蠼螋肠道中碱性内切葡聚糖酶基因的克隆表达及功能分析

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

【目的】从蠼螋肠道细菌菌株Q5中获得一个新型耐碱纤维素酶基因,通过异源表达、酶学性质及功能分析,旨在为以后进一步研究开发高温碱性纤维素酶提供一些理论参考。【方法】采用刚果红平板初筛法,从河南南阳宝天曼国家级自然保护区落叶堆下的昆虫蠼螋肠道中,获得具有分泌较高活性碱性纤维素酶的细菌菌株。基于该菌株的形态学、生理学及16S rRNA序列特征等对高活性菌株进行分类鉴定。并通过设计简并引物,从高活性菌株中克隆出该菌株的纤维素酶基因,并进行序列分析,并导入大肠杆菌BL21中表达。【结果】获得1株具有分泌较高活性碱性内切葡聚糖酶的细菌菌株Q5,经鉴定为甲基营养型芽孢杆菌,进一步从Q5菌株中成功克隆出该菌株的一个全长1500 bp的内切葡聚糖酶基因(GenBank KR067575),在NCBI比对后发现该基因的氨基酸序列与芽孢杆菌菌株LM 4-2的耐碱性β-1,4-内切葡聚糖酶基因(AKE23721.1)有98%的同源性。重组菌经优化培养,细胞破碎后上清液中的酶活力可达3.46 U/mL,是出发菌株Q5(2.05 U/mL)的1.69倍。经正交实验优化后的酶活力为4.99 U/mL。酶学性质研究表明:该酶的最适反应温度与pH值分别为50°C与pH 8.5,在pH 8.0和9.0保温48 h,其酶活力仍然维持到最高酶活的82%和81%;该酶在50°C以下较稳定,60°C以上酶活迅速降低。10 mmol/L的Ca^2+和Mg^2+对酶的活性有明显促进作用,重组酶Ega5的Km和Vmax分别是2.217 mol/mL和9.606μmol/(min·L)。该重组酶对棉花黄萎病病原菌大丽轮枝菌具有显著抑制作用。【结论】本文首次从蠼螋肠道中筛选到了一株产碱性内切葡聚糖酶的细菌菌株并从中克隆出了一个碱性纤维素酶基因,为该酶在碱性条件的应用奠定了理论基础。

[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)(Gen Bank 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 °C 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 °C for 48 h. The enzyme was stable below 50 °C and the activity decreased sharply above 60 °C. The activity of this enzyme was activated by 10 mmol/L Ca^2+和 Mg^2+. The values of Km and Vmax 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.