土壤微生物中新型β-葡萄糖苷酶的挖掘与鉴定
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摘要
【背景】通过培养微生物来获得新β-葡萄糖苷酶因只有少部分微生物可以被培养而受到限制,但宏基因组学技术可以挖掘非培养微生物来源的β-葡萄糖苷酶资源。【目的】利用宏基因组学技术挖掘土壤微生物来源的新型β-葡萄糖苷酶,并对其酶学性质进行初步分析。【方法】构建土壤微生物的宏基因组文库,利用七叶苷平板显色法对所构建的文库进行筛选获得阳性克隆,并对阳性克隆所含的β-葡萄糖苷酶基因进行异源表达和生物化学特性分析。【结果】通过筛选文库中的62万个克隆,获得一个具有β-葡萄糖苷酶活性的克隆,其插入片段中包含一个2 301 bp的ORF(YNBG3),蛋白同源性分析显示其属于β-葡萄糖苷酶第三家族。对YNBG3酶的生化分析确定其最佳反应温度为53°C,最适p H为5.2,有较好的热稳定性,对一定浓度范围内的DMSO、丙酮、乙醇等有机试剂有较好的耐受性,EDTA和尿素可增加该酶的活性。【结论】利用宏基因组学技术获得了一个有较好热稳定性及耐受一定浓度有机试剂和尿素的新β-葡萄糖苷酶。
[Background] Only a small proportion of microbes can be cultured in laboratories, thereby hampering the discovery of novel β-glucosidases from microbes. Novel microbial β-glucosidases can be discovered using culture-independent metagenomic approach. [Objective] Discovery of new β-glucosidase from soil microbes by metagenomic technology. [Methods] Screening a metagenomic library on esculin-containing Luria-Bertani agar plates and the putative β-glucosidase gene derived from the active clone was heterologously expresse d for characterization. [Results] We found a β-glucosidase active clone by screening 620 000 clones in the library and an ORF(YNBG3) whose product is homologous with the third family of β-glucosidase was identified from the active clone. YNBG3 was heterologously expressed and characterized. Its optimum p H is 5.2, and optimal temperature is 53 °C. YNBG3 showed good tolerance to organic solvents including dimethyl sulphoxide, acetone and ethanol, and the activity of YNBG3 was enhanced in presence of EDTA and urea. [Conclusion] A new β-glucosidase, with thermostability and stability in the presence of organic solvents and urea, was discovery in this study.
[Background] Only a small proportion of microbes can be cultured in laboratories, thereby hampering the discovery of novel β-glucosidases from microbes. Novel microbial β-glucosidases can be discovered using culture-independent metagenomic approach. [Objective] Discovery of new β-glucosidase from soil microbes by metagenomic technology. [Methods] Screening a metagenomic library on esculin-containing Luria-Bertani agar plates and the putative β-glucosidase gene derived from the active clone was heterologously expresse d for characterization. [Results] We found a β-glucosidase active clone by screening 620 000 clones in the library and an ORF(YNBG3) whose product is homologous with the third family of β-glucosidase was identified from the active clone. YNBG3 was heterologously expressed and characterized. Its optimum p H is 5.2, and optimal temperature is 53 °C. YNBG3 showed good tolerance to organic solvents including dimethyl sulphoxide, acetone and ethanol, and the activity of YNBG3 was enhanced in presence of EDTA and urea. [Conclusion] A new β-glucosidase, with thermostability and stability in the presence of organic solvents and urea, was discovery in this study.
