) using genetic engineering techniques, cloned and expressed the gene encoding TGase (derived from the bac­ terium Bacillus amyloliquefaciens DSM7) in E. coli. These bacteria are widely used as host cells for the expression of various groups of re­ combinant proteins. Furthermore, E. coli are capable of growing rapidly to high densities. For this purpose, the authors constructed a bicistronic plasmid containing the TGase gene linked to the Streptomyces caniferus prodomain. The specific activity of the resulting transglutaminase was approximately 37 mU/mg protein.

In the case of the studies presented by Han, Ma, Qin, and Liu (2020) the authors cloned the TGase gene isolated from maize leaves and E. coli bacteria were used as the expression system for this enzyme. The pET-28a plasmid was used to construct an expression vector for the maize TGase gene. As a result of the research, it was found that the molecular weight of this enzyme was about 66 kDa. Moreover, this enzyme was characterized by strong immunological properties. The obtained recombinant TGase had a positive effect on casein polymeri­ zation and catalyzed cross-linking of the restructured yogurt.

4.2. Recombinant MTGase expression in other hosts S. lividans is also another ideal host for producing active MTGase because of its ability to convert the inactive pro-MTGase into the active enzyme with its own endogenous proteases. Lin et al. (2008) reported a procedure for the efficient expression of MTGase in S. lividans with a high yield (5.36 U/mL). Recently, Liu et al. (2016) showed that the pro-MTGase from Streptomyces platensis was successfully expressed and correctly processed into active MTGase in S. lividans TK24. On the basis of results, by combining the deletion of the negative element of the promoter and optimization of the gene codon, a yield of 5.73 U/mL of recombinant MTGase was achieved, which was the highest MTGase yield achieved by recombinant S. lividans to date. These studies showed that S. lividans could be a potential host for high-level expression of

MTGase.

B. subtilis is also one of the most well-known host strains for the efficient secretion of enzymes. It is nonpathogenic, and compared to

E. coli, it is generally recognized as safe and is a native MTGase producer.

Recently, Mu, Lu, Qiao, et al. (2018) reported the secretion of S. mobaraensis MTGase from B. subtilis with enzymatic activities similar to that produced from E. coli. The authors showed that the amount of

MTGase secreted and purified successfully from the constitutive system was 63 mg/L with an enzymatic activity of 29.6 U/mg after proteolysis by trypsin.

In a remarkable study, Fu et al. (2020) presented the first report on the extracellular production of active MTGase in B. subtilis. The authors replaced the native pro-peptide with that of S. hygroscopicus to achieve high secretion of MTGase. They also added a self-cleaving intein be­ tween pro-peptide and mature enzyme. By using this strategy, active

MTGase was successfully secreted without protease addition to remove pro-peptide from inactive zymogen. It should be emphasized that the specific activity of this extracellular enzyme was 2.6 U/mg.

Research by ¨Ozçelik et al. (2019) showed that P. pastoris (syn.

Komagataella phaffii) can be used for the production of recombinant transglutamate. The great advantage of P. pastoris yeast as a popular system of recombinant protein expression is its well-known transcrip­ tion mechanism and the ability to post-translational modification of proteins. Moreover, they are characterized by an efficient and strictly regulated promoter. P. pastoris yeast contained a single copy of the gene (derived from the S. mobarensis strain) responsible for MTGase biosyn­ thesis. The highest activity of this enzyme (37640 U/L) was obtained after 72 h of cultivation in a bioreactor at 20 ◦C and pH 7.0. The authors of another publication (Yang & Zhang, 2019) found that the use of

P. pastoris GS115 yeast and the pPIC9K plasmid as an expression vector enabled the production of transglutaminase at the level of 0.7 U/mL.

The obtained enzyme has been successfully used in research aimed at cross-linking soy protein isolates and myofibrillar chicken proteins.

Song, Shao, Guo, Wang, and Cai (2019) evaluated the impact of gene copy number on the biosynthesis of recombinant MTGase by P. pastoris.

The authors concluded that the expression level of active MTGase could be improved by directional increasing copy of target gene. They also reported that the maximum enzyme activity reached 1.41 U/mL under optimal culture condition.

In the studies of Li et al. (2019) the authors compared the effect of using different PFLD1 and PTEF1 promoters on the expression of (maize-­ derived) transglutaminase in the yeast P. pastoris GS115. As a result of the conducted research, higher expression of this enzyme in yeast cells was found due to the induction of the PFLD1 promoter. The trans­ glutaminase activity was 635 U/L. After purification by chromatography (SP Sepharose medium), the enzyme activity was 3.8 U. It is worth noting that the obtained recombinant enzyme effectively increased the gel strength of the acid-induced milk protein concentrate and its stability.

Very little research has been reported about the biosynthesis of

MTGase by Y. lipolytica. In a remarkable study, Liu et al. (2015) cloned and expressed pro-mTGase from S. hygroscopicus in Y. lipolytica. The highest yield of extracellular pro-mTGase was obtained 5.3 U/mL.

4.3. Random and site-directed mutagenesis Amino acid sequences of pro-MTGase and mature enzyme domain can affect the thermostability, solubility, and even the yield of the enzyme; therefore, in recent years, some experiments were conducted to optimize MTGase characteristics by changing amino acid sequences through mutagenesis methods. Marx, Hertel, and Pietzsch (2008)

M. Akbari et al.

Trends in Food Science & Technology 110 (2021) 458–469

463 developed a screening method that included proteolytic activation of the expressed soluble zymogen. They reported that after only one round of mutagenesis, some mutant enzymes with a single amino acid exchange showed significantly increased thermostability at 60 ◦C. In another study, Yokoyama et al. (2010) obtained MTGase with the specific ac­ tivity of 45 U/mg, which was 1.7 times higher than that of the wild-type enzyme (22.7 U/mg). For this purpose, they used a novel method of rotational mutagenesis called water accessible surface hot-space region-oriented mutagenesis (WASH-ROM).

Recently, Jiang et al. (2017) applied the atmospheric and room-temperature plasma (ARTP) mutagenesis to improve the fermen­ tation production of MTGase. The best mutant exhibited a maximum activity of 5.85 U/mL during flask fermentation, which represented a

27% increase as compared to that achieved using the wild-type strain (4.6 U/mL). The enhanced MTGase biosynthesis caused by ARTP mutagenesis may be attributed to alterations in the structural gene of pro-MTGase or the relevant genes regulating the expression of pro-MTGase. In another study, Liu et al. (2019) applied direct evolution strategy to increase the activity and thermostability of MTGase pro­ duced by S. mobaraensis. based on the results, mutant MTGase showed a

1.95 times specific activity of wild-type one at 50 ◦C.

Recently, B¨ohme et al. (2020) investigated the possibility of obtaining thermostable MTGase enzymes derived from S. mobarensis strains. Such enzymes are characterized by a wider range of applica­ tions, which in turn affects the extension of durability and stability of the obtained products. For the overproduction of recombinant proteins, vectors allowing for the induced expression of proteins in E. coli

BL21Gold (DE3) cells were used. The authors, by performing random mutagenesis aimed at substituting amino acids in five positions, ob­ tained the recombinant MTGase protein, which was characterized by greater thermostability at 60 ◦C (t 1/2 = 38 min).

5. Application of MTGase in food products MTGase is one of the important topics of interest in the food industry because of its benefits in practical and commercial utilization. Many researchers have recommended the use of MTGase in various food products such as meat products, cheese, yogurt, and bread, which are discussed below (Fig. 2).

5.1. Application of MTGase in meat products Numerous studies are available regarding the use of MTGase in meat products (Table 3). One of the most widespread applications of MTGase

Fig. 2. The use of transglutaminase in various food products.

Table 3 MTGase application in meat products.

Meat product Functional properties Treatment condition (enzyme concentration, temperature and time of incubation, etc.)

