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当前位置: 首页 > 文章 > 贝莱斯芽孢杆菌的内切葡聚糖酶基因的克隆、表达及其酶学性质分析 中国畜牧杂志 2019 (9) 100-108
Position: Home > Articles > Cloning and Expression of β-1, 4-endoglucanase Gene from Bacillus velezensis and its Enzymatic Properties Chinese Journal of Animal Science 2019 (9) 100-108

贝莱斯芽孢杆菌的内切葡聚糖酶基因的克隆、表达及其酶学性质分析

作  者：
陈龙;吴兴利;闫晓刚;谷巍;徐海燕;钱爱东;王春凤;张芳毓
单  位：
吉林省农业科学院畜牧科学分院
关键词：
内切葡聚糖酶;贝莱斯芽孢杆菌;克隆;表达;酶学性质
摘  要：
本实验利用PCR方法扩增1株贝莱斯芽孢杆菌(Bacillus velezensis)源的内切葡聚糖酶基因(CMCase),插入到p ET32a(+)中构建重组表达大肠杆菌系统,对重组内切葡聚糖酶基因进行生物信息学分析以及酶学性质研究。结果表明:内切葡聚糖酶由499个氨基酸组成,预测分子量为55.02 ku,最大开放阅读框ORF约为1 500 bp。经诱导表达优化后,培养上清中可检测到内切葡聚糖酶酶活力为5.81 IU/mL,菌体中为1.61 IU/mL,诱导后原菌液直接超声破碎处理可达7.41 IU/mL,其酶活力为野生菌株的2.3倍。重组内切葡聚糖酶具有典型内切纤维素酶的特征,其最适酶反应温度和pH分别为50℃和6,在60℃条件下放置1 h相对剩余酶活力可保持在70%以上,在pH 6～10范围内可保持90%以上。Na~+、Mg~(2+)可以促进重组内切葡聚糖酶酶活力,而Co~(2+)、Hg~(2+)、Fe~(2+)等金属离子以及表面活性剂SDS则具有较强的抑制作用。由此可见,本实验在大肠杆菌BL21(DE3)中成功表达了内切葡聚糖酶,且该酶主要进行分泌表达,具有一定的耐热性和良好的pH稳定性。
译  名：
Cloning and Expression of β-1, 4-endoglucanase Gene from Bacillus velezensis and its Enzymatic Properties
作  者：
CHEN Long;WU Xingli;Yan Xiaogang;GU Wei;XU Haiyan;QIAN Aidong;WANG Chunfeng;ZHANG Fangyu;Branch of Animal Husbandry, Jilin Academy of Agricultural Sciences;Shandong BaoLai-LeeLai Bioengineering Co.Ltd;College of Animal Science and Technology, Jilin Agricultural University;
关键词：
Endoglucanase;;Bacillus velezensis;;Clone;;Expression;;Enzymatic property
摘  要：
The β-1, 4-endoglucanase gene were amplified from Bacillus velezensis 157 using PCR methods, and inserted into pET32 a(+) vector to construct the the Escherichia coli expression system and analysis its enzymatic properties. The results showed that β-1, 4-endoglucanase is composed of 499 amino acids with a predicted molecular weight of 55.02 ku and the ORF is about 1 500 bp, which was identified through sequence analysis of the amplified product. In comparison to wild strain(Bacillus velezensis 157), the transformed Escherichia coli exhibited 2.3 times increase in cellulase production. Endoglucanase activity was detected to be 5.81 IU/mL in the supernatant culture, 1.61 IU/mL in the bacterial body, 7.41 IU/mL in the original bacterium fluid suggesting more extracellular production of β-1,4-endoglucanase. Recombinant β-1, 4-endoglucanase belongs to typical endoglucanase. The reaction optimal temperature of β-1, 4-endoglucanase was 50 ℃, and could still maintain above 70% relative enzymatic activity when temperature was 60 ℃ for 1 h. The optimal pH of β-1, 4-endoglucanase was 6, and could maintain over 90% cellulase activity in a pH range from 6-10. In addition, Na+ and Mg~(2+) can induce the activity of recombinant β-1, 4-endoglucanase, while metal ions such as Co~(2+), Hg~(2+), Fe~(2+) and surfactant SDS have a strong inhibitory effect. In conclusion, the β-1, 4-endoglucanase was successfully expressed in Escherichia coli BL21(DE3), and this enzyme can be secreted extracellularly with certain heat resistance and good pH stability.

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