编码草菇中性内切葡聚糖酶Ⅰ基因（eg Ⅰ）的克隆与表达研究
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摘要
本论文以草菇Volvariella volvacea V23总RNA为模板,采用RT-PCR法,克隆了包括自身信号肽在内的中性内切葡聚糖酶Ⅰ基因(egⅠ)的cDNA序列。测序结果表明,cDNA序列全长1167 bp,编码389个氨基酸,推测第1-23位氨基酸为信号肽,第24-389位氨基酸为成熟肽,属于糖苷水解酶第五家族。
将EGⅠ成熟肽编码序列克隆进大肠杆菌表达载体pET-28a,构建了重组表达载体pET/ egⅠ,转化大肠杆菌BL21(DE3)获得了重组菌株BL21/ egⅠ。SDS-PAGE分析表明:EGⅠ在大肠杆菌中实现了胞内表达,分子量约为39.5 kD,但不具有酶活力。
将EGⅠ成熟肽编码序列克隆进分泌型表达载体pPIC9K,构建了重组表达载体pPIC9K/ egⅠ,用SalⅠ线性化后电转化毕赤酵母GS115,经过G418抗性和产酶能力的筛选,得到了一株高产菌株P. pastoris-EGⅠ1,SDS-PAGE分析表明:EGⅠ在毕赤酵母中实现了分泌表达,分子量约为42 kD,大于39.5 kD的理论分子量,原因可能是表达产物在翻译后修饰时发生了糖基化。
对毕赤酵母发酵生产EGⅠ的条件进行了优化,结果显示:在诱导时间为96 h,甲醇诱导浓度为2.0%,最初诱导pH为7.5,BMGY培养基甘油浓度为4.0%,BMMY培养基补加甘油0.75%和表面活性剂吐温20浓度为0.15%时,产酶效率最高,达到4612U/mL。
对EGⅠ的部分酶学性质进行了研究,结果显示:酶的最适反应温度为55℃;最适反应pH为7.5;当温度在55℃以下时,酶有很好的温度稳定性,当超过55℃时快速下降,但是到65℃时仍然保存60%的酶活力;在pH6.5-9.0的范围内,酶液有较好的pH稳定性,其酶活力保存90%以上。
An endoglucanaseⅠ(EGⅠ) cDNA including signal peptide was isolated from V. volvacea V23 by RT-PCR. The full-length cDNA contained an ORF of 1167 bp which encoded a signal peptide of 23 amino-acids and a mature peptide of 366 amino-acids. Similarity analysis showed the EGⅠbelonged to family 5 of glycosyl hydrolases.
The cDNA encoding the mature peptide was cloned into expression plasmid pET-28a, and expressed in Escherichia coli BL21(DE3) by IPTG induction, SDS-PAGE showed that EGⅠwas expressed successfully, the molecular weight of EGⅠwas about 39.5 kD but without enzymatic activity.
The mature peptide cDNA was cloned into the secretive expression vector pPIC9K.The recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after linearized by SalⅠ.After screening, a high yielding recombinant strain P. pastoris-EGⅠ1 was obtained. SDS-PAGE showed the molecular weight of EGⅠwas about 42 kD,it was higher than the theoretic molecular weight which may be due to glycosylation after translation.
The conditions of expressing EGⅠin Pichia pastoris were optimized, The optimum conditions for EGⅠaccumulation were as follows: induced time 96 h by 2.0% methanol; the initial induced pH7.5;the concentration of glycerin in BMGY 4.0%; glycerin addition quantity per 24 h in BMMY 0.75%; the concentration of the surface active agent tween 20 in BMMY 0.15%,the enzyme activity was up to 4612U/mL at these conditions.
Part of the enzymatic properties of EGⅠwere determined, The results revealed that the optimal temperature and optimal pH for enzyme reaction were 55℃and 7.5;the enzyme had good stability when the temperature bellow 55℃while declined quickly above 55℃,but still holded 60% of the enzymatic activity when the temperature reached 65℃.the enzyme had good pH stability in pH6.5-9.0 and holded above 90%enzymatic activity.
