常压室温等离子体快速诱变绿色糖单孢菌筛选木聚糖酶高产菌株及其酶学性质研究
|
摘要
【目的】利用常压室温等离子体快速诱变绿色糖单孢菌,筛选耐热耐碱木聚糖酶高产菌株,并对其进行酶学性质分析,确保其适用于生物制浆漂白工艺。【方法】采用刚果红平板水解圈法结合摇瓶发酵胞外酶测定法进行菌株筛选,并通过DNS木聚糖酶活性测定等方法对来源于不同突变株的木聚糖酶进行酶学性质分析对比。【结果】筛选出遗传稳定性良好的两株木聚糖酶高产菌株AT24和AT22-2,以麦草浆为诱导底物的粗酶液中,突变株AT24及AT22-2所产的木聚糖酶活性分别为512.74、552.70 U/mL,分别为原始菌株S.v的16和17倍的。来源于突变株AT22-2的木聚糖酶的最适反应pH为9.5,最适反应温度为90°C,在50°C 90°C温度范围内具有良好的热稳定性,在100°C条件下处理30 min剩余酶活仍为68%;突变株AT24所产木聚糖酶的最适反应温度为60°C,最适pH为10.0,在60°C 80°C的高温环境下,突变株AT24所产的木聚糖酶具有良好的热稳定性。【结论】突变株AT22-2所产具有耐碱耐高温性质的木聚糖酶,在应用领域尤其在纸浆造纸行业具有较大的潜在应用价值。
[Objective] In order to obtain an industrial strain with higher xylanase production,the original strain of Saccharomonospora viridis was mutated by atmospheric and room temperature plasmas.[Methods] The methods used to screen the mutant strain with higher xylanase production included transparent loop diameter measurement of selective medium with 2% beech-wood xylan and shaking flask fermentation.[Results] It has been demonstrated that both mutants AT22-2 and AT24 maintain good genetic stability.The enzyme activity in the fermentation liquor of AT22-2 and AT24 at 7 d reached 552.70 U/mL and 512.74 U/mL,which were 17 and 16 times higher than that of the original strain,respectively.Furthermore,the optimum pH and temperature values for xylanase activity of AT22-2 were pH 9.5 and 90 °C,respectively,being stable within a temperature range of 50 °C 90 °C,and showing high thermostability at 100 °C for the duration of 30 min.In addition,for another mutant AT24,the optimum pH and temperature values for xylanase activity were pH 10.0 and 60 °C,with a stability within a temperature range of 60 °C 80 °C.[Conclusion] The xylanase with thermostability and alkali-tolerance of mutant AT22-2 is a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.
[Objective] In order to obtain an industrial strain with higher xylanase production,the original strain of Saccharomonospora viridis was mutated by atmospheric and room temperature plasmas.[Methods] The methods used to screen the mutant strain with higher xylanase production included transparent loop diameter measurement of selective medium with 2% beech-wood xylan and shaking flask fermentation.[Results] It has been demonstrated that both mutants AT22-2 and AT24 maintain good genetic stability.The enzyme activity in the fermentation liquor of AT22-2 and AT24 at 7 d reached 552.70 U/mL and 512.74 U/mL,which were 17 and 16 times higher than that of the original strain,respectively.Furthermore,the optimum pH and temperature values for xylanase activity of AT22-2 were pH 9.5 and 90 °C,respectively,being stable within a temperature range of 50 °C 90 °C,and showing high thermostability at 100 °C for the duration of 30 min.In addition,for another mutant AT24,the optimum pH and temperature values for xylanase activity were pH 10.0 and 60 °C,with a stability within a temperature range of 60 °C 80 °C.[Conclusion] The xylanase with thermostability and alkali-tolerance of mutant AT22-2 is a potential candidate for future use in biotechnological applications particularly in the pulp and paper industry.
引文
[1]刘巍,范树田,李心治,等.地衣芽孢杆菌H-1的鉴定及其产木聚糖酶性质的研究[J].工业微生物,1996,269(4):11-15.
[2]徐志兵,张群,夏万燕.酶在纸浆漂白中的应用[J].工业微生物,2002,32(1):54-56.
[3]曾宇成,张树政.海枣曲霉木聚糖酶的提纯和性质[J].微生物学报,1987,27(4):34-349.
[4]包怡红,刘伟丰,毛爱军,等.耐碱性木聚糖酶高产菌株的筛选、产酶条件优化及其在麦草浆生物漂白中的应用[J].农业生物技术学报,2005,13(2):235-240.
[5]刘相梅,祁蒙,曲音波.木聚糖酶基因克隆、表达与分泌及定点诱变研究进展[J].生物工程进展,2001,21(2):28-31.
[6]谢响明,孙晓霞,吴玉英,等.绿色糖单孢菌产木聚糖酶规律及其耐碱耐热性的初步研究[J].生命科学研究,2005,9(1):55-59.