References Low-salt restructured caiman steaks Physico- chemical and sensory attributes

1% in combination with salt replacers (KCl and MgCl2); without incubation

Canto et al. (2014) Low-salt ground meat Cooking loss, textural properties

1% alone or in combination with alginate and fibrimex; cooked at 74 ◦C

Atilgan and Kilic (2017) dry-cured formed ham Sensory parameters

0.05–0.8%, smoked at 20 ◦C for 2 h, and relative humidity of

85% Jira, Sadeghi-Mehr, Brüggemann, and Schw¨agele (2017)

Pork batter Rheological and textural properties, cooking loss

0.5% alone or in combination with 0.5% sodium tripolyphosphate; incubated at 4 ◦C for 4 h

Lesiow, Rentfrow, and Xiong (2017) Protein- enriched restructured beef steaks

Binding strength, textural parameters, cooking loss, thawing loss

1% (w/w) in combination with pea protein isolate, rice protein or lentil flour; incubated at 4 ◦C for

16–18 h Baugreet et al. (2018) Chicken burger Cooking loss, texture value, and sensory parameters

0.2–1% (w/w); baked at 180 ◦C for 5 min Uran and Yilmaz (2018)

Restructured pork Textural properties 3 U/g, incubated at

85 ◦C for 30 min Yang and Zhang (2019) purified myosin from pork

Cross-linking degree 1:20 (enzyme: substrate); at 4 ◦C

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464 in the food industry is the restructuring of meat. According to Canto et al. (2014), MTGase addition to low-salt restructured caiman steaks decreased the cooking loss and improved textural properties without affecting the sensory parameters. This enzyme also permits the use of low-quality raw materials such as collagen, blood proteins, and me­ chanically deboned meat in meat products with a higher nutritional value by supplementing them with deficient amino acids such as exog­ enous lysine (Kieliszek & Misiewicz, 2014).

MTGase improves the texture and gel strength of meat products (red meat and chicken meat) by forming Gln-Lys isopeptide bonds in actin and myosin, which are the two major proteins of the myofibrillar pro­ teins. Uran and Yilmaz (2018) investigated the quality characteristics of chicken burgers produced with MTGase and found that the texture value of burger samples increased with the increase in enzyme concentration when compared with that of the control sample. In another study,

Ahhmed et al. (2007) evaluated the texture of chicken and beef sausages by using MTGase. The authors also investigated the content of Gln-Lys and extractability of myofibrillar proteins and reported that the breaking strength value increased in both meat types containing

MTGase, especially for beef cooked at 80 ◦C, and that treatment with

MTGase significantly increased the Gln-Lys content in both meat types.

It has been demonstrated that the cross-linking activity of MTGase in meat depends on pH, temperature, ionic strength, and protein surface charge. Castro-Briones et al. (2009) showed that the mechanical prop­ erties of restructured beef gels with 0.3% MTGase could be improved by incubating at 50 ◦C for 30 min and cooking at 90 ◦C for 15 min. They suggested that an increase in the mechanical properties of gels incubated at 50 ◦C could be due to sufficient denaturation of muscle proteins. In another study, Sun and Arntfield (2011) reported that appropriate amounts of MTGase increased the gel stiffness and strength of myofi­ brillar protein isolate in chicken meat. The authors also showed that final heating temperature, pH value, and NaCl concentration influenced gel stiffness so that the maximum gel stiffness was achieved at 95 ◦C, pH

6.0, and 0.9 M NaCl.

MTGase has been shown to improve other characteristics of meat products, such as gelation, water-binding, emulsion stability, and cooking loss. Herrero, Cambero, Ord´o˜nez, De La Hoz, and Carmona (2008) examined the effect of MTGase on the structural properties of meat systems. They found that the enzyme significantly changed the secondary structure of myosin heavy chain by remarkably reducing α-helix content and increasing β-sheet content, resulting in the forma­ tion of high-molecular-weight polymers. These structural modifications led to the formation of strong gels with compact and ordered structural properties that allow to improve hardness, springiness, and cohesive­ ness. In another study, Han, Zhang, Fei, Xu, and Zhou (2009) concluded that the addition of MTGase to pork myofibrillar protein gel increased

WHC due to the formation of a more porous microstructure.

According to the results of previous studies, different degrees of response were observed in gelation of meat substrates induced by

MTGase. This difference is probably due to the variation in muscle physiology and morphogenesis, the amount of lysine and glutamine residues that can act as a substrate for the enzyme, and the presence of enzyme inhibitors that may be present in the reaction system. For example, Ahhmed et al. (2009) observed that the activity of MTGase was different in chicken and beef, such that the elasticity of chicken was lower than that of beef, which was due to the presence of aminopepti­ dase H in chicken myofibrils that have a high level of activity against almost all substrates of MTGase.

Several studies have also been conducted to evaluate the effect of

MTGase in improving the interaction of meat proteins and nonmeat proteins. Kilic (2003) investigated the binding effect of MTGase in chicken doner kebab and showed that the effect of the enzyme on the binding properties of doner kebab was more effective when used with sodium caseinate. Sodium caseinate was a better nonmeat protein sub­ strate for MTGase due to a high degree of cross-linking to myosin (Pie­ trasik, Jarmoluk, & Shand, 2007). In another study, Baugreet, Kerry,

Allen, Gallagher, and Hamill (2018) evaluated the effect of MTGase on the physicochemical characteristics of restructured beef steaks enriched with pea protein isolate, rice protein, or lentil flour. They concluded that

MTGase produced better binding in combination with plant proteins.

This result is in close agreement with the observation of Carballo,

Ayo, and Colmenero (2006) who evaluated the effect of MTGase in combination with sodium caseinate (MTG/C) (1.5 g/100 g) on the water binding and textural properties of meat batter in the presence of NaCl (1.5 g/100 g) and sodium tripolyphosphate (0.5 g/100 g) for pork, chicken, and lamb. Products containing the MTG/C system and salts had higher hardness and chewiness, and the efficiency of the MTG/C system as a texture conditioner changed with the meat source. The authors concluded that MTGase with sodium caseinate forms a viscous sol that acts as a glue to bind restructured meat pieces together.

5.2. Application of MTGase in yogurt TGase in yogurt catalyzes the bonding reaction in proteins, leading to the stabilizing effect of transverse, cross-linking covalent bonds. This enzyme is also involved in the cross-linking of adjacent chains or in the formation of a polypeptide chain loop. This leads to changes in protein conformation and thus in gelation stability and water binding capacity, which consequently changes the rheological properties of protein products.

The use of MTGase in yogurt not only improves its nutritional and functional properties by forming a bond between glutamine and lysine, but also decreases the production cost by decreasing the content of skim milk powder, stabilizer, and even fat in the formulation. A previous study reported that the effect of MTGase treatment with 45 U/L almost resembled that of adding 3% skimmed milk powder in yogurt formula­ tion (Mahmood & Sebo, 2012).

MTGase can incorporated into the yogurt in the following two ways: (1) adding MTGase to the raw milk incubated prior to fermentation followed by enzyme inactivation and (2) adding MTGase simultaneously with a starter culture to the milk; in this case, the enzyme is active during storage and may cause negative textural changes during shelf life. Some studies have been used a heating treatment (70–90 ◦C, 1–15 min) to inactivate MTGase in the finished product (Gharibzahedi &

Chronakis, 2018).

MTGase improves gel quality properties of yogurt, such as gel strength/firmness, viscosity, and WHC. Ziarno and Zaręba (2020) re­ ported that the hardness of MTGase-treated set yogurt increased as compared to that of the control yogurt when MTGase was added at 12 h prior to fermentation. Similar results were reported by Cancino, Fuentes,

Kulozik, and B¨onisch (2006) who observed an increase in the firmness of both set and stirred yogurts with increasing enzyme concentration. It is worth mentioning that caseins are more effective substrate than whey proteins for enzymatic modification by MTGase probably due to their open structure and the better accessibility of lysine residues, while whey proteins, i.e., β-lactoglobulins and α-lactalbumins, prevent the forma­ tion of covalent links between them by enzymatic reactions due to their globular dense structure stabilized by disulfide bonds. Therefore, whey proteins need modification before enzymatic cross-linking (Jaros, Hei­ dig, & Rohm, 2007).

Jaros et al. (2007) evaluated the rheological parameters of stirred yogurt produced from skim milk incubated with MTGase. The results showed a significant but shear rate-dependent effect of enzymatic cross-linking on the properties of stirred yogurt, which further enhanced the viscosity at low shear rates or shear stresses. This finding is consis­ tent with the observation of Pakseresht, Mazaheri Tehrani, and Razavi (2017) who reported that yogurts treated with MTGase had significantly higher viscosity values than the untreated sample. This is largely because of conversion of protein monomers into high-molecular-weight polymers by MTGase action.

