粉红聚端孢几丁质酶基因Trchi2在毕赤酵母中的表达及其酶学性质  

Expression of Trichothecium roseum Chitinase Gene Trchi2 in Pichia pastoris and Its Enzyme Characterization

作　　者：于松尼 贾鹏飞 李雅华[1,2] 咸洪泉 YU Song-Ni;JIA Peng-Fei;LI Ya-Hua;XIAN Hong-Quan(College of Life Sciences,Qingdao Agricultural University,Qingdao 266109,China;Shandong Provincial Key Laboratory of Applied Mycology,Qingdao 266109,China)

机构地区：[1]青岛农业大学生命科学学院,青岛266109 [2]山东省应用真菌重点实验室,青岛266109

出　　处：《农业生物技术学报》2022年第7期1385-1393,共9页Journal of Agricultural Biotechnology

基　　金：山东省重点研发计划(2018GNC110024);青岛市科技惠民示范引导专项(21-1-4-ny-5-nsh)。

摘　　要：粉红聚端孢(Trichothecium roseum)是一种重要的菌寄生真菌,能够通过菌寄生作用抑制病原微生物的生长,可以有效防治多种植物病原真菌。几丁质酶(chitinase,chi)在粉红聚端孢寄生和抑菌过程中发挥重要作用,本研究旨在实现粉红聚端孢几丁质酶Trchi2基因在毕赤酵母(Pichia pastoris)中表达,并明确其酶学性质,采用逆转录PCR(reverse transcriptase PCR,RT-PCR)克隆粉红聚端孢几丁质酶Trchi2基因,构建毕赤酵母重组表达载体,转入毕赤酵母GS115,筛选获得的酵母转化子通过甲醇诱导表达,获得Trchi2蛋白,测定其几丁质酶活性并进行酶学性质的研究。本研究成功构建了毕赤酵母重组表达载体pPIC9K-Trchi2,获得了分泌表达几丁质酶Trchi2的工程菌GS115-Trchi2-2-8;诱导培养7 d产酶量最高,酶活力达3.96 U/mL,表达的几丁质酶Trchi2表观分子量为50 kD,酶解胶体几丁质的产物为几丁二糖;在45℃、pH 6.0时酶活性最高,在pH 4~7.5、30~55℃条件下相对稳定;当催化反应体系中存在50 mmol/L的Ag^(+)、Hg^(2+)、Cu^(2+)、Fe^(2+)时,Trchi2酶活性受到强烈抑制。本研究对几丁质酶Trchi2的基本性质进行了研究,为其结构功能的深入发掘和生产应用提供了理论依据。Trichothecium roseum is a kind of mycoparasites,which can inhibit the growth of Sclerotinia sclerotiorum and other pathogenic bacteria.Chitin is the major component of the most fungal cell wall,the chitinase of T.roseum plays an important role in the parasitism and bacteriostasis.In order to achieve the expression of the chitinase Trchi2 gene from T.roseum and clarify its enzymological properties,T.roseum chitinase gene Trchi2 was cloned by reverse transcriptase PCR(RT-PCR),the recombinant expression vector was constructed and transferred into Pichia pastoris GS115.The yeast transformants were screened and identified by PCR,and the engineered yeast was cultured with methanol to obtain the protein Trchi2 and studied its enzymatic properties.The results showed that the recombinant plasmid pPIC9K-Trchi2 was successfully constructed,and the engineered yeast GS115-Trchi2-2-8 that expressed chitinase Trchi2 were obtained.The enzyme production was the highest after induction for 7 d,the enzyme activity reached 3.96 U/mL,the apparent molecular weight of chitinase Trchi2 was 50 kD.The main product of chitin decomposition by chitinase Trchi2 was N,N-diacetyl-chitobiose.The optimum temperature and pH of enzyme reaction were45℃and 6.0,respectively.Under 30 to 55℃,the enzyme reaction was relatively stable.The enzyme solution treated at 60℃for 1 h still had 25%relative activity.While pH between 4 to 7.5,the relative activity was stable at more than 50%.When there were Ag^(+),Hg^(2+),Cu^(2+) and Fe^(2+) at a concentration of 50 mmol/L in catalytic system,activity was strongly inhibited,and the relative activity was less than 5%.Na^(+),K^(+) and Ca^(2+) had no significant effect on chitinase Trchi2.The activity range of the expressed chitinase was similar to that of most fungal chitinases,with a wide range of temperature adaptability.This study lays a foundation for the further study of chitinase Trchi2 and also promotes the production and application of chitinase.

关 键 词：粉红聚端孢 几丁质酶Trchi2 毕赤酵母 酶学性质 

分 类 号：S182[农业科学—农业基础科学]