[7]Máximo C,Ferriera MC,Duarte J.Some propertiesof eucalyptus kraft pulp treated with xylanase fromAspergillus niger[J].World Journal ofMicrobiology and Biotechnology,1998,14(3):365-367.
[8]Nakamura S,Wakabayashi K,Nakai R,et al.Purification and some properties of an alkalinexylanase from alkaliphilic Bacillus sp.strain41M-1[J].Applied and EnvironmentalMicrobiology,1993,59(7):2311-2316.
[9]Tabernero C,Sanchez J,Perez P,et al.Cloning andDNA sequencing of xyaA,a gene encoding anendo-β-1,4-xylanase from an alkalophilic Bacillusstrain(N137)[J].Applied and EnvironmentalMicrobiology,1995,61(6):2420-2424.
[10]Dey D,Hinge J,Shendye A,et al.Purification andproperties of extracellular endoxylanases fromalkaliphilic thermophilic Bacillus sp.[J].CanadianJournal of Microbiology,1992,38(5):436-442.
[11]Christakopoulos P,Nerinckx W,Kekos D,et al.Purification and characterization of two lowmolecular mass alkaline xylanases from Fusariumoxysporum F3[J].Journal of Biotechnology,1996,51(2):181-189.
[12]金丽华,方明月,张翀,等.常压室温等离子体快速诱变产油酵母的条件及其突变株的特性[J].生物工程学报,2011,27(3):461-467.
[13]张秋卓,蔡伟民.纤维素酶高产菌株的复合交替诱变选育[J].工业微生物,2008,38(6):32-37.
[14]全桂静,赵航.半纤维素酶高产菌株的筛选及产酶条件的研究[J].沈阳化工学院学报,2010,24(1):20-23,57.
[15]Lopez C,Blanco A,Javier pastor FI.Xylanaseproduction by a new alkali-tolerant isolate ofBacillus[J].Biotechnology Letter,1998,20(3):243-246.
[16]Sunna A,Bergquist PL.A gene encoding a novelextremely thermostable 1,4-beta-xylanase isolateddirectly from an environmental DNA sample[J].Extremophiles,2003,7(1):63-70.
[17]Bataillon M,Nunes CP,Castillon N,et al.Purification and characterization of a moderatelythermostable xylanase from Bacillus sp.strainSPS-0[J].Enzyme and Microbial Technology,2000,26(2/4):187-192.
[18]张龙翔,张庭芳,李令媛.生化实验方法和技术[M].北京:高等教育出版社,1997:138-140.
[19]陈红歌,刘新育,张世敏,等.木聚糖酶高产菌株的诱变[J].微生物学通报,2004,31(6):33-36.
[20]Wu M,Li SC,Yao JM,el al.Mutant of axylanase-produciing strain of Aspergillus niger insolid state fermentation by low energy ionimplantation[J].World Journal of Microbiology andBiotechnology,2005,21(6/7):1045-1049.
[21]Pompl R,Jamitzky F,Shimizu T,et al.The effect oflow-temperature plasma on bacteria as observed byrepeated AFM imaging[J].New Journal of Physics,2009,11(11):115023.
[22]Morfill GE,Kong MG,Zimmermann JL.Focus onplasma medicine[J].New Journal of Physics,2009,11(11):115011.
[23]Wang LY,Huang ZL,Li G,et al.Novel mutationbreeding method for Streptomyces avermitilis usingan atmospheric pressure glow discharge plasma[J].Journal of Applied Microbiology,2010,108(3):851-858.
[24]Wang LY.Studies on the mechanisms andapplications of the atmospheric room temperatureplasmas acting on the microbes[D].Beijing:Tsinghua University,2010.
[25]张义生,慈元钊.碱性木聚糖酶在麦草浆漂白生产中的应用试验[J].湖北造纸,2002(2):8-9.
[26]葛本灵.碱性木聚糖酶在硫酸盐竹浆漂白生产实践中的应用[J].华东纸业,2010,41(1):34-37.
[27]袁勤生.现代酶学[M].上海:华东理工大学出版社,2007:81-91.
[28]Ninawe S,Kuhad RC.Bleaching of wheatstraw-rich soda pulp with xylanase from athermoalkalophilic Streptomyces cyaneus SN32[J].Bioresource Technology,2006,97(18):2291-2295.
[29]Taibi Z,Saoudi B,Boudelaa M,et al.Purificationand biochemical characterization of a highlythermostable xylanase from Actinomadura sp.strain Cpt20 isolated from poultry compost[J].Applied Biochemistry and Biotechnology,2012,166(3):663-679.
[30]Zhengchu L,Xiaoyang D,Juzuo Z,et al.Screeningof a xylanase high-producing strain and its rapidseparation and purification[J].Annals ofMicrobiology,2011,61(4):901-906.
