Cloning, expression and function of an alkaline resistance endo-glucanase gene isolated from guts of earwig

Abstract:

[Objective] To provide some theoretical references for further research and development of alkaline cellulase via obtaining a novel alkaline resistance cellulase in bacteria from intestines of earwig, and heterologously express, characterize the functions of the enzyme. [Methods] First, we isolated the bacterial strains from earwig gut samples in Nanyang Baotianman National Reserve Area, Henan province, China by primarily screening according to Congo red plate methods. Then, we obtained and identified the bacterial strains with high alkaline cellulase activities by phenotypic and genotypic characteristics. We designed degenerate primes according to the known endoglucanase gene sequences in GenBank to carry out PCR, analyzed the cloned sequence, and expressed the enzyme in Escherichia coli BL21. [Results] We obtained one bacterial strain with high alkaline cellulase activities named strain Q5. The bacterium was classified to be Bacillus methylotrophicus. The full length of a cellulase gene cDNA (1500 bp) (GenBank KR067575) coding region was successfully cloned. The homogeneous analysis demonstrated that the deduced amino acid of the gene showed 98% similarities with the alkaline β-1,4 endoglycosidase from Bacillus sp. 2190 (ALE32753.1). The activity of the recombined endoglucanase was 3.46 U/mL, which was 1.69 times higher than that of the wild Bacillus methylotrophicus Q5 (2.05 U/mL). The highest cellulase activity reached 4.99 U/mL after orthogonal experiment. The properties of the recombined enzyme were determined. The optimum temperature and pH value were 50℃ and pH 8.5, respectively. The enzyme maintained over 80% of the original enzyme activity at pH 8.0 and pH 9.0 after incubated at 50℃ for 48 h. The enzyme was stable below 50℃ and the activity decreased sharply above 60℃. The activity of this enzyme was activated by 10 mmol/L Ca²⁺和Mg²⁺. The values of K_m and V_max were 2.217 mol/mL and 9.606 μmol/(min·L), respectively. The enzyme showed obviously inhibiting the growth of the cotton pathogenic fungi Verticillum dahliae. [Conclusion] It was the first time we got an alkaline resistance endoglucanasegene from Bacillus methylotrophicus isolated from guts of earwig. Our findings will lay a theoretical foundation for the application in alkaline environments.