Rahila, Kumar, Mann, and Koli (2016) showed that MTGase addition to low-fat set dahi (Indian traditional yogurt) increased WHC up to

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465 17.4% in skim milk dahi and up to 19% in partially skimmed milk dahi.

In another study, Chen et al. (2018) stated that milk protein concentrate (MPC) modified with MTGase greatly enhanced the gel strength, vis­ cosity, and WHC of stirred yogurt. They also investigated the gelling mechanism of modified MPC and concluded that optimal MPC gel properties were achieved after MTGase treatment at pH 7.25 and 35 ◦C for 1 h using 2.5 U/g enzyme.

Some studies have been conducted to evaluate the effect of MTGase treatment on the growth behavior of starter culture in yogurt. Neve,

Lorenzen, Mautner, Schlimme, and Heller (2001) showed a minor imbalance in the associative growth of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus. The authors reported that viable counts for the lactobacilli in MTGase-treated yogurt were slightly lower than those in the untreated sample. This was attributed to the decreased availability of nitrogen sources such as low-molecular-weight peptides and amino acids needed for lactic acid bacteria to produce proteinases. In contrast, Dinkci (2012) reported that MTGase incorpo­ ration at different concentrations (0.74, 1.29, and 1.85 U/g protein) had no significant effect on the viable counts of strained yogurt bacteria.

In most studies, MTGase incorporation did not have any unfavorable effect on the sensory attributes of yogurt (Domagała, 2013; S¸anlı, Sez­ gin, Deveci, S¸enel, & Benli, 2011). However, Ozer, Kirmaci, Oztekin,

Hayaloglu, and Atamer (2007) reported a negative correlation between

MTGase levels and acetaldehyde content in yogurts. This was attributed to the slow metabolic activity of the starter bacteria in the presence of

MTGase or an interfering enzyme along with the mechanism of acetal­ dehyde synthesis from amino acids.

Some studies have been performed on the microstructural properties of yogurt treated by MTGase. The microstructure of yogurt treated with the enzyme is denser and more compact than that of the control, which could be attributed to improved gel firmness and less syneresis (Farns­ worth, Li, Hendricks, & Guo, 2006). Ziarno and Zaręba (2020) reported that MTGase addition increased network density in the yogurt gel and thus reduced the pore size because of the polymerization of caseins and whey proteins.

5.3. Application of MTGase in cheese Several experiments have been conducted on the use of MTGase in cheese production. Significant improvements in the yield and WHC, texture, rheology, and sensory properties of cheese, without changes in its chemical composition, are considered to be crucial advantages of

MTGase treatment (Gharibzahedi et al., 2018; Romeih & Walker, 2017).

However, the use of MTGase is uncommon in the production of hard cheese, mainly due to the delayed ripening process resulting from the slow microbial proteolytic activity. This is particularly observed when

MTGase remains active in the curd, with continuous cross-linking ac­ tivity during cheese ripening (Metwally, Badran, Emara, & Ali, 2014).

Overall, several approaches have been proposed for producing nat­ ural cheese with MTGase (Fig. 3): (1) adding MTGase to the raw milk prior to pasteurization, which deactivates the enzyme (Prakasan,

Chawla, & Sharma, 2015); (2) adding MTGase simultaneously with starter culture (Darnay, Kr´alik, Oros, Koncz, & Firtha, 2017); (3) adding

MTGase before rennet addition (Cozzolino et al., 2003); (4) adding

MTGase simultaneously with rennet to the milk (Cozzolino et al., 2003;

Pierro et al., 2010; Sayadi, Madadlou, & Khosrowshahi, 2013); (5) adding MTGase after rennet action (Cozzolino et al., 2003; De S´a &

Bordignon-Luiz, 2010; Hu et al., 2013); and (6) adding MTGase after cutting of the coagulum (Pierro et al., 2010).

The step of MTGase addition in cheese manufacturing can affect milk coagulation time (Cozzolino et al., 2003). MTGase addition before the addition of rennet retards the coagulation time due to competitive re­ actions between MTGase and chymosin during coagulation. In this case,

MTGase probably catalyzes κ-casein cross-linking because of their pe­ ripheral position in casein micelles. Consequently, the tendency of mi­ celles to coagulate on the micelle surface decreases. However, ¨Ozer,

Guyot, and Kulozik (2012) concluded that the coagulation time decreased with the decrease in the initial pH of milk to 6.3 and increased coagulation temperature when MTGase was pre-incubated with raw milk.

Di Pierro et al. (2010) demonstrated that the addition of MTGase after incubation of milk with chymosin and the cutting of the coagulum produced a higher cheese yield (~26%) and protein content (16.8%) than that for the control cheese without MTGase. The addition of

MTGase simultaneously with clotting enzyme improved the cheese yield and protein content by only ~10.1% and 0.6%, respectively. This sug­ gested the entrapment of the whey proteins β-lactoglobulin and α-lact­ albumin into the curd caseins by cross-linking activity of the enzyme, resulting in a concurrent increase in both the yield and nutritional value of the produced cheese product (Cozzolino et al., 2003).

Fig. 3. Production scheme for natural cheese using MTGase.

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466 Some studies have investigated the use of MTGase in processed cheese production. De S´a and Bordignon-Luiz (2010) concluded that the addition of MTGase after rennet addition was more effective to improve physical properties (reduced syneresis index and increased consistency index) of processed cheese as compared to that of MTGase addition before or simultaneously with rennet. Furthermore, MTGase treatment did not affect the meltability of processed Kashar cheese as reported by

Topcu, Bulat, and ¨Ozer (2020). B¨onisch, Heidebach, and Kulozik (2008) investigated the effect of MTGase on the coagulation properties of rennet by using yeast extract as a source of glutathione (GSH). They found that the simultaneous action of MTGase + GSH and rennet increased curd yield. This may be due to enhanced serum binding of the gel network stabilized by the formation of cross-links between whey proteins and curd proteins.

Conflicting results have been reported by several researchers regarding the effect of MTGase incorporation into cheese structure.

Mazuknaite, Guyot, Leskauskaite, and Kulozik (2013) concluded that the hardness of MTGase-treated cottage cheese produced without rennet addition was significantly higher than that of the control sample.

Consistent with this finding, Darnay et al. (2017) observed that the addition of MTGase to semi-hard cheese led to increased hardness. In contrast, Sayadi et al. (2013) concluded that MTGase treatment decreased hardness as compared to that of the control low-fat cheese.

The authors evaluated the effect of using MTGase simultaneously with chymosin and with or without fortification with whey protein isolate on the textural properties of low-fat Iranian white cheese. To the best of our knowledge, the differences in cheese formulation (such as fat, protein, and specially rennet content) and the step of rennet addition may ac­ count for these conflicting results.

Treatment of cheese with MTGase leads to the formation of a microstructure containing small particles by intermolecular or intra­ molecular cross-links, which minimizes the interspace volume. There­ fore, this microstructure with a high number of Gln-Lys isopeptide bonds can significantly improve the resistance of finished product against the deformation forces (Mahmood & Sebo, 2012).

5.4. Application of MTGase in bread Changes in dietary trends affect the search for technological solu­ tions for both consumers and producers. One suggestion is the use of

TGase. Its use can contribute to obtain new, high-quality bread. The search for new technological solutions and the production of high- quality and attractive products should interest the consumers. The use of enzymatic preparations is a modern solution. The most important features of enzyme preparations are accelerated production processes, increased quality, and extended durability. TGase offers such possibil­ ities, because of which it has become an attractive baking additive.

MTGase produces advantageous effects during breadmaking that are similar to those rendered by traditional oxidizing improvers such as glucose oxidase, likely due to the formation of disulfide cross-links (Gerrard et al., 1998). MTGase is commonly used as a cross-linker of proteins in dough handling processes (Table 4) to increase dough strength, and it can convert weak gluten to stronger gluten by its effect on the rheological behavior of the dough (Ceresino, Kuktaite, Sato,

Hedenqvist, & Johansson, 2019; Scarnato et al., 2017; Steffolani,

Ribotta, P´erez, & Le´on, 2010).

