具有极高热稳定性的β-(2,6)果聚糖蔗糖酶的挖掘、鉴定及应用  

Characterization of an Extreme Thermostable Levansucrase and Its Application

作　　者：张晓琦 徐炜 倪大伟 张文立 光翠娥[1] 沐万孟[1,2] ZHANG Xiaoqi;XU Wei;NI dawei;ZHANG Wenli;GUANG Cuie;MU Wanmeng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China;International Joint Laboratory on Food Safety,Jiangnan University,Wuxi 214122,China)

机构地区：[1]食品科学与技术国家重点实验室,江南大学,江苏无锡214122 [2]江南大学食品安全国际合作联合实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2023年第4期57-68,共12页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31922073);江苏省自然科学基金项目(BK20210463)。

摘　　要：挖掘具有高热稳定性的β-(2,6)果聚糖蔗糖酶(levansucrase,LS)并进行β-(2,6)果聚糖的高效合成。以分子动力学模拟的方式筛选出具有潜在高热稳定性的β-(2,6)果聚糖蔗糖酶。将目的酶在大肠杆菌Escherichia coli BL21(DE3)中进行重组及诱导表达,并通过镍柱亲和层析进行纯化。在不同温度下进行保温实验以验证LS的热稳定性,并通过条件优化获得产物的高转化率。Cedi-LS在65℃时显示出最高活性,远高于已鉴定的其他LS。同时,在45℃下保温72 h,重组酶Cedi-LS能够保留90%以上的相对活性;在55℃下保温60 h,其保留活性仍可达到初始活性的60%以上,表现出优异的热稳定性。在反应过程中,Cedi-LS可同时生成低聚糖、低相对分子质量的β-(2,6)果聚糖和高相对分子质量的β-(2,6)果聚糖。当温度从65℃降至35℃时,Cedi-LS倾向于产生HMW-levan,其相对分子质量可达8.4×10^(6)。在pH 5.5和35℃的条件下,以质量分数30%的蔗糖为底物,加酶量为20μg/mL,蔗糖转化为β-(2,6)果聚糖的平衡转化率为41.4%。作者挖掘了一株具有高热稳定性的LS,其能够高效生产一系列具有不同相对分子质量的果聚糖。β-(2,6)levansucrase(LS)with high thermostability was identified and the efficient synthesis of levan by levansucrase was studied.High-temperature molecular dynamics simulation was used to screen the LS with potential high thermostability.The target enzyme was heterogeneously expressed in Escherichia coli BL21(DE3)and purified through one-step Ni2+-affinity purification.The thermostability of LS was determined by incubating the enzyme at different temperatures,and the highest conversion was obtained through the optimization of conditions.The Cedi-LS showed the highest activity at 65℃,which was much higher than the other identified LSs.Meanwhile,Cedi-LS remained more than 90%active when incubated at 45℃for 72 h,and it also remained more than 60%of its initial activity at 55℃for 60 h,exhibiting excellent thermostability.The Cedi-LS produced oligosaccharides,low molecular weight(LMW)β-(2,6)levan,and high molecular weight(HMW)β-(2,6)levan simultaneously during reaction.When the temperature decreased from 65℃to 35℃,Cedi-LS tended to produce HMW-levan with a Mw of 8.4×10^(6).At pH 5.5 and 35℃,the equilibrium conversion ratio from sucrose to levan was 41.4%with 30%sucrose and 20μg/mL enzyme.In summary,an extreme thermostable LS was screened which could efficiently produce levan with different molecular weights.

关 键 词：β-(2 6)果聚糖蔗糖酶 热稳定性 β-(2 6)果聚糖 产物特异性 

分 类 号：Q814[生物学—生物工程]