摘要
葡萄糖苷酶在食品、医药、生物质转化等领域具有重要的应用价值,因此发掘适应性强、性质优良的β-葡萄糖苷酶是国内外研究热点。本研究从嗜热古菌Infirmifilum uzonense中成功克隆出一个GH3家族的β-葡萄糖苷酶基因,命名为Iubgl3。基因序列分析显示Iubgl3全长为2109 bp,编码702个氨基酸,理论分子量为77.0 kDa。将该基因在大肠杆菌中进行克隆表达并对纯化后的IuBgl3进行酶学性质研究。结果显示,重组酶IuBgl3最适pH 5.0,最适温度85℃。该酶具有良好的热稳定性,80℃处理2 h后仍能保持85%以上的酶活力。其具有优良的pH稳定性,在pH 4.0-11.0范围内处理1 h,仍维持85%以上的酶活力。通过底物特异性测定发现,该酶对对硝基苯-β-D-吡喃葡萄糖苷(p-nitrophenyl β-D-glucoside,p NPG)和对硝基苯-β-D-吡喃木糖苷(p-nitrophenyl β-D-xylopyranoside,p NPX)均有很高的水解能力,是典型的双功能酶。以p NPG为底物时的动力学参数K_(m)和V_(max)分别为0.38 mmol和248.55μmol/(mg·min),催化效率k_(cat)/K_(m)=6149.20 s^(-1)mmol^(-1)。大多数金属离子对IuBgl3的酶活力没有显著影响,SDS可导致酶完全失活,而EDTA却能提高30%的酶活力。本研究丰富了高温古菌GH3家族的β-葡萄糖苷酶基因,获得了一个稳定性优良的高温酸性双功能酶,具有良好的工业应用前景。
glucosidase has important applications in food,medicine,biomass conversion and other fields.Therefore,exploring β-glucosidase with strong stability and excellent properties is a research hotspot.In this study,a GH3 family β-glucosidase gene named Iubgl3 was successfully cloned from Infirmifilum uzonense.Sequence analysis showed that the full length of Iubgl3 was 2106 bp,encoding702 amino acids,with a theoretical molecular weight of 77.0 kDa.The gene was cloned and expressed in E.coli and the enzymatic properties of purified IuBgl3 were studied.The results showed that the optimal pH and temperature for p NPG hydrolysis were 5.0 and 85℃,respectively.The enzyme has good thermal stability,and more than 85% of enzyme activity can be retained after being treated at 80℃ for 2 h.This enzyme has good pH stability and more than 85% of its activity can be retained after being treated at pH 4.0-11.0 for 1 h.It was found that the enzyme had high hydrolysis ability to p-nitrophenyl β-D-glucoside(p NPG)and p-nitrophenyl β-D-xylopyranoside(p NPX).When p NPG was used as the substrate,the kinetic parameters K_(m)and V_(max)were 0.38 mmol and 248.55μmol/(mg·min),respectively,and the catalytic efficiency k_(cat)/K_(m)was 6149.20 s^(-1)mmol^(-1).Most metal ions had no significant effect on the enzyme activity of IuBgl3.SDS completely inactivated the enzyme,while EDTA increased the enzyme activity by 30%.This study expanded the β-glucosidase gene diversity of the thermophilic archaea GH3 family and obtained a thermostable acid bifunctional enzyme with good industrial application potential.
作者
刘鑫涵
沈风飞
石鹏君
刘慧芹
LIU Xinhan;SHEN Fengfei;SHI Pengjun;LIU Huiqin(College of Horticulture and Landscape,Tianjin Agricultural University,Tianjin 300392,China;College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,Jiangxi,China;Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha 410205,Hunan,China;Institute of Food Science and Technology,Chinese Academy of Agricultural Sciences,Beijing 100193,China)
出处
《生物工程学报》
CAS
CSCD
北大核心
2022年第12期4644-4657,共14页
Chinese Journal of Biotechnology
基金
国家自然科学基金(31760438,32072166)
中国农业科学院农产品加工研究所“N专项”(CAAS-ASTIP-2021-IFST-SN2021-11)
天津市高校中青年骨干创新人才培养计划(J01009030709)
天津市企业科技特派员项目(20YDTPJC01330)
宁夏特殊生境微生物资源开发与利用重点实验室开放课题(2019TSWZ01)。
关键词
嗜热古菌
GH3家族
Β-葡萄糖苷酶
酶学性质
双功能酶
thermophilic archaea
GH3 family
β-glucosidase
enzymatic properties
bifunctional enzymes