Scarnato et al. (2017) evaluated the effects of the application of

MTGase on the rheological properties of bread. The authors concluded that low levels of MTGase improved crust and crumb properties. This result is in accordance with the study of Gerrard et al. (1998) who evaluated the effects of MTGase on dough properties and crumb strength of white pan bread. They reported that MTGase addition increased the relaxation time as a quantitative indicator of dough development.

MTGase can form disulfide linkages through covalent cross-links that are very important in dough development. In another study, Sal­ menkallio-Marttila, Roininen, Autio, and L¨ahteenm¨aki (2004) reported that MTGase treatment made the oat bread harder and gummier than the bread baked without the enzyme.

Seravalli, Iguti, Santana, and Filho (2011) reported that MTGase treatment increased the amount of water added to the finished product.

This finding is consistent with the observation of Gerrard et al. (1998) who reported increased water absorption of bread containing MTGase by 6%. This action represents a potential cost-saving for using MTGase in the baking industry. It seems that change in gluten structure by cross-links increases WHC. Another reason can be explained by a side activity of MTGase: hydrolysis of glutamine residues to glutamic acid residues in the protein increases the hydrophilicity of the gluten pro­ teins, allowing a higher affinity for water.

It should be noted that the effect of MTGase markedly relies on the level of enzyme concentration, the quality of wheat flour, and the pro­ tein source used in the bread formulation (Moore, Heinbockel, Dockery,

Ulmer, & Arendt, 2006; Steffolani et al., 2010). Both Steffolani et al. (2010) and Scarnato et al. (2017) observed a negative effect of higher

MTGase levels (0.5%) on volume presumably because of additional cross-linking, resulting in an over-strong dough.

Several studies have been conducted on the production gluten-free bread by MTGase treatment alone or in combination with other in­ gredients such as whey protein, caseinate, and soy proteins (Shin, Gang,

& Song, 2010), egg protein and skim milk powder (Moore et al., 2006), and hydroxypropylmethyl cellulose (HPMC) and soybean protein isolate (Marco & Rosell, 2008). According to Mohammadi, Azizi, Neyestani,

Hosseini, and Mortazavian (2015), the addition of 1 U/g protein of

MTGase caused appropriate crumb texture, and increase in MTGase concentration yielded higher crumb hardness in gluten-free bread based on rice flour. This agrees with the finding of Pongjaruvat, Methacanon,

Seetapan, Fuongfuchat, and Gamonpilas (2014) who found that increasing MTGase concentration increased crumb hardness and chew­ iness. In another study, Dłu˙zewska, Marciniak-Lukasiak, and Kurek

Table 4 Different bread formulations treated with various concentrations of MTGase.

Bread product Enzyme concentration Formulation of the control sample

Functional properties References Pan bread dough 0.03, 0.05, 0.1, 0.15, and

0.17% (flour weight basis) 100 g wheat flour and maize resistant starch mixture, 60 g

MiliRO water, 2 g salt Dough: rheological properties measured by empirical and fundamental methods, gelatinization

Sanchez et al., (2014) Gluten-free bread 0.1 and 10 U/g protein

200 g rice flour, 150 g corn starch, 50 g soy flour, 24 g sodium caseinate, 20 g sugar, 10 g inulin, 7 g salt, 7g dried instant yeast, and 1 g DATEM

Dough: water absorption, development time, resistance and degree of softening, and yield

Mohammadi et al., (2015) Bread: crumb texture, staling, specific volume, and yield

Gluten-free bread 1 and 10 U/g protein 190 g potato starch, 570 g corn starch, 70 g corn flour, 51 g sugar, 15 g salt, 12–24 g yeast, 7.5 or 15 g xanthan gum

Bread: specific mass, specific volume, crumb porosity, moisture, and crumb hardness

Dłu˙zewska et al. (2015) Wheat bread 0.5, 1, 2, and 5 U/g flour

30% sourdough and 70% (wheat flour, water, sugar, salt, baker’s yeast, and olive oil)

Bread: textural properties, crumb morphological features, and shelf-life

Scarnato et al. (2017) Fresh bread 0.5, 0.1, and 0.2% (flour weight basis)

100 g flour, 48 g water, 3.4 g oil, 3.2 g yeast, 1.5 g sugar, and

1.9 g salt Bread: specific volume, textural parameters, color, moisture content and water activity

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467 (2015) showed that the addition of 1 U/g MTGase to gluten-free bread with soy or whey protein improved several physicochemical and sensory attributes of the finished product.

Several studies have suggested that MTGase action in food products might affect the incidence of celiac disease. MTGase can deamidate gluten and thus mimic endogenous tissue TGase, which plays a sub­ stantial role in the pathogenesis of celiac disease by catalyzing deami­ dation of glutamine (Aaron & Torsten, 2019; Amirdivani et al., 2018;

Lerner & Matthias, 2015). Therefore, a safety concern has emerged regarding the treatment of gluten-free bakery products by MTGase.

However, Heil et al. (2017) proved that standard bakery concentrations of MTGase in wheat bread (2–8 U/Kg of flour) have no effects on celiac disease incidence, although it cannot be excluded that higher doses might be correlated with it.

6. Food safety and legislation MTGase has been approved as “Generally Recognized as Safe (GRAS)” with Approval No. GRN 000095 by the Food and Drug

Administration (2001). This enzyme is permitted as a processing aid for food use in many other countries besides EU member states, including, but not limited to, USA, Canada, Brazil, Japan, Korea, China, and

Thailand (Giosafatto et al., 2018). Furthermore, in May 2014, a report of

“Labeling foodstuffs made with the enzyme transglutaminase” was published, which indicated that MTGase as a processing aid is not an ingredient and under the current law must not be labeled in the list of ingredients (Ajinomoto, 2019). In Europe, some regulatory authorities have decided to inform consumers about the MTGase food additive and its labeling (e.g. in Switzerland). According to Aloisi et al. (2016), the use of transglutaminase as a food additive is allowed in some countries.

In accordance with the requirements of EU law on food labeling (Regulation (EU) No 1169/2011 of the European Parliament and of the

Council of October 25, 2011 on the provision of food information to consumers), there is no obligation to mention the ingredients used in the list of ingredients.

7. Conclusions Presently, MTGase is widely used in the food industry because of its unique properties. This enzyme can improve the functional properties of proteins in food systems by forming isopeptide bonds between gluta­ mine and lysine residues. Commercial MTGase is produced by fermen­ tation of S. mobaraensis on the industrial scale, but this system has some drawbacks, including low enzyme yield and problems related to the presence of proteases that can hydrolyze the target proteins. Thus, it is necessary to develop an efficient system for MTGase biosynthesis without the abovementioned limitations. Over the last decades, much progress has been made to improve MTGase properties by using genetic manipulation. For example, some genetically modified strains were improved for obtaining higher enzyme yields, and some strains could also produce efficient MTGases that are more heat stable than wild-type

MTGase. It seems that the biotechnological approaches can be a suitable alternative of conventional fermentation to produce MTGase in the near future.

Declaration of competing interest Authors declare no conflict of interest.

References Aaron, L., & Torsten, M. (2019). Microbial transglutaminase: A new potential player in celiac disease. Clinical Immunology, 199, 37–43. https://doi.org/10.1016/j. clim.2018.12.008

Ahhmed, A. M., Kawahara, S., Ohta, K., Nakade, K., Soeda, T., & Muguruma, M. (2007).

Differentiation in improvements of gel strength in chicken and beef sausages induced by transglutaminase. Meat Science, 76(3), 455–462. https://doi.org/10.1016/j. meatsci.2007.01.002

Ahhmed, A. M., Kuroda, R., Kawahara, S., Ohta, K., Nakade, K., Aoki, T., et al. (2009).

Dependence of microbial transglutaminase on meat type in myofibrillar proteins cross-linking. Food Chemistry, 112(2), 354–361. https://doi.org/10.1016/j. foodchem.2008.05.078

Ajinomoto©. (2019). Gorny law labeling of TG in final product 14 May 2014_en.pdf.

Available: https://www.transglutaminase.com/regulatory-status/labelling-of-foods tuffs-made-enzyme-transglutaminase accessed april 25, 2019.

