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β-N-Acetylglucosaminidases (GlcNAcases) are important for many biological functions and industrial applications. In this study, a glycoside hydrolase family 20 GlcNAcase from Shinella sp. JB10 was expressed in Escherichia coli BL21 (DE3). Compared to many GlcNAcases, the purified recombinant enzyme (rJB10Nag) exhibited a higher specificity activity (538.8 µmol min -1  mg -1 ) or V max (1030.0 ± 82.1 µmol min -1  mg -1 ) toward p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetylchitobiose (specificity activity of 35.4 µmol min -1  mg -1 ) and a higher N-acetylglucosaminide tolerance (approximately 50% activity in 70.0 mM N-acetylglucosaminide). The degree of synergy on enzymatic degradation of chitin by a commercial chitinase and rJB10Nag was as high as 2.35. The enzyme was tolerant to most salts, especially 3.0-15.0% (w/v) NaCl and KCl. These biochemical characteristics make the JB10 GlcNAcase a candidate for use in many potential applications, including processing marine materials and the bioconversion of chitin waste. Furthermore, the enzyme has the highest proportions of alanine (16.5%), glycine (10.5%), and random coils (48.8%) with the lowest proportion of α-helices (24.9%) among experimentally characterized GH 20 GlcNAcases from other organisms.

β-N-Acetylglucosaminidases serve important biological functions and various industrial applications. A glycoside hydrolase family 3 β-N-acetylglucosaminidase gene was cloned from Sphingobacterium sp. HWLB1 and expressed in Escherichia coli BL21 (DE3). The purified recombinant enzyme (rNag3HWLB1) showed apparent optimal activity at pH 7.0 and 40 °C. In the presence of 0.5-20.0 % (w/v) NaCl, the activity and stability of rNag3HWLB1 were slightly affected or not affected. The enzyme could even retain 73.6 % activity when 30.0 % (w/v) NaCl was added to the reaction mixture. The half-life of the enzyme was approximately 10 min at 37 °C without the addition of NaCl. However, the enzyme was stable at 37 °C in the presence of 3.0 % (w/v) NaCl. A large negatively charged surface in the catalytic pocket of the enzyme was observed and might contribute to NaCl tolerance and thermostability improvement. The degree of synergy between a commercial endochitinase and rNag3HWLB1 on chitin enzymatic degradation ranged from 3.11 to 3.74. This study is the first to report the molecular and biochemical properties of a NaCl-tolerant β-N-acetylglucosaminidase.

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0-30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0-30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5-2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.

A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment's performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35-40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.

A glycoside hydrolase family 32 exo-inulinase gene was cloned from Sphingomonas sp. JB13 and expressed in Escherichia coli BL21 (DE3). The purified recombinant enzyme (rInuAJB13) showed an apparently optimal activity at pH 5.5 and 55 °C and remained activity at 10-70 °C. The addition of most metal ions and chemical reagents showed little or no effect (retaining more than 76.5 % activity) on the enzyme activity, notably the addition of surfactants SDS, CTAB, Tween 80, and Triton X-100. Most local liquid detergents, including Balin, Walch, Ariel, Tide, Tupperware, and Bluemoon, also showed little or no effect (retaining more than 77.8 % activity) on the enzyme activity. rInuAJB13 exhibited 135.3-163.6 % activity at the NaCl concentration of 1.0-4.5 M. After incubation with up to 57.0 mg mL(-1) trypsin and 90.0 mg mL(-1) proteinase K at 37 °C for 60 min (pH 7.2), rInuAJB13 retained more than 80 % of its initial activity. The enzyme presents a high proportion (28.0 %) of amino acid residues G, A, and V. This paper is the first to report a detergent-, salt-, and protease-tolerant exo-inulinase.

A glycosyl hydrolase family 10 endoxylanase from Bacillus sp. HJ14 was grouped in a separated cluster with another six Bacillus endoxylanases which have not been characterized. These Bacillus endoxylanases showed less than 52% amino acid sequence identity with other endoxylanases and far distance with endoxylanases from most microorganisms. Signal peptide was not detected in the endoxylanase. The endoxylanase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant enzyme (rXynAHJ14) was characterized. rXynAHJ14 was apparent optimal at 62.5 °C and pH 6.5 and retained more than 55% of the maximum activity when assayed at 40-75 °C, 23% at 20 °C, 16% at 85 °C, and even 8% at 0 °C. Half-lives of the enzyme were more than 60 min, approximately 25 and 4 min at 70, 75, and 80 °C, respectively. The enzyme exhibited more than 62% xylanase activity and stability at the concentration of 3-30% (w/v) NaCl. No xylanase activity was lost after incubation of the purified rXynAHJ14 with trypsin and proteinase K at 37 °C for 60 min. Different components of oligosaccharides were detected in the time-course hydrolysis of beechwood xylan by the enzyme. During the simulated intestinal digestion phase in vitro, 11.5-19.0, 15.3-19.0, 21.9-27.7, and 28.2-31.2 μmol/mL reducing sugar were released by the purified rXynAHJ14 from soybean meal, wheat bran, beechwood xylan, and rapeseed meal, respectively. The endoxylanase might be an alternative for potential applications in the processing of sea food and saline food and in aquaculture as agastric fish feed additive.