将EGⅠ成熟肽编码序列克隆进大肠杆菌表达载体pET-28a,构建了重组表达载体pET/ egⅠ,转化大肠杆菌BL21(DE3)获得了重组菌株BL21/ egⅠ。SDS-PAGE分析表明:EGⅠ在大肠杆菌中实现了胞内表达,分子量约为39.5 kD,但不具有酶活力。
将EGⅠ成熟肽编码序列克隆进分泌型表达载体pPIC9K,构建了重组表达载体pPIC9K/ egⅠ,用SalⅠ线性化后电转化毕赤酵母GS115,经过G418抗性和产酶能力的筛选,得到了一株高产菌株P. pastoris-EGⅠ1,SDS-PAGE分析表明:EGⅠ在毕赤酵母中实现了分泌表达,分子量约为42 kD,大于39.5 kD的理论分子量,原因可能是表达产物在翻译后修饰时发生了糖基化。
对毕赤酵母发酵生产EGⅠ的条件进行了优化,结果显示:在诱导时间为96 h,甲醇诱导浓度为2.0%,最初诱导pH为7.5,BMGY培养基甘油浓度为4.0%,BMMY培养基补加甘油0.75%和表面活性剂吐温20浓度为0.15%时,产酶效率最高,达到4612U/mL。
对EGⅠ的部分酶学性质进行了研究,结果显示:酶的最适反应温度为55℃;最适反应pH为7.5;当温度在55℃以下时,酶有很好的温度稳定性,当超过55℃时快速下降,但是到65℃时仍然保存60%的酶活力;在pH6.5-9.0的范围内,酶液有较好的pH稳定性,其酶活力保存90%以上。
An endoglucanaseⅠ(EGⅠ) cDNA including signal peptide was isolated from V. volvacea V23 by RT-PCR. The full-length cDNA contained an ORF of 1167 bp which encoded a signal peptide of 23 amino-acids and a mature peptide of 366 amino-acids. Similarity analysis showed the EGⅠbelonged to family 5 of glycosyl hydrolases.
The cDNA encoding the mature peptide was cloned into expression plasmid pET-28a, and expressed in Escherichia coli BL21(DE3) by IPTG induction, SDS-PAGE showed that EGⅠwas expressed successfully, the molecular weight of EGⅠwas about 39.5 kD but without enzymatic activity.
The mature peptide cDNA was cloned into the secretive expression vector pPIC9K.The recombinant plasmid was introduced into Pichia pastoris GS115 by electroporation after linearized by SalⅠ.After screening, a high yielding recombinant strain P. pastoris-EGⅠ1 was obtained. SDS-PAGE showed the molecular weight of EGⅠwas about 42 kD,it was higher than the theoretic molecular weight which may be due to glycosylation after translation.
The conditions of expressing EGⅠin Pichia pastoris were optimized, The optimum conditions for EGⅠaccumulation were as follows: induced time 96 h by 2.0% methanol; the initial induced pH7.5;the concentration of glycerin in BMGY 4.0%; glycerin addition quantity per 24 h in BMMY 0.75%; the concentration of the surface active agent tween 20 in BMMY 0.15%,the enzyme activity was up to 4612U/mL at these conditions.
Part of the enzymatic properties of EGⅠwere determined, The results revealed that the optimal temperature and optimal pH for enzyme reaction were 55℃and 7.5;the enzyme had good stability when the temperature bellow 55℃while declined quickly above 55℃,but still holded 60% of the enzymatic activity when the temperature reached 65℃.the enzyme had good pH stability in pH6.5-9.0 and holded above 90%enzymatic activity.
引文
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[5] Peter Zugenmaier.Conformation and packing of various crystalline cellulose fibers[J],prog.polym.sci.2001,26:1341-1417.