Aloisi, I., Cai, G., Serafini-Fracassini, D., & Duca, S. D. (2016). Transglutaminase as polyamine mediator in plant growth and differentiation. Amino Acids, 48(10),

2467–2478. https://doi.org/10.1007/s00726-016-2235-y

Amirdivani, S., Khorshidian, N., Fidelis, M., Granato, D., Koushki, M. R.,

Mohammadi, M., et al. (2018). Effects of transglutaminase on health properties of food products. Current Opinion in Food Science, 22, 74–80. https://doi.org/10.1016/j. cofs.2018.01.008

Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., et al. (1989).

Purification and characteristics of a novel transglutaminase derived from microorganisms. Agricultural & Biological Chemistry, 53(10), 2613–2617. https://doi. org/10.1080/00021369.1989.10869735

Atilgan, E., & Kilic, B. (2017). Effects of microbial transglutaminase, fibrimex and alginate on physicochemical properties of cooked ground meat with reduced salt level. Journal of Food Science & Technology, 54(2), 303–312. https://doi.org/

10.1007/s13197-016-2463-x Baugreet, S., Kerry, J. P., Allen, P., Gallagher, E., & Hamill, R. M. (2018).

Physicochemical characteristics of protein-enriched restructured beef steaks with phosphates, transglutaminase, and elasticised package Forming. Journal of Food

Quality, 1–11. https://doi.org/10.1155/2018/4737602, 2018.

B¨ohme, B., Moritz, B., Wendler, J., Hertel, T. C., Ihling, C., Brandt, W., et al. (2020).

Enzymatic activity and thermoresistance of improved microbial transglutaminase variants. Amino Acids, 52(2), 313–326. https://doi.org/10.1007/s00726-019-02764- 9

B¨onisch, M. P., Heidebach, T. C., & Kulozik, U. (2008). Influence of transglutaminase protein cross-linking on the rennet coagulation of casein. Food Hydrocolloids, 22(2),

288–297. https://doi.org/10.1016/j.foodhyd.2006.11.015

Boukid, F., Carini, E., Curti, E., Bardini, G., Pizzigalli, E., & Vittadini, E. (2018).

Effectiveness of vital gluten and transglutaminase in the improvement of physico- chemical properties of fresh bread. LWT-Food Science and Technology, 92, 465–470. https://doi.org/10.1016/j.lwt.2018.02.059

Cancino, B., Fuentes, P., Kulozik, U., & B¨onisch, M. (2006). Effect of the protein addition on the structure of set style and stirred yoghurt with and without the use of transglutaminase. Desalination, 1(200), 531–532.

Canto, A. C., Lima, B. R. C., Suman, S. P., Lazaro, C. A., Monteiro, M. L. G., Conte- Junior, C. A., et al. (2014). Physico-chemical and sensory attributes of low-sodium restructured caiman steaks containing microbial transglutaminase and salt replacers.

Meat Science, 96(1), 623–632. https://doi.org/10.1016/j.meatsci.2013.08.003

Carballo, J., Ayo, J., & Colmenero, F. J. (2006). Microbial transglutaminase and caseinate as cold set binders: Influence of meat species and chilling storage. LWT- Food Science and Technology, 39(6), 692–699. https://doi.org/10.1016/j. lwt.2005.03.020

Castro-Briones, M., Calder´on, G. N., Velazquez, G., Rubio, M. S., V´azquez, M., &

Ramírez, J. A. (2009). Mechanical and functional properties of beef products obtained using microbial transglutaminase with treatments of pre-heating followed by cold binding. Meat Science, 83(2), 229–238. https://doi.org/10.1016/j. meatsci.2009.05.002

Ceresino, E. B., de Melo, R. R., Kuktaite, R., Hedenqvist, M. S., Zucchi, T. D.,

Johansson, E., et al. (2018). Transglutaminase from newly isolated Streptomyces sp.

CBMAI 1617: Production optimization, characterization and evaluation in wheat protein and dough systems. Food Chemistry, 241, 403–410. https://doi.org/10.1016/ j.foodchem.2017.09.010

Ceresino, E. B., Kuktaite, R., Sato, H. H., Hedenqvist, M. S., & Johansson, E. (2019).

Impact of gluten separation process and transglutaminase source on gluten based dough properties. Food Hydrocolloids, 87, 661–669. https://doi.org/10.1016/j. foodhyd.2018.08.035

Chen, L., Li, Y., Han, J., Yuan, D., Lu, Z., & Zhang, L. (2018). Influence of transglutaminase-induced modification of milk protein concentrate (MPC) on yoghurt texture. International Dairy Journal, 78, 65–72. https://doi.org/10.1016/j. idairyj.2017.10.001

Cozzolino, A., Di Pierro, P., Mariniello, L., Sorrentino, A., Masi, P., & Porta, R. (2003).

Incorporation of whey proteins into cheese curd by using transglutaminase.

Biotechnology and Applied Biochemistry, 38(3), 289–295. https://doi.org/10.1042/

BA20030102 Cui, L., Du, G., Zhang, D., Liu, H., & Chen, J. (2007). Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus. Food Chemistry,

105(2), 612–618. https://doi.org/10.1016/j.foodchem.2007.04.020

Darnay, L., Kr´alik, F., Oros, G., Koncz, ´A., & Firtha, F. (2017). Monitoring the effect of transglutaminase in semi-hard cheese during ripening by hyperspectral imaging.

Journal of Food Engineering, 196, 123–129. https://doi.org/10.1016/j. jfoodeng.2016.10.020

De S´a, E. M. F., & Bordignon-Luiz, M. T. (2010). The effect of transglutaminase on the properties of milk gels and processed cheese. International Journal of Dairy

Technology, 63(2), 243–251. https://doi.org/10.1111/j.1471-0307.2010.00568.x

Dinkci, N. (2012). The influence of transglutaminase treatment on functional properties of strained yoghurt. Journal of Animal and Veterinary Advances, 11(13), 2238–2246.

Domagała, J., Wszołek, M., Tamime, A. Y., & Kupiec-Teahan, B. (2013). The effect of transglutaminase concentration on the texture, syneresis and microstructure of set- M. Akbari et al.

Trends in Food Science & Technology 110 (2021) 458–469

468 type goat’s milk yoghurt during the storage period. Small Ruminant Research, 112 (1–3), 154–161. https://doi.org/10.1016/j.smallrumres.2012.12.003

Duarte, L. S., Bars´e, L. Q., Dalberto, P. F., da Silva, W. T. S., Rodrigues, R. C.,

Machado, P., et al. (2020a). Cloning and expression of the Bacillus amyloliquefaciens transglutaminase gene in E. coli using a bicistronic vector construction. Enzyme and

Microbial Technology, 134, 109468. https://doi.org/10.1016/j. enzmictec.2019.109468

Duarte, L., Matte, C. R., Bizarro, C. V., & Ayub, M. A. (2020b). Transglutaminases: Part

I—origins, sources, and biotechnological characteristics. World Journal of

Microbiology and Biotechnology, 36(1), 15. https://doi.org/10.3390/biom4010117

Dłu˙zewska, E., Marciniak-Lukasiak, K., & Kurek, N. (2015). Effect of transglutaminase additive on the quality of gluten-free bread. CyTA - Journal of Food, 13(1), 80–86. https://doi.org/10.1080/19476337.2014.917336

Eshra, D. H., El-Iraki, S. M., & Bakr, T. A. (2015). Isolation and identification of

Actinomycetes transglutaminase producing strains. International Journal of Current

Science, 18, 76–88.

Farnsworth, J. P., Li, J., Hendricks, G. M., & Guo, M. R. (2006). Effects of transglutaminase treatment on functional properties and probiotic culture survivability of goat milk yogurt. Small Ruminant Research, 65(1–2), 113–121. https://doi.org/10.1016/j.smallrumres.2005.05.036

Fatima, S. W., Tiwari, R., & Khare, S. K. (2019). Utilization of agro-industrial waste for production of transglutaminase from Streptomyces mobaraensis. Bioresource

Technology, 287, 121391. https://doi.org/10.1016/j.biortech.2019.121391

Food and Drug Administration. (2001). Transglutaminase GRAS notification’,

Washington, D.C. Available:https://www.fda.gov/downloads/food/ingredientspac kaginglabeling/gras/noticeinventory/ucm267031.pdf accessed april 25, 2019.

Fu, L., Wang, Y., Ju, J., Cheng, L., Xu, Y., Yu, B., et al. (2020). Extracellular production of active-form Streptomyces mobaraensis transglutaminase in Bacillus subtilis. Applied

Microbiology and Biotechnology, 104(2), 623–631.