引文
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[21]Suzuki K,Sumitani J,Nam YW,et al.Crystal structures of glycoside hydrolase family 3β-glucosidase 1 from Aspergillus aculeatus[J].Biochemical Journal,2013,452(2):211-221
[22]Fang W,Fang ZM,Liu JJ,et al.Cloning and characterization of aβ-glucosidase from marine metagenome[J].Chinese Journal of Biotechnology,2009,25(12):1914-1920(in Chinese)房伟,方泽民,刘娟娟,等.新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质[J].生物工程学报,2009,25(12):1914-1920
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[24]Gu XQ,Wang SL,Wang SC,et al.Identification and characterization of two novel esterases from a metagenomic library[J].Food Science and Technology Research,2015,21(5):649-657
[25]Li YK,Chir J,Tanaka S,et al.Identification of the general acid/base catalyst of a family 3β-glucosidase from Flavobacterium meningosepticum[J].Biochemistry,2002,41(8):2751-2759
[26]Pellerin P,Brillouet JM.Purification and properties of an exo-(1→3)-beta-D-galactanase from Aspergillus niger[J].Carbohydrate Research,1994,264(2):281-291
[27]Rodríguez ME,Lopes C,Valles S,et al.Selection and preliminary characterization ofβ-glycosidases producer Patagonian wild yeasts[J].Enzyme and Microbial Technology,2007,41(6/7):812-820
[28]Yi XN,Cheng W,Ding SH,et al.Construction of an engineered strain producing highβ-glucosidase activity and its application in the production of 2,6-Dimethoxybenzoquinone[J].Food Science,2016,38(8):69-73(in Chinese)易晓男,程炜,丁树慧,等.高产β-葡萄糖苷酶工程菌株的构建及其在2,6-二甲氧基对苯醌収酵制备中的应用[J].食品科学,2016,38(8):69-73
[29]Peng LS,Zhang YX,Yan Q,et al.Separation,purification,and enzymatic properties of thermo-tolerantβ-glucosidase from Tridchoderma viride[J].Biotechnology Bulletin,2016,32(9):189-196(in Chinese)彭利沙,张永祥,闫青,等.绿色木霉耐热β-葡萄糖苷酶分离纯化及酶学性质研究[J].生物技术通报,2016,32(9):189-196
[30]Zhou HC,Lei PD,Ding Y.Research advance onβ-glucosidase of tea plant[J].Journal of Tea Science,2016,36(2):111-118(in Chinese)周汉琛,雷攀登,丁勇.茶树β-葡萄糖苷酶研究迚展[J].茶叶科学,2016,36(2):111-118
[31]Lu J,Du LQ,Pang H,et al.Construction of metagen omic library of microbes from sugar enriching soil and identification ofβ-glucosidase genes[J].Genomics and Applied Biology,2013,32(1):30-35(in Chinese)陆坚,杜丼琴,庞浩,等.高糖土壤微生物宏基因组文库的构建及β-葡萄糖苷酶基因鉴定[J].基因组学与应用生物学,2013,32(1):30-35
[32]Hou YH,Yan SC,Wu WJ,et al.Gene cloning and characterization of aβ-glucosidase gene from Coptotermes formosanus Shiraki[J].Journal of Northeast Forestry University,2017,45(3):96-101(in Chinese)侯亚会,严善春,吴文静,等.台湾乳白蚁β-葡萄糖苷酶基因的克隆与分析[J].东北林业大学学报,2017,45(3):96-101
[33]Jiang CJ,Hao ZY,Zeng R,et al.Characterization of a novel serine protease inhibitor gene from a marine metagenome[J].Marine Drugs,2011,9(9):1487-1501
[34]Chen J,Hao WW,Wang CM,et al.Screening and identification ofβ-glucosidase-producing fungi,and purification and enzymatic analysis[J].Food Science,2013,34(5):191-196(in Chinese)陈静,郝伟伟,王春梅,等.产β-葡萄糖苷酶真菌的筛选鉴定、纯化及酶学性质分析[J].食品科学,2013,34(5):191-196
[35]Tang J,Su D,Yuan S,et al.Cloning and enzymatic characteristics of three newβ-glucosidases in soil metagenomic library[J].Guizhou Agricultural Sciences,2013,41(4):14-18(in Chinese)唐婧,苏迪,袁帅,等.土壤宏基因组文库中3个新β-葡萄糖苷酶基因的克隆及其酶学特性[J].贵州农业科学,2013,41(4):14-18
[36]Liu DH,Hao YM,Yue DD,et al.Isolation of a strain withβ-glucosidase and its enzymatic properties[J].China Brewing,2013,32(6):47-51,60(in Chinese)刘德海,郝益民,岳丹丹,等.一株产β-葡萄糖苷酶菌株的筛选及酶学性质研究[J].中国酿造,2013,32(6):47-51,60
[37]Su ZF.Bioinformation and expression patterns analysis of GH1β-glucosidase in Arabidopsis and rice[D].Tai’an:Master’s Thesis of Shandong Agricultural University,2014(in Chinese)苏振峰.GH1β-葡萄糖苷酶在拟南芥和水稻中的生物信息学及表达模式分析[D].泰安:山东农业大学硕士学位论文,2014