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[8] Lemos M A,Teixeira J A,Domingues M R M,et al.The enhancement of the cellulolytic activity of cellobiohydrolaseI and endoglucanase by the addition of cellulose bindingdomains derived from Trichoderma reesei[J].Enzyme and Microbial Technology,2003,2:35-40.
[9]孟雷,陈冠军,王怡等.纤维素酶的多型性[J].纤维素科学与技术,2002,10(2):47- 55.
[10]陈春岚,李楠.细菌纤维素酶研究进展[J].广西轻工业,2007,1:18-20.
[11]秦浙新,王雪奇.纤维素酶的发酵研究进展[J].广西轻工业,2008,1:10-11.
[12]汪金萍,徐尔尼,史立康.高产纤维素酶生产方法的研究[J].食品科技,2007,2:73-75.
[13]胡春霞,宋会仪.真菌纤维素酶及其在饲料中的应用[J].饲料工业,2006,27(16):15-17.
[14] NINA A ,MARJA I ,ANU S ,et al .ACEI of Trichoderma reesei is a repressor of cellulose and xylanase expression[J].Applied and Environmental Microbiology ,2003 ,69 (1):56-65.
[15]刘颖,林亲录.纤维素酶制取与应用研究进展[J].中国食物与营养,2006,4:33-34.
[16] Smant G,Stokkemans J P,Yan Y T, et al.Endogenous cellulases in animals:isolation of beta-1,4-endoglucanase genes from two species of plant-parasitic cyst nematodes[J].Proc Natl Acad Sci USA,1998,95(9):4906-4911.
[17] Watanabe H,Noda H,Tokuda G, et al.A cellulose gene of termite origin[J]. Nature, 1998,394(6691):330-331.
[18] Tilbeurgh H,Tomme P,Claeystens M,et al.Limited proteolysis of the cellobiohydrolaseⅠfrom T.reesei[J].FEBS Lett,1986,204(2):223-227.
[19] Srisodsuk M.Mode of action of Trichoderma reesei CBH 1 on crystalline cellulose[J]. Enz Microb Tech,1994,23:213-219.
[20]杨永彬,黄谚谚,林跃鑫.纤维素酶的结构及分子多样性[J].生命的化学, 2004, 24 (3): 221-222.
[21]宋贤良,温其标,朱江.纤维素酶法水解的研究进展[J].郑州工程学院学报,2001, 22(4): 67-70.
[22]刘树立,王华,王春艳等.纤维素酶分子结构及作用机理的研究进展[J].食品科技, 2007,7:13-14.
[23] Juy M,Amit A G,Alzari P M,et al Crystal structure of a thermostable becterial cellulose degrading enzyme[J]. Nature,1992,357 (6373):89-91.
[24] Tomme P ,Warren RA ,Gilkes NR. Cellulose hydrolysis by bacteria and fungi [J] . Adv Microb Physiol ,1995 ,37 :1-81.
[25]张鸿雁,陈锡时.微生物纤维素酶分子生物学研究进展[J].生物技术,2003.13(3):41-42.
[26]张传富,顾文杰,彭科峰等.微生物纤维素酶的研究现状[J].生物信息学,2007,1:34-35.
[27] J .Morgado ,J. Andreaus, DG Kilburn et al. Interactions of cotton with CBD peptides. Enzyme Microb. Technol . 1999 ,25:639 - 643.
[28]阎伯旭,齐飞,张颖舒.纤维素酶分子结构和功能研究进展[J].生物化学与生物物理进展,1999,26(3):235-237.
[29] Reese E T. Polysaccharases and the hydrolysis of insoluble substrates[J]. Proc Sess, 1976, (6):9-12.
[30] Wood T M.The cellulase of fusarium solani purification and specificity of the (1,4)- glucosidase components[J].Biochem,1971,121:353.
[31]夏黎明.可再生纤维素资源酶法降解的研究进展[J].林产化工通讯,1999,33(1):23-28.
[32]刘燕,张宏福,孙哲.纤维素酶的分子生物学和基因工程研究进展[J].饲料工业,2007,28(18):12-13.
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