RESUMO
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.
Assuntos
Proteínas de Bactérias , Expressão Gênica , Methylobacteriaceae , Trealase , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Methylobacteriaceae/enzimologia , Methylobacteriaceae/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Trealase/biossíntese , Trealase/química , Trealase/genética , Trealase/isolamento & purificaçãoRESUMO
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.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Proteínas de Bactérias/química , Suplementos Nutricionais/análise , Methylobacteriaceae/enzimologia , Amido/química , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hidrólise , Cinética , Methylobacteriaceae/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
In our previous study, the chitosanase AqCoA and the chitooligosaccharides it produced were found to exhibit significant protective effects against fungal diseases. In this study, we enhanced the expression of AqCoA using the novel pMC-GAP that enables stable transformation of Escherichia coli, and built an integrated model based on the gene copy number, molecular chaperones, and protein production of AqCoA. In terms of gene dosage, the highest hydrolase activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that of the strain with only one copy (0.18 U/ml). In addition, we found the chaperones such as PDI, ERO1, HAC1, YDJ1, SSE1, SSA4, and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1, and YDJ1/SSO2 pairs synergistically increased the expression levels by 61%, 31%, and 42%, respectively. Finally, we investigated the combined effects of gene copy numbers and molecular chaperones on protein expression. The highest activity reached 2.32 U/ml in the strain with six integrated molecular chaperone expression cassettes and sixteen copies of the target gene, which was 13-fold higher than that of the control strain with only one copy (GAP-1AqCoA). Combined optimization of gene dosage and molecular chaperone combinations significantly increased the expression level of AqCoA, providing a powerful strategy to improve the expression of other heterologous proteins in P. pastoris.