| ?-mannanase(EC 3.1.2.78)is a hemicellulose hydrolase which is capable of degrading mannan,it can hydrolyze ?-1,4 glycosidic bond of mannan by the manner of incision enzyme and generates mannan-oligosaccharides(MOS)composed of 2 to 10 monosaccharide molecules.At present,?-mannanase has been widely used in food,medicine,textile,papermaking,and petroleum exploitation industries because of its important functional role as an enzyme preparation.?-mannanase is found in nature widely,such as the intestinal secretions of some marine mollusks and the seeds of certain legumes.However,microorganisms are the main source of production of?-mannanase currently.The enzyme activity,character,mode of action and the primary structure of the enzyme protein from various microorganisms are different.The microbial-derived P-mannanase has advantages of high enzyme activity,low cost,convenient extraction.And it has wider pH range,temperature range and better thermostability,acid and alkali resistance,and substrate specificity than that derived from animals and plants.Therefore microbial-derived ?-mannanase is highly valued by researchers in this field.However,using microbial enzyme production capacity to produce ?-mannanase by direct fermentation is often limited by the low initial enzyme activity,high fermentation cost,and insecure biological and food safety,which make it difficult to carry out large-scale industrial production.With the deep study of genetic engineering and protein engineering technology,the research on P-mannanase production has been transferred to the construction of enzyme-producing engineering bacteria and molecular modification of the enzyme.In this study,?-mannanase gene was cloned from the genome of Bacillus subtilis 168 with 6 histidine tag added,and heterologous expression of the fusion protein was carried out in Escherichia coli and Corynebacterium glutamicum respectively.Analysis of SDS-PAGE and enzyme activity assay showed that the expression of recombinant enzyme in E.coli was mostly in the form of inactive inclusion bodies,while it was in the form of soluble protein in C.glutamicum.The recombinant protein produced by C.glutamicum was isolated and purified by Ni-NTA resin.The results showed that the recombinant protein was eluted under the condition of NTA250.SDS-PAGE showed that the purified recombinant protein was clear and single,and its molecular size was about 41 kDa.At the same time,the enzyme activity of?-mannanase was determined by DNS chromogenic method,and the enzymatic properties such as thermostability,acid and alkali resistance of the recombinant enzyme and the effect of metal ions on enzyme activity were analyzed.The results showed that the enzyme activity of recombinase was up to 1065.02 U/mL after purified by Ni-NTA resin,the optimum pH of recombinant ?-mannanase was 6,the optimum reaction temperature was 55?.and the enzyme activity remained nearly 50%under the reaction condition of 70?.At the same time,the recombinant enzyme retained half of the highest enzyme activity after storage for 6h at 60?.At the same time,the recombinant enzyme was stable and its enzyme activity remained 70%after 24 h storage under the pH4?8.However,the activity of recombinant enzyme decreased rapidly in the extreme acid environment of pH 2,and it was nearly inactivated after 2 h.In summary,this research used Corynebacterium glutamicum as a novel expression system to express ?-mannanase for the first time,and it successfully expressed ?-mannanase with high enzyme activity,and the recombinant enzyme exists as soluble protein.The recombinant enzyme has good pH adaptability and thermostability,which will greatly increase the breadth of production and application of recombinant ?-mannanase.Therefore,C.glutamicum can substitute Escherichia coli as a novel genetically engineered strain expressing ?-mannanase in certain degree,thereby providing a new idea and experimental basis for the industrial production of P-mannanase. |
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