Thr22 plays an important role in the efficient catalytic process of Bacillus subtilis chitosanase BsCsn46A

Chitosan is composing of glucosamine monomers linked by β-1, 4-glycosidic bonds[1], [2]. Chitooligosaccharides (COS) are hydrolyzed products of chitosan and have a variety of bioactivities, such as antitumor[3], immune enhancing[4], antioxidizing[5], free radical scavenging[6], and antimicrobial activities[7]. Biodegradation, chemical and physical methods have been reported for the preparation of COS[8]. Compared to the chemical and physical methods used for the degradation of chitosan, biodegradation is more environmentally friendly and controllable[9]. Chitosanase (EC3.2.1.132) is a kind of glycoside hydrolase (GH) which can specifically hydrolyze chitosan to produce COS[10]. According to the CAZy (http://www.cazy.org), the reported chitosanases are classified into GH8[11], 46[12], 75[13] and 80[14] families. GH3, GH5 and GH7 have chitosanase activity, but can also hydrolyze other substrates, such as cellulose and xylan[15]. Among them, the research on GH46 chitosanase is the most in-depth. The crystal structure of GH46 family chitosanase is dumbbell-shaped. The crack in the middle is the substrate binding region containing two acidic amino acids as catalytic residues (Asp and Glu), one of which acts as a proton donor and the other as nucleophile/base[16], [17]. Lv et al. demonstrated that the substrate binding pocket of non-processed chitosanase underwent three conformational changes of "open-closed-open" when binding to the substrate, and most of the amino acid sites associated with substrate binding were polar residues[17].

As mentioned above, the catalytic hydrolysis mechanism of GH46 family chitosanases has been well elucidated, but little is known about the function of amino acids near the catalytic center, especially those near catalytic residues. However, a lot of studies have shown that amino acids near the active center have significant effects on the catalytic activity and substrate selectivity of the enzyme. Yang et al. confirmed that loop3 in the active center of GH12 family glucanases of Aspergillus Niger had a significant effect on the catalytic efficiency[18]. Cheng et al. designed the entrance of the substrate tunnel to enhance the catalytic activity of thermophilic nitrile hydratase[19]. Liang et al. extended the substrate spectrum of carotenoid cracking dioxygenase by designing substrate channel and significantly improved the catalytic activity of the enzyme[20].

Previously, we enhanced the catalytic activity of Bacillus subtilis chitosanase by saturation mutagenesis of Proline 121. In this study, we identified a non-conserved site Thr22 near the catalytic site of Glu19 by comparing the chitosanase protein sequences of different Bacillus species. Site-directed saturation mutation was carried out to obtain the mutants. The enzymatic properties of the mutants were studied and the role of the site in chitosanase catalysis was studied by bioinformatics analysis.

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