嗜冷德沃斯氏菌来源GH1家族β-葡萄糖苷酶Bgl59的原核表达及酶学性质分析  

Prokaryotic expression and characterization of the GH1β-glucosidase Bgl59 from Devosia psychrophila

作　　者：刘颖 董盼盼[1] 孙丽芳 吴琳娇 李兰兰 吴允昆 LIU Ying;DONG Panpan;SUN Lifang;WU Linjiao;LI Lanlan;WU Yunkun(College of Life Sciences,Fujian Normal University,Fuzhou 350108,Fujian,China)

机构地区：[1]福建师范大学生命科学学院,福建福州350108

出　　处：《微生物学报》2024年第8期2955-2966,共12页Acta Microbiologica Sinica

基　　金：福建省自然科学基金(2019J01280,2021J01171)。

摘　　要：β-葡萄糖苷酶在食品、医学以及生物能源等多个领域有着广泛的应用,因此挖掘新型高效的β-葡萄糖苷酶是十分必要的。【目的】对嗜冷德沃斯氏菌(Devosia psychrophile)来源的GH1家族的葡萄糖苷酶原核表达并测定其酶学性质。【方法】通过人工合成技术得到D.psychrophila来源的β-葡萄糖苷酶的编码基因,命名为bgl59。将该基因转化到大肠杆菌(Escherichia coli)BL21(DE3)中,诱导基因表达,对得到的蛋白进行纯化并测定其酶学性质。【结果】Bgl59的分子量为48.8 kDa,最适温度为55℃,最适pH为6.0;Bgl59在pH 5.0-8.5范围内处理1 h后仍保持80%以上酶活;在8种供试底物中,Bgl59对4-硝基苯基-β-D-葡萄糖苷(4-nitrophenyl-β-D-glucopyranoside,pNPG)有着最高的水解能力,其K_(m)为3.090 mmol/L,V_(max)为194μmol/(min·mg),kcat为159 s^(-1);1 mmol/L的Ca^(2+)、Co^(2+)对Bgl59具有明显的激活作用,0.1%的SDS会使酶活全部丧失;0.10 mol/L葡萄糖和0.30 mol/L木糖具有促酶活作用,可分别使Bgl59酶活提高74%和91%;在1.25 mol/L葡萄糖或2.00 mol/L木糖存在的条件下,仍可保持50%以上酶活。【结论】Bgl59的酶学性质优异,具有良好的pH稳定性,对金属离子或化学试剂都具有一定耐受能力,是少见的葡萄糖激活型β-葡萄糖苷酶,具有优良的糖促活性和耐受性,在未来的工业生产以及应用中具有潜在研究价值。β-glucosidases have been widely used in food,medicine,bioenergy and other fields,and thus it is necessary to explore new and efficientβ-glucosidases.[Objective]To realize the prokaryotic expression of a GH1 glucosidase derived from Devosia psychrophila and characterize the enzymatic properties of the expressed protein.[Methods]The gene encoding theβ-glucosidase derived from D.psychrophila was synthesized,named bgl59,and then transformed into Escherichia coli BL21(DE3).After the gene expression was induced,and the obtained protein was purified and characterized for the enzymatic properties.[Results]Bgl59 had a molecular weight of 48.8 kDa,with the highest activity at 55℃and pH 6.0.After treatment for 1 h within the range of pH 5.0-8.5,Bgl59 maintained the relative activity over 80%.Bgl59 had the highest hydrolysis ability for 4-nitrophenyl-β-D-glucopyranoside(pNPG)among the eight substrates tested,with the K_(m)of 3.090 mmol/L,V_(max)of 194μmol/(min·mg),and kcat of 159 s^(-1).The presence of 1 mmol/L of Ca^(2+)and Co^(2+)had a significant activating effect on Bgl59,while the presence of 0.1%SDS resulted in a complete loss of enzyme activity.The presence of 0.10 mol/L glucose and 0.30 mol/L xylose increased the activity of Bgl59 by 74%and 91%,respectively.Moreover,the enzyme remained the relative activity above 50%even when being cultured with 1.25 mol/L glucose or 2.00 mol/L xylose.[Conclusion]Bgl59 exhibits outstanding enzymatic properties,robust pH stability,and tolerance to metal ions and chemical reagents.It is a rare glucose-activatedβ-glucosidase with exceptional tolerance to glucose,holding significant potential for future industrial production and application.

关 键 词：Β-葡萄糖苷酶 酶学性质 GH1家族 葡萄糖耐受 

分 类 号：Q78[生物学—分子生物学] Q55