[2]Handelsman J,Rondon MR,Brady SF,et al.Molecular bio-logical access to the chemistry of unknown soil microbes:A new frontier for natural products[J].Chemistry&Biology,1998,5(10):R245-R249
[3]Hugenholtz P,Tyson GW.Microbiology:metagenomics[J].Nature,2008,455(7212):481-483
[4]Martin MF,Liras P.Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites[J].Annual Review of Medicine,1989,43(1):173-206
[5]Torsvik VL,Goksoyr J.Determination of bacterial DNA in soil[J].Soil Biology and Biochemistry,1978,10(1):7-12
[6]Qian CL,Liu N,Yan X,et al.Engineering a high-performance,metagenomic-derived novel xylanase with improved soluble protein yield and thermostability[J].Enzyme and Microbial Technology,2015,70:35-41
[7]Sathya TA,Khan M.Diversity of glycosyl hydrolase enzymes from metagenome and their application in food industry[J].Journal of Food Science,2014,79(11):R2149-R2156
[8]Mai ZM,Su HF,Li LZ,et al.Construction of a mangrove soil metagenome library and identification of two novelβ-glucosidase genes[J].Biotechnology Bulletin,2014(6):168-172(in Chinese)麦志茂,苏宏飞,李丼珍,等.红树林土壤微生物宏基因组文库的构建及两种新颖的β-葡萄糖苷酶基因的鉴定[J].生物技术通报,2014(6):168-172
[9]Willis JD,Oppert B,Oppert C,et al.Identification,cloning,and expression of a GHF9 cellulase from Tribolium castaneum(Coleoptera:Tenebrionidae)[J].Journal of Insect Physiology,2011,57(2):300-306
[10]Li XM,Ma HL.Comparison of twoβ-Glucosidase-producing strains from the intestinal tract of vegetarians and optimizaiton of fermentation conditions forβ-Glucosidase production[J].Food Science,2016,37(7):123-127(in Chinese)李笑梅,马慧玱.两株素食者肠道菌产β-葡萄糖苷酶考察及产酶条件优化[J].食品科学,2016,37(7):123-127
[11]Li YH.Research progress onβ-glucosidase[J].Journal of Anhui Agricultural University,2002,29(4):421-425(in Chinese)李进华.β-葡萄糖苷酶的研究迚展(综述)[J].安徽农业大学学报,2002,29(4):421-425
[12]Wood TM,Mc Crae SI.Purification and some properties of the extracellularβ-glucosidase of the cellulolytic fungus Trichoderma koningii[J].Journal of General and Applied Microbiology,1980,128:2973-2982
[13]Harada KM,Tanaka K,Fukuda Y,et al.Degradation of rice bran hemieellulose by Paenibacillus sp.strain HC1:gene cloning,characterization and function ofβ-D-glueosidase as an enzyme involved indegradation[J].Archives of Microbiology,2005,184:215
[14]Li YB,Wu GL.Current studies on theβ-glucosidase and the enzyme immobilization[J].Journal of Tea Business,2004,26(1):2l-22(in Chinese)李英波,吴国林.β-葡萄糖苷酶及酶固定化研究迚展[J].茶叶通报,2004,26(1):2l-22
[15]Pan LH,Luo JP.Advance in research and application ofβ-D-glucosidase[J].Food Science,2006,27(12):803-807(in Chinese)潘利华,罗建平.β-葡萄糖苷酶的研究及应用迚展[J].食品科学,2006,27(12):803-807
[16]Shao JH.The application ofβ-glucosidase[J].Chemistry of Life,2005,25(1):22-24(in Chinese)邵金辉.β-葡萄糖苷酶在工农医领域的应用[J].生命的化学,2005,25(1):22-24
[17]Wang X,Shen C,Zhou YB.Relationship betweenβ-glucosidase and tea flavor,and tea pest and disease resistence[J].Tea Communication,2014,41(4):8-12(in Chinese)王晓,沈程,周跃斌.β-葡萄糖苷酶与茶增香及抗病虫害的研究迚展[J].茶叶通讯,2014,41(4):8-12
[18]Mielenz J.Ethanol production from biomass:technology and commercialization status[J].Current Opinion in Biotechnology,2001,4(3):324-329
[19]Lynd LR,Weimer PJ,van Zyl WH,et al.Microbial cellulose utilization:fundamentals and biotechnology[J].Microbiology and Molecular Biology Reviews,2002,66(3):506-577
[20]Bhat MK.Cellulases and related enzymes in biotechnology[J].Biotechnology Advances,2000,18(5):355-383
[21]Suzuki K,Sumitani J,Nam YW,et al.Crystal structures of glycoside hydrolase family 3β-glucosidase 1 from Aspergillus aculeatus[J].Biochemical Journal,2013,452(2):211-221
