高产纤维素酶突变株的筛选及其产酶条件优化
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摘要
通过常压室温等离子体技术诱变里氏木霉RUT-C30,筛选高产纤维素酶突变株,并对其产酶进行优化,提高纤维素酶的产量。筛选得到高产纤维素酶突变株后,进行全基因组测序分析突变型,并对产酶培养基和培养条件进行优化。结果表明:经过筛选获得高产纤维素酶突变株JNDY-13,其摇瓶发酵最高滤纸酶活可达2.21 IU/mL,为出发菌株的2.21倍,优化后JNDY-13在5 L罐中流加发酵所产最高滤纸酶活为5.40 IU/mL;测序结果显示JNDY-13基因组中共有752个突变发生,其中半乳糖激酶基因中被插入的18个碱基可能是突变株纤维素酶活力增加的原因。
Mutants of Trichoderma reesei RUT-C30 induced by atmospheric and room temperature plasma(ARTP) were screened for cellulase production. Whole-genome sequencing was performed to analyze the mutation types of the selected mutant. Also, we investigated the optimization of fermentation medium and conditions for improved production of cellulase.Mutant JNDY-13 was found to produce the highest cellulase activity. Through shake-flask fermentation, the filter paper activity(FPA) of JNDY-13 was 2.21 IU/m L, which was 2.21 times higher than that of T. reesei RUT-C30. The highest FPA of 5.40 IU/m L was achieved through fed-batch fermentation in a 5-L fermentor under optimized conditions. A total of 752 mutations were identified in JNDY-13, and the 18-base pair insertion in the galactokinase gene may account for the increased cellulase activity.
Mutants of Trichoderma reesei RUT-C30 induced by atmospheric and room temperature plasma(ARTP) were screened for cellulase production. Whole-genome sequencing was performed to analyze the mutation types of the selected mutant. Also, we investigated the optimization of fermentation medium and conditions for improved production of cellulase.Mutant JNDY-13 was found to produce the highest cellulase activity. Through shake-flask fermentation, the filter paper activity(FPA) of JNDY-13 was 2.21 IU/m L, which was 2.21 times higher than that of T. reesei RUT-C30. The highest FPA of 5.40 IU/m L was achieved through fed-batch fermentation in a 5-L fermentor under optimized conditions. A total of 752 mutations were identified in JNDY-13, and the 18-base pair insertion in the galactokinase gene may account for the increased cellulase activity.
引文
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[5]顾方媛,陈朝银,石家骥,等.纤维素酶的研究进展与发展趋势[J].微生物学杂志,2008,28(1):83-87.DOI:10.3969/j.issn.1005-7021.2008.01.018.
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[7]M E S S N E RR,K U B I C E K-P R A N ZEM,G S U RA,e ta l.Cellobiohy drolase II is the main conidial-bound cellulasein Trichoder mareesei,and otherT richoder mastrains[J]. Archives of Microbiology, 1991, 155(6):601-606. DOI:10.1007/BF00245356.
[8]陆晨.高产纤维素酶菌株的筛选及其产酶条件的优化[D].长沙:中南林业科技大学, 2012:51-53.
[9]ZHANGX,ZHANGC,ZHOUQQ,etal.Quantitative evaluation of DNA damage and mutationrate by at mospheric and room temperature plasma(ARTP)and conventional mutagenesis[J]. Applied Microbiology&Biotechnology,2015,99(13):5639.DOI:10.1007/s00253-015-6678-y.
[10]程艳,堵国成,周景文,等.高通量筛选诱变菌株降低黄酒发酵氨基甲酸乙酯前体积累[J].微生物学报, 2017, 57(10):1517-1526.DOI:10.13343/j.cnki.wsxb.20160472.
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[12]KONGX,HE A,ZHAOJ,etal.Efficient acetone-butanol-ethanol(ABE)production by abutanol-tolerantmutant of Clostridium beijerinckii,inafermentation-pervaporation coupledprocess[J].Biochemical Engineering Journal,2016,105:90-96.DOI:10.1016/j.bej.2015.09.013.
[13]AHAMED A, VERMETTE P. Effect of culture medium composition on Trichoder mareesei’s morphology and cellulase production[J].Bioresource Technology,2009,100(23):5979-5987.DOI:10.1016/j.biortech.2009.02.070.
[14]RAPP P, GROTE E, WAGNER F. Formation and location of 1,4-betaglucanases and 1,4-beta-glucosidases from Penicillium janthinellum[J].Applied&Environmental Microbiology, 1981, 41(4):857-866.