Gerrard, J. A., Fayle, S. E., Wilson, A. J., Newberry, M. P., Ross, M., & Kavale, S. (1998).

Dough properties and crumb strength of white pan bread as affected by microbial transglutaminase. Journal of Food Science, 63(3), 472–475. https://doi.org/10.1111/ j.1365-2621.1998.tb15766.x

Gharibzahedi, S. M. T., & Chronakis, I. S. (2018). Crosslinking of milk proteins by microbial transglutaminase: Utilization in functional yogurt products. Food

Chemistry, 245, 620–632. https://doi.org/10.1016/j.foodchem.2017.10.138

Gharibzahedi, S. M. T., Koubaa, M., Barba, F. J., Greiner, R., George, S., & Roohinejad, S. (2018). Recent advances in the application of microbial transglutaminase crosslinking in cheese and ice cream products: A review. International Journal of

Biological Macromolecules, 107, 2364–2374. https://doi.org/10.1016/j. ijbiomac.2017.10.115

Giosafatto, C. V. L., Al-Asmar, A., & Mariniello, L. (2018). Transglutaminase protein substrates of food interest. In Kuddus M. Enzymes in food technology: Improvement and innovation (pp. 293–317). Singapore: Springer Nature Singapore Private Limited.

Glodowsky, A. P., Ruberto, L. A., Martorell, M. M., Mac Cormack, W. P., & Levin, G. J. (2020). Cold active transglutaminase from antarctic Penicillium chrysogenum: Partial purification, characterization and potential application in food technology.

Biocatalysis and Agricultural Biotechnology, 29, 101807. https://doi.org/10.1016/j. bcab.2020.101807

Guerra-Rodríguez, E., & V´azquez, M. (2014). Evaluation of a novel low-cost culture medium containing exclusively milk, potato and glycerol for microbial transglutaminase production by Streptomyces mobaraensis. Chemical Engineering

Research and Design, 92(4), 784–791. https://doi.org/10.1016/j.cherd.2013.06.027

Gundersen, M. T., Keillor, J. W., & Pelletier, J. N. (2014). Microbial transglutaminase displays broad acyl-acceptor substrate specificity. Applied Microbiology and

Biotechnology, 98(1), 219–230. https://doi.org/10.1007/s00253-013-4886-x

Han, X., Ma, C., Qin, L., & Liu, L. (2020). Cloning and expression of maize transglutaminase gene in Escherichia coli and its action over dairy proteins. Journal of

Food Processing and Preservation, 44(10), Article e14756. https://doi.org/10.1111/ jfpp.14756

Han, M., Zhang, Y., Fei, Y., Xu, X., & Zhou, G. (2009). Effect of microbial transglutaminase on NMR relaxometry and microstructure of pork myofibrillar protein gel. European Food Research and Technology, 228(4), 665–670. https://doi. org/10.1007/s00217-008-0976-x

Heil, A., Ohsam, J., van Genugten, B., Diez, O., Yokoyama, K., Kumazawa, Y., et al. (2017). Microbial transglutaminase used in bread preparation at standard bakery concentrations does not increase immunodetectable amounts of deamidated gliadin.

Journal of Agricultural and Food Chemistry, 65(32), 6982–6990. https://doi.org/

10.1021/acs.jafc.7b02414 Herrero, A. M., Cambero, M. I., Ord´o˜nez, J. A., De La Hoz, L., & Carmona, P. (2008).

Raman spectroscopy study of the structural effect of microbial transglutaminase on meat systems and its relationship with textural characteristics. Food Chemistry, 109 (1), 25–32. https://doi.org/10.1016/j.foodchem.2007.12.003

Hu, Y. N., Ge, K. S., Jiang, L., Guo, H. Y., Luo, J., Wang, F., et al. (2013). Effect of transglutaminase on yield, compositional and functional properties of low-fat

Cheddar cheese. Food Science and Technology Research, 19(3), 359–367. https://doi. org/10.3136/fstr.19.359

Jaros, D., Heidig, C., & Rohm, H. (2007). Enzymatic modification through microbial transglutaminase enhances the viscosity of stirred yogurt. Journal of Texture Studies,

38(2), 179–198. https://doi.org/10.1111/j.1745-4603.2007.00093.x

Javitt, G., Ben-Barak-Zelas, Z., Jerabek-Willemsen, M., & Fishman, A. (2017).

Constitutive expression of active microbial transglutaminase in Escherichia coli and comparative characterization to a known variant. BMC Biotechnology, 17(1), 23. https://doi.org/10.1186/s12896-017-0339-4

Jiang, Y., Shang, Y. P., Li, H., Zhang, C., Pan, J., Bai, Y. P., et al. (2017). Enhancing transglutaminase production of Streptomyces mobaraensis by iterative mutagenesis breeding with atmospheric and room-temperature plasma (ARTP). Bioresources and

Bioprocessing, 4(1), 37. https://doi.org/10.1186/s40643-017-0168-2

Jin, M., Huang, J., Pei, Z., Huang, J., Gao, H., & Chang, Z. (2016). Purification and characterization of a high-salt-resistant microbial transglutaminase from

Streptomyces mobaraensis. Journal of Molecular Catalysis B: Enzymatic, 133, 6–11. https://doi.org/10.1016/j.molcatb.2016.07.005

Jira, W., Sadeghi-Mehr, A., Brüggemann, D. A., & Schw¨agele, F. (2017). Production of dry-cured formed ham with different concentrations of microbial transglutaminase:

Mass spectrometric analysis and sensory evaluation. Meat Science, 129, 81–87. https://doi.org/10.1016/j.meatsci.2017.02.021

Juettner, N. E., Classen, M., Colin, F., Hoffmann, S. B., Meyners, C., Pfeifer, F., et al. (2018). Features of the transglutaminase-activating metalloprotease from

Streptomyces mobaraensis DSM 40847 produced in Escherichia coli. Journal of

Biotechnology, 281, 115–122. https://doi.org/10.1016/j.jbiotec.2018.07.004

Kieliszek, M., & Bła˙zejak, S. (2017). In R. C. Ray, & C. M. Rosell (Eds.), Microbial transglutaminase and applications in food industry. Book chapter in: Microbial Enzyme

Technology in Food Applications (pp. 180–194). https://doi.org/10.1201/

9781315368405 Kieliszek, M., & Misiewicz, A. (2014). Microbial transglutaminase and its application in the food industry. A review. Folia Microbiologica, 59(3), 241–250. https://doi.org/

10.1007/s12223-013-0287-x Kilic, B. (2003). Effect of microbial transglutaminase and sodium caseinate on quality of chicken d¨oner kebab. Meat Science, 63(3), 417–421. https://doi.org/10.1016/S0309- 1740(02)00102-X

Lerner, A., & Matthias, T. (2015). Possible association between celiac disease and bacterial transglutaminase in food processing: A hypothesis. Nutrition Reviews, 73(8),

544–552. https://doi.org/10.1093/nutrit/nuv011 Lesiow, T., Rentfrow, G. K., & Xiong, Y. L. (2017). Polyphosphate and myofibrillar protein extract promote transglutaminase-mediated enhancements of rheological and textural properties of PSE pork meat batters. Meat Science, 128, 40–46. https:// doi.org/10.1016/j.meatsci.2017.02.002

Lin, S. J., Hsieh, Y. F., Lai, L. A., Chao, M. L., & Chu, W. S. (2008). Characterization and large-scale production of recombinant Streptoverticillium platensis transglutaminase.