[22]Fang W,Fang ZM,Liu JJ,et al.Cloning and characterization of aβ-glucosidase from marine metagenome[J].Chinese Journal of Biotechnology,2009,25(12):1914-1920(in Chinese)房伟,方泽民,刘娟娟,等.新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质[J].生物工程学报,2009,25(12):1914-1920
[23]Xia W.Diversity and molecular engineering of glycoside hvdrolvase family 3β-glucosidase from thermophilic fungi[D].Hangzhou:Doctoral Dissertation of Zhejiang University,2016(in Chinese)夏伟.嗜热真菌第3家族β-葡萄糖苷酶的多样性和分子改造[D].杭州:浙江大学博士学位论文,2016
[24]Gu XQ,Wang SL,Wang SC,et al.Identification and characterization of two novel esterases from a metagenomic library[J].Food Science and Technology Research,2015,21(5):649-657
[25]Li YK,Chir J,Tanaka S,et al.Identification of the general acid/base catalyst of a family 3β-glucosidase from Flavobacterium meningosepticum[J].Biochemistry,2002,41(8):2751-2759
[26]Pellerin P,Brillouet JM.Purification and properties of an exo-(1→3)-beta-D-galactanase from Aspergillus niger[J].Carbohydrate Research,1994,264(2):281-291
[27]Rodríguez ME,Lopes C,Valles S,et al.Selection and preliminary characterization ofβ-glycosidases producer Patagonian wild yeasts[J].Enzyme and Microbial Technology,2007,41(6/7):812-820
[28]Yi XN,Cheng W,Ding SH,et al.Construction of an engineered strain producing highβ-glucosidase activity and its application in the production of 2,6-Dimethoxybenzoquinone[J].Food Science,2016,38(8):69-73(in Chinese)易晓男,程炜,丁树慧,等.高产β-葡萄糖苷酶工程菌株的构建及其在2,6-二甲氧基对苯醌収酵制备中的应用[J].食品科学,2016,38(8):69-73
[29]Peng LS,Zhang YX,Yan Q,et al.Separation,purification,and enzymatic properties of thermo-tolerantβ-glucosidase from Tridchoderma viride[J].Biotechnology Bulletin,2016,32(9):189-196(in Chinese)彭利沙,张永祥,闫青,等.绿色木霉耐热β-葡萄糖苷酶分离纯化及酶学性质研究[J].生物技术通报,2016,32(9):189-196
[30]Zhou HC,Lei PD,Ding Y.Research advance onβ-glucosidase of tea plant[J].Journal of Tea Science,2016,36(2):111-118(in Chinese)周汉琛,雷攀登,丁勇.茶树β-葡萄糖苷酶研究迚展[J].茶叶科学,2016,36(2):111-118
[31]Lu J,Du LQ,Pang H,et al.Construction of metagen omic library of microbes from sugar enriching soil and identification ofβ-glucosidase genes[J].Genomics and Applied Biology,2013,32(1):30-35(in Chinese)陆坚,杜丼琴,庞浩,等.高糖土壤微生物宏基因组文库的构建及β-葡萄糖苷酶基因鉴定[J].基因组学与应用生物学,2013,32(1):30-35
[32]Hou YH,Yan SC,Wu WJ,et al.Gene cloning and characterization of aβ-glucosidase gene from Coptotermes formosanus Shiraki[J].Journal of Northeast Forestry University,2017,45(3):96-101(in Chinese)侯亚会,严善春,吴文静,等.台湾乳白蚁β-葡萄糖苷酶基因的克隆与分析[J].东北林业大学学报,2017,45(3):96-101
[33]Jiang CJ,Hao ZY,Zeng R,et al.Characterization of a novel serine protease inhibitor gene from a marine metagenome[J].Marine Drugs,2011,9(9):1487-1501
[34]Chen J,Hao WW,Wang CM,et al.Screening and identification ofβ-glucosidase-producing fungi,and purification and enzymatic analysis[J].Food Science,2013,34(5):191-196(in Chinese)陈静,郝伟伟,王春梅,等.产β-葡萄糖苷酶真菌的筛选鉴定、纯化及酶学性质分析[J].食品科学,2013,34(5):191-196
[35]Tang J,Su D,Yuan S,et al.Cloning and enzymatic characteristics of three newβ-glucosidases in soil metagenomic library[J].Guizhou Agricultural Sciences,2013,41(4):14-18(in Chinese)唐婧,苏迪,袁帅,等.土壤宏基因组文库中3个新β-葡萄糖苷酶基因的克隆及其酶学特性[J].贵州农业科学,2013,41(4):14-18
[36]Liu DH,Hao YM,Yue DD,et al.Isolation of a strain withβ-glucosidase and its enzymatic properties[J].China Brewing,2013,32(6):47-51,60(in Chinese)刘德海,郝益民,岳丹丹,等.一株产β-葡萄糖苷酶菌株的筛选及酶学性质研究[J].中国酿造,2013,32(6):47-51,60
[37]Su ZF.Bioinformation and expression patterns analysis of GH1β-glucosidase in Arabidopsis and rice[D].Tai’an:Master’s Thesis of Shandong Agricultural University,2014(in Chinese)苏振峰.GH1β-葡萄糖苷酶在拟南芥和水稻中的生物信息学及表达模式分析[D].泰安:山东农业大学硕士学位论文,2014