[15]IVANOVAC,RAMONIJ, AOUAM T,etal.Genomese quencing and transcrip tomeanalysis ofT richoder mareesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction[J]. Biotechnology for Biofuels, 2017, 10(1):209. DOI:10.1186/s13068-017-0897-7.
[16]GHOSE T K. Measurement of cellulase activities[J]. Pure&Applied Chemistry, 2009, 59(2):257-268. DOI:10.1351/pac198759020257.
[17]KOWALSKIZE,GIESECKEWH. Ahighperformanceliquid chromatographic method for the fluorimetric determination of lactose,galactose and glucose innormal and abnormal milk of cows[J].Onderstepoort Journal of Veterinary Research, 1986, 53(4):225.
[18]方浩.里氏木霉纤维二糖水解酶II基因的克隆与表达[D].杭州:浙江大学, 2014:30-40.
[19]ZHAO Y,CIMPOIAR,LIUZ,etal.Orthogonaloptimizationof carboxydothermus hydrogenoformans, culture medium for hydrogen productionfromcarbonmonoxidebybiologicalwater-gasshift reaction[J]. International Journal of Hydrogen Energy, 2011, 36(17):10655-10665. DOI:10.1016/j.ijhydene.2011.05.134.
[20]CARDER J H. Detection and quantitation of cellulase by Congo red stainingofsubstratesinacup-platediffusionassay[J]. Analytical Biochemistry, 1986, 153(1):75. DOI:10.1016/0003-2697(86)90063-1.
[21]LAMB J, LOY T. Seeing red:the use of Congo Red dye to identify cookedanddamagedstarchgrainsinarchaeologicalresidues[J].Journalof ArchaeologicalScience,2005,32(10):1433-1440.DOI:10.1016/j.jas.2005.03.020.
[22]MANE S P, MODISE T, SOBRAL B W. Analysis of high-throughput sequencing data[J]. Methods in Molecular Biology, 2011, 678(5):1.DOI:10.1007/978-1-60761-682-5_1.
[23]DRUZHININAIS,KUBICEKCP.Geneticengineeringof Trichodermareeseicellulasesandtheirproduction[J].Microbial Biotechnology, 2017, 10:1485-1499. DOI:10.1111/1751-7915.12726.
[24]SEIBOTH B, HARTL L, PAIL M, et al. The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on D-galactose[J]. Molecular Microbiology, 2004, 51(4):1015-1025. DOI:10.1046/j.1365-2958.2003.03901.x.
[25]PRASETYO J, SUMITA S, OKUDA N, et al. Response of cellulase activityinpH-controlledculturesofthefilamentousfungus Acremonium cellulolyticus[J]. Applied Biochemistry&Biotechnology,2010, 162(1):52. DOI:10.1086/597525.
[26]HENDYN A,WILKECR,BLANCHHW.Enhancedcellulase production in fed-batch culture of Trichoderma reesei, C30[J]. Enzyme&MicrobialTechnology,1984,6(2):73-77.DOI:10.1016/0141-0229(84)90038-3.
[27]LIC,YANGZ,ZHANGRH,etal.EffectofpHoncellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis[J]. Journal of Biotechnology, 2013,168(4):470. DOI:10.1016/j.jbiotec.2013.10.003.
[28]SUH D H, BECKER T C, SANDS J A, et al. Effects of temperature onxylanasesecretionbyTrichodermareesei[J].Biotechnology&Bioengineering, 1988, 32(6):821-825. DOI:10.1002/bit.260320614.
[29]张晓烜,李景富,王傲雪.里氏木霉产纤维素酶条件的优化[J].东北农业大学学报,2008(7):29-33.DOI:10.3969/j.issn.1005-9369.2008.07.008.
[30]JANAS P, TARGO?SKI Z. Effect of temperature on the production of cellulases, xylanases and lytic enzymes by selected Trichoderma reesei mutants[J]. ActaMycologica,1995,30(2):255-264.DOI:10.5586/am.1995.022.
[31]WASEDA,MCMANAMEYWJ,RAYMAHASAYS,etal.Comparisons between cellulase production byAspergillus fumigatus inagitatedvesselsandinanair-liftfermentor[J].Biotechnology&Bioengineering, 1985, 27(8):1166-1172. DOI:10.1002/bit.260270811.
[32]姜伯玲.重离子辐照诱变选育纤维素酶高产菌株及其发酵工艺的研究[D].兰州:中国科学院研究生院(近代物理研究所), 2016:40-42.
[33]张素敏.纤维素酶高产菌株—里氏木霉的选育及发酵生产条件研究[D].福州:福州大学, 2013:65-66.
[34]HANG Z X, RAO Q L, YU S Y. The Influence of PH and dissolved oxygen tension(DOT)on mycelium growth and cellulase production by Trichoderma reesei[J]. Advanced Materials Research, 2011, 236/237/238:1005-1013. DOI:10.4028/www.scientific.net/AMR.236-238.1005.