Journal of Industrial Microbiology & Biotechnology, 35(9), 981–990. https://doi.org/

10.1007/s10295-008-0373-2 Liu, Y., Huang, L., Shan, M., Sang, J., Li, Y., Jia, L., et al. (2019). Enhancing the activity and thermostability of Streptomyces mobaraensis transglutaminase by directed evolution and molecular dynamics simulation. Biochemical Engineering Journal, 151,

107333. https://doi.org/10.1016/j.bej.2019.107333 Liu, S., Wang, M., Du, G., & Chen, J. (2016). Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization. BMC Biotechnology, 16(1), 75. https://doi.org/10.1186/s12896-016- 0304-7

Liu, S., Wan, D., Wang, M., Madzak, C., Du, G., & Chen, J. (2015). Overproduction of pro- transglutaminase from Streptomyces hygroscopicus in Yarrowia lipolytica and its biochemical characterization. BMC Biotechnology, 15(1), 75. https://doi.org/

10.1186/s12896-015-0193-1 Liu, S., Zhang, D., Wang, M., Cui, W., Chen, K., Du, G., et al. (2011). The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide. Microbial Cell Factories, 10(1), 112. https://doi.org/10.1186/1475-2859-10-112

Li, C., & Xiong, Y. L. (2019). Mild oxidation promotes myosin S2 cross-linking by microbial transglutaminase. Food Chemistry, 287, 390–397. https://doi.org/

10.1016/j.foodchem.2019.02.097 Li, H., Zhang, T., Li, J., Li, H., Xu, Y., & Yu, J. (2019). Expression of Zea mays transglutaminase in Pichia pastoris under different promoters and its impact on properties of acidified milk protein concentrate gel. Journal of the Science of Food and

Agriculture, 99(10), 4518–4523. https://doi.org/10.1002/jsfa.9688

Mahmood, W. A., & Sebo, N. H. (2012). Improvement of yogurt properties by microbial transglutaminase. Jordan Journal of Agricultural Sciences, 173(802), 1–21.

Marco, C., & Rosell, C. M. (2008). Breadmaking performance of protein enriched, gluten- free breads. European Food Research and Technology, 227(4), 1205–1213. https://doi. org/10.1007/s00217-008-0838-6

Mariniello, L., Di Pierro, P., Giosafatto, C. V. L., Sorrentino, A., & Porta, R. (2008).

Transglutaminase in food biotechnology. In R. Porta, P. Di Pierro, & L. Mariniello (Eds.), Recent research developments in food biotechnology. Enzymes as additives or processing aids (pp. 185–211). Trivandrum, Kerala: Research Signpost.

Martins, I. M., Matos, M., Costa, R., Silva, F., Pascoal, A., Estevinho, L. M., et al. (2014).

Transglutaminases: Recent achievements and new sources. Applied Microbiology and

Biotechnology, 98(16), 6957–6964. https://doi.org/10.1007/s00253-014-5894-1

Marx, C. K., Hertel, T. C., & Pietzsch, M. (2007). Soluble expression of a pro- transglutaminase from Streptomyces mobaraensis in Escherichia coli. Enzyme and

Microbial Technology, 40(6), 1543–1550. https://doi.org/10.1016/j. enzmictec.2006.10.036

Marx, C. K., Hertel, T. C., & Pietzsch, M. (2008). Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat-sensitive variants. Journal of Biotechnology, 136(3–4), 156–162. https://doi.org/10.1016/j. jbiotec.2008.06.005

Mazuknaite, I., Guyot, C., Leskauskaite, D., & Kulozik, U. (2013). Influence of transglutaminase on the physical and chemical properties of acid milk gel and cottage type cheese. Journal of Food Agriculture and Environment, 11, 119–124.

Metwally, A. M. M., Badran, S. M., Emara, E. A., & Ali, H. H. E. (2014). The use of transglutaminase enzyme in processing ras cheese. Mansoura Journal of Food and

Dairy Sciences, 5(10), 717–723. https://doi.org/10.21608/jfds.2014.53207

Mohammadi, M., Azizi, M. H., Neyestani, T. R., Hosseini, H., & Mortazavian, A. M. (2015). Development of gluten-free bread using guar gum and transglutaminase.

M. Akbari et al.

Trends in Food Science & Technology 110 (2021) 458–469

469 Journal of Industrial and Engineering Chemistry, 21, 1398–1402. https://doi.org/

10.1016/j.jiec.2014.06.013 Moore, M. M., Heinbockel, M., Dockery, P., Ulmer, H. M., & Arendt, E. K. (2006).

Network formation in gluten-free bread with application of transglutaminase. Cereal

Chemistry, 83(1), 28–36. https://doi.org/10.1094/CC-83-0028

Mu, D., Lu, J., Qiao, M., Kuipers, O. P., Zhu, J., Li, X., et al. (2018). Heterologous signal peptides-directing secretion of Streptomyces mobaraensis transglutaminase by Bacillus subtilis. Applied Microbiology and Biotechnology, 10, 5533–5543. https://doi.org/

10.1007/s00253-018-9000-y Neve, H., Lorenzen, P. C., Mautner, A., Schlimme, E., & Heller, K. J. (2001). Effects of transglutaminase treatment on the production of set skim milk yoghurt:

Microbiological aspects. Kieler Milchwirtschaftliche Forschungsberichte, 53(4),

347–361, 2001.

Noda, S., Miyazaki, T., Tanaka, T., Ogino, C., & Kondo, A. (2012). Production of

Streptoverticillium cinnamoneum transglutaminase and cinnamic acid by recombinant

Streptomyces lividans cultured on biomass-derived carbon sources. Bioresource

Technology, 104, 648–651. https://doi.org/10.1016/j.biortech.2011.10.045

¨Ozer, B., Guyot, C., & Kulozik, U. (2012). Simultaneous use of transglutaminase and rennet in milk coagulation: Effect of initial milk pH and renneting temperature.

International Dairy Journal, 24(1), 1–7. https://doi.org/10.1016/j. idairyj.2011.10.002

Ozer, B., Kirmaci, H. A., Oztekin, S., Hayaloglu, A., & Atamer, M. (2007). Incorporation of microbial transglutaminase into non-fat yogurt production. International Dairy

Journal, 17(3), 199–207. https://doi.org/10.1016/j.idairyj.2006.02.007

¨Ozçelik, A. T., Ers¨oz, F., & ˙Inan, M. (2019). Extracellular production of the recombinant bacterial transglutaminase in Pichia pastoris. Protein Expression and Purification, 159,

83–90. https://doi.org/10.1016/j.pep.2019.03.003 Pakseresht, S., Mazaheri Tehrani, M., & Razavi, S. M. A. (2017). Optimization of low-fat set-type yoghurt: Effect of altered whey protein to casein ratio, fat content and microbial transglutaminase on rheological and sensorial properties. Journal of Food

Science & Technology, 54(8), 2351–2360. https://doi.org/10.1007/s13197-017- 2675-8

Pierro, P. D., Mariniello, L., Sorrentino, A., Giosafatto, C. V. L., Chianese, L., & Porta, R. (2010). Transglutaminase-induced chemical and rheological properties of cheese.

Food Biotechnology, 24(2), 107–120. https://doi.org/10.1080/08905431003784465

Pietrasik, Z., Jarmoluk, A., & Shand, P. J. (2007). Effect of non-meat proteins on hydration and textural properties of pork meat gels enhanced with microbial transglutaminase. LWT-Food Science and Technology, 40(5), 915–920. https://doi. org/10.1016/j.lwt.2006.03.003

Pongjaruvat, W., Methacanon, P., Seetapan, N., Fuongfuchat, A., & Gamonpilas, C. (2014). Influence of pregelatinised tapioca starch and transglutaminase on dough rheology and quality of gluten-free jasmine rice breads. Food Hydrocolloids, 36,

143–150. https://doi.org/10.1016/j.foodhyd.2013.09.004

Portilla-Rivera, O. M., T´ellez-Luis, S. J., de Le´on, J. A. R., & V´azquez, M. (2009).

Production of microbial transglutaminase on media made from sugar cane molasses and glycerol. Food Technology and Biotechnology, 47(1), 19–26.

Prakasan, V., Chawla, S. P., & Sharma, A. (2015). Effect of transglutaminase treatment on functional properties of paneer. International Journal of Current Microbiology and

Applied Sciences, 4(5), 227–238.

Rahila, M. P., Kumar, R., Mann, B., & Koli, P. S. (2016). Enzymatic modification of milk proteins for the preparation of low fat dahi. Journal of Food Processing and

Preservation, 40(5), 1038–1046. https://doi.org/10.1111/jfpp.12684

Rickert, M., Strop, P., Lui, V., Melton-Witt, J., Farias, S. E., Foletti, D., et al. (2016).

