Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog

γ-Glutamyl transpeptidase (GGT; EC 2.3.2.2) is an enzyme that is widely distributed in nature and is known to catalyze the hydrolysis of γ-glutamyl bonds in glutamine, as well as the transfer of the released γ-glutamyl group to amino acids or short peptides [1]. Because of its diverse applications in various biotechnology fields, the biochemical, genetic, and molecular biological properties of microbial GGTs have been extensively studied [1], [2]. After the formation of a γ-glutamyl-GGT intermediate, it then reacts with water to produce glutamate by hydrolysis or with amino acids or peptides to produce the corresponding γ-glutamyl peptides by transpeptidation. Among microbial GGTs, protein engineering of Bacillus GGTs has been performed to enhance post-translational auto-processing [1], [3], [4], overproduction [5], [6], and enzyme activity and stability [1], [4], [7], [8], [9], for the biocatalytic synthesis of valuable γ-glutamyl peptides. However, there is limited information on enhancing the hydrolysis ability of GGTs for producing glutamate from glutamine [7], [10].

In traditional Japanese soy sauce and soybean fermentation under high salt conditions (c. 17%single bond18% NaCl), glutaminases produced by koji molds such as A. oryzae and A. sojae are key hydrolytic enzymes for flavor enhancement, as they convert L-glutamine to L-glutamic acid [11], [12]. However, fungal glutaminases, including GGTs, are non-salt-tolerant enzymes, and their hydrolytic activity markedly decreases under high salt conditions [11], [12]. Salt-tolerant (halotolerant) enzymes are defined as those that retain their activity and stability under a broad range of salt concentrations [13]. Therefore, various salt-tolerant glutaminases, including GGT, have been isolated and characterized from bacteria and yeasts [14] for application in wheat and soybean fermentation instead of A. oryzae GGTs. While several Bacillus GGTs are categorized as a salt-tolerant type [10], [15], [16], [17], Bacillus spp. proliferates very rapidly during koji fermentation, which is the process of culturing A. oryzae on cooked soybean, rice, and barley [18]. Moreover, Bacillus spp. not only antagonistically prevent the growth of A. oryzae, but also produce an off-flavor [18]. Thus, the salt-tolerant GGT-producing Bacillus spp. and their crude GGT preparations cannot be utilized for koji fermentation with A. oryzae. Furthermore, B. subtilis GGT shows low sequence identity (c. 30%) with A. oryzae extracellular GGTs [19], which makes it difficult to improve the salt-tolerance of A. oryzae GGTs by protein engineering based on reported sequence and structural data analysis of bacterial GGTs.

Recently, a salt-tolerant GGT, termed ASggtA, has been isolated and characterized from a xerophilic mold, A. sydowii MA0196 [19]. ASggtA has high salt-tolerance compared with previously reported Aspergillus GGTs [19]. The deduced amino acid sequence of ASggtA showed high identity (73% [435 / 593 amino acids]) with that of AOggtA [19], making ASggtA a potential salt-tolerant counterpart for enhancing salt-tolerance in AOggtA. Halophilic and salt-tolerant enzymes generally contain a relatively high proportion of acidic amino acids (Asp and Glu) compared with homologous non-halophilic/salt-tolerant proteins [13], [20], [21], [22]. Thus, protein surface engineering by multiple substitution with Asp and/or Glu residues has been applied for improvement of the salt-tolerance of highly desirable enzymes, such as glucose dehydrogenase, carbonic anhydrase, and xylanase [23], [24], [25]. However, no clear differences in protein surface properties were detected between AOggtA and ASggtA mature proteins [19]. There is considerable different in the terms of N-terminal region for revealing salt-tolerant mechanism and/or for protein engineering in non-salt-tolerant AOggtA, described below (see Fig. 1(a)).

In this study, we designed a chimeric enzyme, in which the N-terminus of salt-tolerant ASggtA was connected by protein fusion to the C-terminus of non-salt-tolerant AOggtA. The chimera and its parental GGTs were heterologously expressed, to elucidate the role of the N-terminal region for enzymatic stability and tolerance under high salt conditions. In addition, differences in thermo- and pH-stabilities and catalytic properties were characterized and compared between the chimera and its parental enzymes.

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