[35]SHAHRIARINOURM,MOHDNOOR AW, ARIFF AB,etal.Kinetics of cellulase production by Aspergillus terreus at various levels of dissolved oxygen tension in a stirred tank bioreactor[J]. Bioresources,2011, 6(4):4909-4921. DOI:10.15376/biores.6.4.4909-4921.
[2]ANG S K, SHAZA E M, ADIBAH Y, et al. Production of cellulases and xylanase by Aspergillus fumigatus, SK1 using untreated oil palm trunk through solid state fermentation[J]. Process Biochemistry, 2013,48(9):1293-1302. DOI:10.1016/j.procbio.2013.06.019.
[3]KARMAKAR M, RAY R R. Current trends in research and application of microbial cellulases[J].Research Journal of Microbiology,2011,6(1):41-53. DOI:10.3923/jm.2011.41.53.
[4]BR??KNER C, AUSTB??D, N?SSET J A, et al. Equine pre-caecal and tota ltract digesti bility of in dividual carbohy drate fractions and their effect on caecal pH response[J]. Archives of Animal Nutrition,2012, 66(6):490-506. DOI:10.1080/1745039X.2012.740311.
[5]顾方媛,陈朝银,石家骥,等.纤维素酶的研究进展与发展趋势[J].微生物学杂志,2008,28(1):83-87.DOI:10.3969/j.issn.1005-7021.2008.01.018.
[6]李雪峰,侯红萍.选育高产纤维素酶菌种的研究进展[J].酿酒科技,2010(5):92-94. DOI:10.13746/j.njkj.2010.05.004.
[7]M E S S N E RR,K U B I C E K-P R A N ZEM,G S U RA,e ta l.Cellobiohy drolase II is the main conidial-bound cellulasein Trichoder mareesei,and otherT richoder mastrains[J]. Archives of Microbiology, 1991, 155(6):601-606. DOI:10.1007/BF00245356.
[8]陆晨.高产纤维素酶菌株的筛选及其产酶条件的优化[D].长沙:中南林业科技大学, 2012:51-53.
[9]ZHANGX,ZHANGC,ZHOUQQ,etal.Quantitative evaluation of DNA damage and mutationrate by at mospheric and room temperature plasma(ARTP)and conventional mutagenesis[J]. Applied Microbiology&Biotechnology,2015,99(13):5639.DOI:10.1007/s00253-015-6678-y.
[10]程艳,堵国成,周景文,等.高通量筛选诱变菌株降低黄酒发酵氨基甲酸乙酯前体积累[J].微生物学报, 2017, 57(10):1517-1526.DOI:10.13343/j.cnki.wsxb.20160472.
[11]MANDELS M, MEDEIROS J E, ANDREOTTI R E, et al. Enzymatic hydrolysis of cellulose:evaluation of cellulase culture filtrates under useconditions[J].Biotechnology&Bioengineering,1981,23(9):2009-2026. DOI:10.1002/bit.260230907.
[12]KONGX,HE A,ZHAOJ,etal.Efficient acetone-butanol-ethanol(ABE)production by abutanol-tolerantmutant of Clostridium beijerinckii,inafermentation-pervaporation coupledprocess[J].Biochemical Engineering Journal,2016,105:90-96.DOI:10.1016/j.bej.2015.09.013.
[13]AHAMED A, VERMETTE P. Effect of culture medium composition on Trichoder mareesei’s morphology and cellulase production[J].Bioresource Technology,2009,100(23):5979-5987.DOI:10.1016/j.biortech.2009.02.070.
[14]RAPP P, GROTE E, WAGNER F. Formation and location of 1,4-betaglucanases and 1,4-beta-glucosidases from Penicillium janthinellum[J].Applied&Environmental Microbiology, 1981, 41(4):857-866.
[15]IVANOVAC,RAMONIJ, AOUAM T,etal.Genomese quencing and transcrip tomeanalysis ofT richoder mareesei QM9978 strain reveals a distal chromosome translocation to be responsible for loss of vib1 expression and loss of cellulase induction[J]. Biotechnology for Biofuels, 2017, 10(1):209. DOI:10.1186/s13068-017-0897-7.
[16]GHOSE T K. Measurement of cellulase activities[J]. Pure&Applied Chemistry, 2009, 59(2):257-268. DOI:10.1351/pac198759020257.
[17]KOWALSKIZE,GIESECKEWH. Ahighperformanceliquid chromatographic method for the fluorimetric determination of lactose,galactose and glucose innormal and abnormal milk of cows[J].Onderstepoort Journal of Veterinary Research, 1986, 53(4):225.
