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Chitosanases hydrolyze chitosan into chitooligosaccharides (COSs) with various biological activities, which are widely employed in many areas including plant disease management. In this study, the novel chitosanase AqCsn1 belonging to the glycoside hydrolase family 46 (GH46) was cloned from Aquabacterium sp. A7-Y and heterologously expressed in Escherichia coli BL21 (DE3). AqCsn1 displayed the highest hydrolytic activity towards chitosan with 95% degree of deacetylation at 40 °C and pH 5.0, with a specific activity of 13.18 U/mg. Product analysis showed that AqCsn1 hydrolyzed chitosan into (GlcN)

A new glycoside hydrolase family 2 (GH2) β-galactosidase encoding gene galM was cloned from Microvirga sp. strain MC18 and overexpressed in Escherichia coli. The recombinant β-galactosidase GalM showed optimal activity at pH 7.0 and 50 °C, with a stability at pH 6.0-9.0 and 20-40 °C, which are conditions suitable for the diary environment. The K

A pectinase-producing bacterial isolate, identified as Paenibacillus xylanexedens SZ 29, was screened by using the soil dilution plate with citrus pectin and congo red. A pectin methylesterase gene (Pxpme) was cloned and expressed in Escherichia coli. The gene coded for a protein with 334 amino acids and a calculated molecular mass of 36.76 kDa. PxPME showed the highest identity of 32.4% with the characterized carbohydrate esterase family 8 pectin methylesterase from Daucus carota. The recombined PxPME showed a specific activity with 39.38 U/mg against citrus pectin with >65% methylesterification. The optimal pH and temperature for PxPME activity were 5.0 and 45 °C. Its Km and Vmax value were determined to be 1.43 mg/mL and 71.5 μmol/mg·min, respectively. Moreover, PxPME could increase the firmness of pineapple cubes by 114% when combined with CaCl2. The acidic and mesophilic properties make PxPME a potential candidate for application in the fruit processing.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Trehalase catalyzes the hydrolysis of trehalose into two glucose molecules and is present in nearly all tissues in various forms. In this study, a putative bacterial trehalase gene, encoding a glycoside hydrolase family 15 (GH15) protein was identified in Microvirga sp. strain MC18 and heterologously expressed in E. coli. The specific activity of the purified recombinant trehalase MtreH was 24 U/mg, with Km and Vmax values of 23.45 mg/mL and 184.23 μmol/mg/min, respectively. The enzyme exhibited optimal activity at 40 °C and pH 7.0, whereby Ca2+ had a considerable positive effects on the catalytic activity and thermostability. The optimized enzymatic reaction conditions for the bioconversion of trehalose using rMtreH were determined as 40 °C, pH 7.0, 10 h and 1% trehalose concentration. The characterization of this bacterial trehalase improves our understanding of the metabolism and biological role of trehalose in prokaryotic organism.

The lamC gene encoding a novel β-(1,3)-glucanase was cloned from Corallococcus sp. EGB and successfully expressed in the industrial yeast Pichia pastoris. The mature protein without the initial 26 residues of signal peptide, designated LamC27, was found to be composed of fascin-like module and laminarinase-like catalytic module. The purified recombinant enzyme (rLamC27) with a calculated molecular mass of 45.3 kDa displays activities toward a broad range of β-linked polysaccharides, including laminarin, curdlan, pachyman, lichenan, and CMC. Enzymological characterization showed that rLamC27 performes its optimal activity under the condition of 45 °C and pH 7.0, respectively, and preferentially catalyzes the hydrolysis of glucans with a β-1,3-linkage, which is similar to the LamC previously expressed in E. coli. TherLamC27 enzyme was activated by Mn2+ and Ba2+, while it was inhibited by Cu2+, Zn2+, and Co2+. Moreover, rLamC27 was strongly inhibited by 10 mM EDTA with 7.5% of its original activity remiaining, and weakly by SDS and Triton X-100. In antifungal assay, rLamC27 was conformed to possess lytic and antifungal activity against rice blast fungus. Specifically, a significant decrease germ tube and appressorium formation ratios from 94% to 59% and 97%–51%, respectively, were observed following exposure to rLamC27. H2DCFDA and CFW staining further demonstrated that the fungistasis capability of rLamC27 could be contributed by its ability to hydrolyze components of the cell wall. All these favorable properties indicate a promising potential for using rLamC27 as a biological antifungal agent in areas such as plant protection and food preservation.

The gene encoding a novel glucoamylase (GlucaM) from the Corallococcus sp. strain EGB was cloned and heterologous expressed in Escherichia coli BL21(DE3), and the enzymatic characterization of recombinant GlucaM (rGlucaM) was determined in the study. The glucaM had an open reading frame of 1938 bp encoding GlucaM of 645 amino acids with no signal peptide. GlucaM belongs to glycosyl hydrolase family 15 and shares the highest identity 96% with the GH15 glucoamylase of Corallococcus coralloides DSM 2259. The rGlucaM with His-tag was purified by the Ni2+-NTA resin, with a specific activity from 3.4 U/mg up to 180 U/mg, and the molecular weight of rGlucaM was approximately 73 kDa on SDS-PAGE. The Km and Vmax of rGlucaM for soluble starch were 1.2 mg/mL and 46 U/mg, respectively. rGlucaM was optimally active at pH 7.0 and 50 °C and had highly tolerance to high concentrations of salts, detergents, and various organic solvents. rGlucaM hydrolyzed soluble starch to glucose, and hydrolytic activities were also detected with amylopectin, amylase, glycogen, starch (potato), α-cyclodextrin, starch (corn and potato). The analysis of hydrolysis products shown that rGlucaM with α-(1–4),(1–6)-D-glucan glucohydrolase toward substrates. These characteristics indicated that the GlucaM was a new member of glucoamylase family and a potential candidate for industrial application.