夏颖,赵杰,夏黎明

• 1:浙江大学生物质化工教育部重点实验室浙江大学化学工程与生物工程学院

摘要(Abstract)：

刺糙青霉(Penicillium echinulatum)可产生一种耐热、耐碱的新型内切-β-葡聚糖酶(EGL1),具有重要的工业应用价值。鉴于野生型刺糙青霉的产酶水平低,将该菌的内切-β-葡聚糖酶基因经过密码子优化后,在里氏木霉(Trichoderma reesei)中进行重组与表达,采用里氏木霉强启动子Pcbh1(纤维二糖水解酶Ⅰ)及其信号肽,以pCAMBIA1300为载体骨架构建重组质粒pCB-PET,并采用根瘤农杆菌介导转化技术将重组质粒pCB-PET导入里氏木霉的分生孢子中,在潮霉素抗性平板上筛选获得5个优良的重组转化子。将5个转化子重复传代培养10个批次后,分别提取染色体DNA进行PCR验证,均可检测到目的基因Egl1n(密码子优化后的Egl1)条带,说明该基因已稳定地整合到里氏木霉基因组中。采用SDS-PAGE蛋白电泳对转化子培养液进行检测,获得了与目的基因表达产物相符的蛋白条带(约41 kDa),表明Egl1n已在重组里氏木霉中成功实现了胞外表达。重组转化子在30℃,摇瓶转速200 r×min~(-1)条件下培养48 h,取发酵上清液在pH 8.0,60℃条件下,测得内切-β-葡聚糖酶活力为382.6 U×mL~(-1),是出发菌株的22.5倍。酶学性质研究结果表明:该酶耐热性能较好,在70℃以下性能稳定,其最适催化温度为60℃;该酶在pH 5.0~10.5稳定性较好、具有明显的催化活性,其最适pH为8.0,属于碱性纤维素酶。从而成功地实现了刺糙青霉内切-β-葡聚糖酶基因在里氏木霉中的重组与胞外表达,有关研究结果在里氏木霉纤维素酶的定向进化及其工业化应用中具有重要的促进作用。

关键词(KeyWords)： 内切-β-葡聚糖酶;碱性纤维素酶;里氏木霉;基因重组;表达

Abstract:

Keywords:

基金项目(Foundation): 浙江省重点科技创新团队资助项目(2011R50002)

作者(Author): 夏颖,赵杰,夏黎明

参考文献(References)：

• [1]Saloheima M,Pakula T.The cargo and the transport system:secreted proteins and protein secretion in Trichoderma reesei(Hypocrea jecorina)[J].Microbiology,2012,158(1):46-57.
• [2]Heikinheimo L,Buchert J,Miettinen-Oinonen A,et al.Treating denim fabrics with Trichoderma reesei cellulases[J].Textile Research Journal,2000,70(11):969-973.
• [3]Bhat M K.Cellulases and related enzymes in biotechnology[J].Biotechnology Advances,2000,18(5):355-383.
• [4]Miettinen-Oinonen A,Londesborough J,Joutsjoki V,et al.Three cellulases from Melanoarpus Albomyces with applications in the textile industry[J].Enzyme and Microbial Technology,2004,34(1):332-341.
• [5]Zhang Y H P,Himmel M E,Mielenz J R.Outlook for cellulose improvement:screening and selection strategies[J].Biotechnology Advances,2006,24(5):452-481.
• [6]Kumar R,Singh S,Singh O V.Bioconversion of lignocellulosic biomass:biochemical and molecular perspectives[J].Journal of Industrial Microbiology and Biotechnology,2008,35(5):377-391.
• [7]Dillon A J P,Zorgi C,Camassola M,et al.Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase andβ-glucosidase activities[J].Applied Microbiology and Biotechnology,2008,70(6):740-746.
• [8]Camassola M,De Bittencourt L R,Shenem N T,et al.Characterization of the cellulase complex of Penicillium echinulatum[J].Biocatalysis and Biotransformation,2004,22(5-6):391-396.
• [9]Rau M,Heidemann C,Pascoalin A M,et al.Application of cellulases from Acrophialophora nainiana and Penicillium echinulatum in textile processing of cellulosic fibres[J].Biocatalysis and Biotransformation,2008,26(5):383-390.
• [10]Wang B B,Xia L M.High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei[J].Bioresource Technology,2011,102(6):4568-4572.
• [11]Kiiskinen L L,Kruus K,Bailey M,et al.Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme[J].Microbiology-Sgm,2004,150(9):3065-3074.
• [12]Liu T,Wang T H,Li X,et al.Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene(cbh1)promoter optimization[J].Acta Biochimica et Biophysica Sinica,2008,40(2):158-165.
• [13]Xia L M,Shen X L.High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue[J].Bioresource Technology,2004,91(3):259-262.
• [14]Jyothishwaran G,Kotresha D,Selvaraj T,et al.A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens[J].Current Science,2007,93(6):770-772.
• [15]Sun W L,Liu L X,Hu X L,et al.Generation and identification of DNA sequence flanking T-DNA integration site of Trichoderma atroviride mutants with high dichlorvos-degrading capacity[J].Bioresource Technology,2009,100(23):5941-5946.
• [16]Michielse C B,Hooykaas P J J,Vanden Hondel C A,et al.Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori[J].Nature Protocols,2008,3(10):1671-1678.
• [17]Jyothishwaran G,Kotresha D,Selvaraj T,et al.A modified freeze-thaw method for efficient transformation of Agrobacterium tumefaciens[J].Current Science,2007,93(6):770-772.
• [18]HUANG Zhen-yan(黄振艳),XIA Li-ming(夏黎明).Study on the soluble inducer for cellulase production derived from glucose by glucosylase(利用葡萄糖转苷酶制备纤维素酶可溶性诱导物的研究)[J].Journal of Chemical Engineering of Chinese Universities(高校化学工程学报),2009,23(2):270-274.
• [19]Miller G L.Use of dinitrosalicylic acid reagent for the determination of reducing sugar[J].Analytical Chemistry,1959,31(3):426-428.
• [20]Jin X,Xia L M.Heterologous expression of an endo-b-1,4-glucanase gene from the anaerobic fungus Orpinomyces PC-2 in Trichoderma reesei[J].World Journal of Microbiology and Biotechnology,2011,27(12):2913-2920.
• [21]Fang H,Xia L M.Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw[J].Biomass and Bioenergy,2015,78(1):99-109.