[18]方浩.里氏木霉纤维二糖水解酶II基因的克隆与表达[D].杭州:浙江大学, 2014:30-40.
[19]ZHAO Y,CIMPOIAR,LIUZ,etal.Orthogonaloptimizationof carboxydothermus hydrogenoformans, culture medium for hydrogen productionfromcarbonmonoxidebybiologicalwater-gasshift reaction[J]. International Journal of Hydrogen Energy, 2011, 36(17):10655-10665. DOI:10.1016/j.ijhydene.2011.05.134.
[20]CARDER J H. Detection and quantitation of cellulase by Congo red stainingofsubstratesinacup-platediffusionassay[J]. Analytical Biochemistry, 1986, 153(1):75. DOI:10.1016/0003-2697(86)90063-1.
[21]LAMB J, LOY T. Seeing red:the use of Congo Red dye to identify cookedanddamagedstarchgrainsinarchaeologicalresidues[J].Journalof ArchaeologicalScience,2005,32(10):1433-1440.DOI:10.1016/j.jas.2005.03.020.
[22]MANE S P, MODISE T, SOBRAL B W. Analysis of high-throughput sequencing data[J]. Methods in Molecular Biology, 2011, 678(5):1.DOI:10.1007/978-1-60761-682-5_1.
[23]DRUZHININAIS,KUBICEKCP.Geneticengineeringof Trichodermareeseicellulasesandtheirproduction[J].Microbial Biotechnology, 2017, 10:1485-1499. DOI:10.1111/1751-7915.12726.
[24]SEIBOTH B, HARTL L, PAIL M, et al. The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on D-galactose[J]. Molecular Microbiology, 2004, 51(4):1015-1025. DOI:10.1046/j.1365-2958.2003.03901.x.
[25]PRASETYO J, SUMITA S, OKUDA N, et al. Response of cellulase activityinpH-controlledculturesofthefilamentousfungus Acremonium cellulolyticus[J]. Applied Biochemistry&Biotechnology,2010, 162(1):52. DOI:10.1086/597525.
[26]HENDYN A,WILKECR,BLANCHHW.Enhancedcellulase production in fed-batch culture of Trichoderma reesei, C30[J]. Enzyme&MicrobialTechnology,1984,6(2):73-77.DOI:10.1016/0141-0229(84)90038-3.
[27]LIC,YANGZ,ZHANGRH,etal.EffectofpHoncellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis[J]. Journal of Biotechnology, 2013,168(4):470. DOI:10.1016/j.jbiotec.2013.10.003.
[28]SUH D H, BECKER T C, SANDS J A, et al. Effects of temperature onxylanasesecretionbyTrichodermareesei[J].Biotechnology&Bioengineering, 1988, 32(6):821-825. DOI:10.1002/bit.260320614.
[29]张晓烜,李景富,王傲雪.里氏木霉产纤维素酶条件的优化[J].东北农业大学学报,2008(7):29-33.DOI:10.3969/j.issn.1005-9369.2008.07.008.
[30]JANAS P, TARGO?SKI Z. Effect of temperature on the production of cellulases, xylanases and lytic enzymes by selected Trichoderma reesei mutants[J]. ActaMycologica,1995,30(2):255-264.DOI:10.5586/am.1995.022.
[31]WASEDA,MCMANAMEYWJ,RAYMAHASAYS,etal.Comparisons between cellulase production byAspergillus fumigatus inagitatedvesselsandinanair-liftfermentor[J].Biotechnology&Bioengineering, 1985, 27(8):1166-1172. DOI:10.1002/bit.260270811.
[32]姜伯玲.重离子辐照诱变选育纤维素酶高产菌株及其发酵工艺的研究[D].兰州:中国科学院研究生院(近代物理研究所), 2016:40-42.
[33]张素敏.纤维素酶高产菌株—里氏木霉的选育及发酵生产条件研究[D].福州:福州大学, 2013:65-66.
[34]HANG Z X, RAO Q L, YU S Y. The Influence of PH and dissolved oxygen tension(DOT)on mycelium growth and cellulase production by Trichoderma reesei[J]. Advanced Materials Research, 2011, 236/237/238:1005-1013. DOI:10.4028/www.scientific.net/AMR.236-238.1005.
[35]SHAHRIARINOURM,MOHDNOOR AW, ARIFF AB,etal.Kinetics of cellulase production by Aspergillus terreus at various levels of dissolved oxygen tension in a stirred tank bioreactor[J]. Bioresources,2011, 6(4):4909-4921. DOI:10.15376/biores.6.4.4909-4921.
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