Production of soluble and active microbial transglutaminase in Escherichia coli for site-specific antibody drug conjugation. Protein Science, 25(2), 442–455. https://doi. org/10.1002/pro.2833

Romeih, E., & Walker, G. (2017). Recent advances on microbial transglutaminase and dairy application. Trends in Food Science & Technology, 62, 133–140. https://doi.org/

10.1016/j.tifs.2017.02.015 Salmenkallio-Marttila, M., Roininen, K., Autio, K., & L¨ahteenm¨aki, L. (2004). Effects of gluten and transglutaminase on microstructure, sensory characteristics and instrumental texture of oat bread. Agricultural and Food Science, 13(1–2), 138–150.

Sanchez, D. B., Puppo, M. C., A˜non, M. C., Ribotta, P. D., Le´on, A. E., & Tadini, C. C. (2014). Effect of maize resistant starch and transglutaminase: A study of fundamental and empirical rheology properties of pan bread dough. Food and

Bioprocess Technology, 7(10), 2865–2876. https://doi.org/10.1007/s11947-013- 1246-x

S¸anlı, T., Sezgin, E., Deveci, O., S¸enel, E., & Benli, M. (2011). Effect of using transglutaminase on physical, chemical and sensory properties of set-type yoghurt.

Food Hydrocolloids, 25(6), 1477–1481. https://doi.org/10.1016/j. foodhyd.2010.09.028

Santhi, D., Kalaikannan, A., Malairaj, P., & Arun Prabhu, S. (2017). Application of microbial transglutaminase in meat foods: A review. Critical Reviews in Food Science and Nutrition, 57(10), 2071–2076. https://doi.org/10.1080/10408398.2014.945990

Sato, R., Minamihata, K., Ariyoshi, R., Taniguchi, H., & Kamiya, N. (2020). Recombinant production of active microbial transglutaminase in E. coli by using self-cleavable zymogen with mutated propeptide. BioRxiv. https://doi.org/10.1101/

2020.02.25.965541 Sayadi, A., Madadlou, A., & Khosrowshahi, A. (2013). Enzymatic cross-linking of whey proteins in low fat Iranian white cheese. International Dairy Journal, 29(2), 88–92. https://doi.org/10.1016/j.idairyj.2012.10.006

Scarnato, L., Montanari, C., Serrazanetti, D. I., Aloisi, I., Balestra, F., Del Duca, S., et al. (2017). New bread formulation with improved rheological properties and longer shelf-life by the combined use of transglutaminase and sourdough. LWT-Food Science and Technology, 81, 101–110. https://doi.org/10.1016/j.lwt.2017.03.042

Seravalli, E. A. G., Iguti, A. M., Santana, I. A., & Filho, F. F. (2011). Effects of application of transglutaminase in wheat proteins during the production of bread. Procedia Food

Science, 1, 935–942. https://doi.org/10.1016/j.profoo.2011.09.141

Shin, M., Gang, D. O., & Song, J. Y. (2010). Effects of protein and transglutaminase on the preparation of gluten-free rice bread. Food Science and Biotechnology, 19(4),

951–956. https://doi.org/10.1007/s10068-010-0133-8

Singh, P., & Kumar, S. (2019). Microbial enzyme in food biotechnology. In Kuddus M.

Enzymes in food biotechnology, 4th edition. Academic press (pp. 19–28).

Song, X., Shao, C., Guo, Y., Wang, Y., & Cai, J. (2019). Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris. BMC Biotechnology, 54, 19. https://doi.org/10.1186/s12896-019-0542-6

Sorde, K. L., & Ananthanarayan, L. (2019). Isolation, screening, and optimization of bacterial strains for novel transglutaminase production. Preparative Biochemistry &

Biotechnology, 49(1), 64–73. https://doi.org/10.1080/10826068.2018.1536986 de Souza, C. F. V., Rodrigues, R. C., & Ayub, M. A. Z. (2009). Effects of oxygen volumetric mass transfer coefficient on transglutaminase production by Bacillus circulans BL32.

Biotechnology and Bioprocess Engineering, 14(5), 571–576. https://doi.org/10.1007/ s12257-008-0076-6

Steffolani, M. E., Ribotta, P. D., P´erez, G. T., & Le´on, A. E. (2010). Effect of glucose oxidase, transglutaminase, and pentosanase on wheat proteins: Relationship with dough properties and bread-making quality. Journal of Cereal Science, 51(3),

366–373. https://doi.org/10.1016/j.jcs.2010.01.010

Sun, X. D., & Arntfield, S. D. (2011). Gelation properties of chicken myofibrillar protein induced by transglutaminase crosslinking. Journal of Food Engineering, 107(2),

226–233. https://doi.org/10.1016/j.jfoodeng.2011.06.019

T´ellez-Luis, S. J., Gonz´alez-Cabriales, J. J., Ramírez, J. A., & V´azquez, M. (2004).

Production of transglutaminase by Streptoverticillium ladakanum NRRL-3191 grown on media made from hydrolysates of sorghum straw. Food Technology and

Biotechnology, 42(1), 1–4.

Topcu, A., Bulat, T., & ¨Ozer, B. (2020). Process design for processed Kashar cheese (a pasta-filata cheese) by means of microbial transglutaminase: Effect on physical properties, yield and proteolysis. LWT- Journal of Food Science and Technology, 125,

109226. https://doi.org/10.1016/j.lwt.2020.109226 Turker, I., Domurcuk, G., Tokatli, M., Isleroglu, H., & Koc, B. (2016). Enhancement of microbial transglutaminase production from Streptomyces sp. Ukrainian food Journal,

5(2), 306–313. https://doi.org/10.24263/2304-974X-2016-5-2-10

Umakoshi, M., Hirasawa, T., Furusawa, C., Takenaka, Y., Kikuchi, Y., & Shimizu, H. (2011). Improving protein secretion of a transglutaminase-secreting Corynebacterium glutamicum recombinant strain on the basis of 13C metabolic flux analysis. Journal of

Bioscience and Bioengineering, 112(6), 595–601. https://doi.org/10.1016/j. jbiosc.2011.08.011

Uran, H., & Yilmaz, I. (2018). A research on determination of quality characteristics of chicken burgers produced with transglutaminase supplementation. Food Science and

Technology, 38(1), 19–25. https://doi.org/10.1590/1678-457x.33816

Wang, L., Yu, B., Wang, R., & Xie, J. (2018). Biotechnological routes for transglutaminase production: Recent achievements, perspectives and limits. Trends in Food Science &

Technology, 81, 116–120. https://doi.org/10.1016/j.tifs.2018.09.015

Xavier, J. R., Ramana, K. V., & Sharma, R. K. (2017). Screening and statistical optimization of media ingredients for production of microbial transglutaminase.

Defence Life Science Journal, 2(2), 216–225. https://doi.org/10.14429/dlsj.2.11369

Yang, X., & Zhang, Y. (2019). Expression of recombinant transglutaminase gene in Pichia pastoris and its uses in restructured meat products. Food Chemistry, 291, 245–252. https://doi.org/10.1016/j.foodchem.2019.04.015

Yokoyama, K., Utsumi, H., Nakamura, T., Ogaya, D., Shimba, N., Suzuki, E., et al. (2010).

Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis. Applied

Microbiology and Biotechnology, 87(6), 2087–2096. https://doi.org/10.1007/s00253- 010-2656-6

Zhang, L., Zhang, L., Han, X., Du, M., Zhang, Y., Feng, Z., et al. (2012). Enhancement of transglutaminase production in Streptomyces mobaraensis as achieved by treatment with excessive MgCl2. Applied Microbiology and Biotechnology, 93(6), 2335–2343. https://doi.org/10.1007/s00253-011-3790-5

Zhu, Y., Rinzema, A., Tramper, J., & Bol, J. (1996). Medium design based on stoichiometric analysis of microbial transglutaminase production by

Streptoverticillium mobaraense. Biotechnology and Bioengineering, 50(3), 291–298.

Zhu, Y., & Tramper, J. (2018). Novel applications for microbial transglutaminase beyond food processing. Trends in Biotechnology, 26(10), 559–565. https://doi.org/10.1016/ j.tibtech.2008.06.006

Ziarno, M., & Zaręba, D. (2020). The effect of the addition of microbial transglutaminase before the fermentation process on the quality characteristics of three types of yogurt. Food Science and Biotechnology, 29(1), 109–119. https://doi.org/10.1007/ s10068-019-00640-6

M. Akbari et al.