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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 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.

The exploration and utilization of microbial salt-tolerant enzymatic and genetic resources are of great significance in the field of biotechnology and for the research of the adaptation of microorganisms to extreme environments. The presence of new salt-tolerant genes and enzymes in the microbial metagenomic library of the gastrointestinal tract has been confirmed through metagenomic technology. This paper aimed to identify and characterize enzymes that confer salt tolerance in the gastrointestinal tract microbe. By screening the fecal metagenomic library, 48 salt-tolerant clones were detected, of which 10 salt-tolerant clones exhibited stronger tolerance to 7% (wt/vol) NaCl and stability in different concentrations of NaCl [5%–9% (wt/vol)]. High-throughput sequencing and biological information analysis showed that 91 potential genes encoded proteins and enzymes that were widely involved in salt tolerance. Furthermore, two trehalose-6-phosphate hydrolase genes, namely, tre_P2 and tre_P3 , were successfully cloned and expressed in Escherichia coli BL21 (DE3). By virtue of the substrate of p -nitrophenyl-α-D-glucopyranoside (p NPG) which can be specifically hydrolyzed by trehalose-6-phosphate hydrolase to produce glucose and p -nitrophenol, the two enzymes can act optimally at pH 7.5 and 30°C. Steady-state kinetics with p NPG showed that the K M and K cat values were 15.63 mM and 10.04 s–1 for rTRE_P2 and 12.51 mM and 10.71 s–1 for rTRE_P3, respectively. Characterization of enzymatic properties demonstrated that rTRE_P2 and rTRE_P3 were salt-tolerant. The enzymatic activity increased with increasing NaCl concentration, and the maximum activities of rTRE_P2 and rTRE_P3 were obtained at 4 and 3 M NaCl, respectively. The activities of rTRE_P2 increased by approximately 43-fold even after 24 h of incubation with 5 M NaCl. This study is the first to report the identification as well as molecular and biochemical characterization of salt-tolerant trehalose-6-phosphate hydrolase from the metagenomic library of the gastrointestinal tract. Results indicate the existence of numerous salt-tolerant genes and enzymes in gastrointestinal microbes and provide new insights into the salt-tolerant mechanisms in the gastrointestinal environment. [ABSTRACT FROM AUTHOR]

A GH43 bifunctional β-xylosidase encoding gene (XylRBM26) was cloned from Massilia sp. RBM26 and successfully expressed in Escherichia coli. Recombinant XylRBM26 exhibited β-xylosidase and α- l -arabinofuranosidase activities. When 4-nitrophenyl-β- d -xylopyranoside was used as a substrate, the enzyme reached optimal activity at pH 6.5 and 50°C and remained stable at pH 5.0–10.0. Purified XylRBM26 presented good salt tolerance and retained 96.6% activity in 3.5 M NaCl and 77.9% initial activity even in 4.0 M NaCl. In addition, it exhibited high tolerance to xylose with Ki value of 500 mM. This study was the first to identify and characterize NaCl-tolerant β-xylosidase/α- l -arabinofuranosidase from the gut microbiota. The enzyme's salt, xylose, and alkali stability and resistance to various chemicals make it a potential biocatalyst for the saccharification of lignocellulose, the food industry, and industrial processes conducted in sea water. [ABSTRACT FROM AUTHOR]