[2]徐志兵,张群,夏万燕.酶在纸浆漂白中的应用[J].工业微生物,2002,32(1):54-56.
[3]曾宇成,张树政.海枣曲霉木聚糖酶的提纯和性质[J].微生物学报,1987,27(4):34-349.
[4]包怡红,刘伟丰,毛爱军,等.耐碱性木聚糖酶高产菌株的筛选、产酶条件优化及其在麦草浆生物漂白中的应用[J].农业生物技术学报,2005,13(2):235-240.
[5]刘相梅,祁蒙,曲音波.木聚糖酶基因克隆、表达与分泌及定点诱变研究进展[J].生物工程进展,2001,21(2):28-31.
[6]谢响明,孙晓霞,吴玉英,等.绿色糖单孢菌产木聚糖酶规律及其耐碱耐热性的初步研究[J].生命科学研究,2005,9(1):55-59.
[7]Máximo C,Ferriera MC,Duarte J.Some propertiesof eucalyptus kraft pulp treated with xylanase fromAspergillus niger[J].World Journal ofMicrobiology and Biotechnology,1998,14(3):365-367.
[8]Nakamura S,Wakabayashi K,Nakai R,et al.Purification and some properties of an alkalinexylanase from alkaliphilic Bacillus sp.strain41M-1[J].Applied and EnvironmentalMicrobiology,1993,59(7):2311-2316.
[9]Tabernero C,Sanchez J,Perez P,et al.Cloning andDNA sequencing of xyaA,a gene encoding anendo-β-1,4-xylanase from an alkalophilic Bacillusstrain(N137)[J].Applied and EnvironmentalMicrobiology,1995,61(6):2420-2424.
[10]Dey D,Hinge J,Shendye A,et al.Purification andproperties of extracellular endoxylanases fromalkaliphilic thermophilic Bacillus sp.[J].CanadianJournal of Microbiology,1992,38(5):436-442.
[11]Christakopoulos P,Nerinckx W,Kekos D,et al.Purification and characterization of two lowmolecular mass alkaline xylanases from Fusariumoxysporum F3[J].Journal of Biotechnology,1996,51(2):181-189.
[12]金丽华,方明月,张翀,等.常压室温等离子体快速诱变产油酵母的条件及其突变株的特性[J].生物工程学报,2011,27(3):461-467.
[13]张秋卓,蔡伟民.纤维素酶高产菌株的复合交替诱变选育[J].工业微生物,2008,38(6):32-37.
[14]全桂静,赵航.半纤维素酶高产菌株的筛选及产酶条件的研究[J].沈阳化工学院学报,2010,24(1):20-23,57.
[15]Lopez C,Blanco A,Javier pastor FI.Xylanaseproduction by a new alkali-tolerant isolate ofBacillus[J].Biotechnology Letter,1998,20(3):243-246.
[16]Sunna A,Bergquist PL.A gene encoding a novelextremely thermostable 1,4-beta-xylanase isolateddirectly from an environmental DNA sample[J].Extremophiles,2003,7(1):63-70.
[17]Bataillon M,Nunes CP,Castillon N,et al.Purification and characterization of a moderatelythermostable xylanase from Bacillus sp.strainSPS-0[J].Enzyme and Microbial Technology,2000,26(2/4):187-192.
[18]张龙翔,张庭芳,李令媛.生化实验方法和技术[M].北京:高等教育出版社,1997:138-140.
[19]陈红歌,刘新育,张世敏,等.木聚糖酶高产菌株的诱变[J].微生物学通报,2004,31(6):33-36.
[20]Wu M,Li SC,Yao JM,el al.Mutant of axylanase-produciing strain of Aspergillus niger insolid state fermentation by low energy ionimplantation[J].World Journal of Microbiology andBiotechnology,2005,21(6/7):1045-1049.
[21]Pompl R,Jamitzky F,Shimizu T,et al.The effect oflow-temperature plasma on bacteria as observed byrepeated AFM imaging[J].New Journal of Physics,2009,11(11):115023.
[22]Morfill GE,Kong MG,Zimmermann JL.Focus onplasma medicine[J].New Journal of Physics,2009,11(11):115011.
[23]Wang LY,Huang ZL,Li G,et al.Novel mutationbreeding method for Streptomyces avermitilis usingan atmospheric pressure glow discharge plasma[J].Journal of Applied Microbiology,2010,108(3):851-858.
[24]Wang LY.Studies on the mechanisms andapplications of the atmospheric room temperatureplasmas acting on the microbes[D].Beijing:Tsinghua University,2010.
[25]张义生,慈元钊.碱性木聚糖酶在麦草浆漂白生产中的应用试验[J].湖北造纸,2002(2):8-9.
[26]葛本灵.碱性木聚糖酶在硫酸盐竹浆漂白生产实践中的应用[J].华东纸业,2010,41(1):34-37.
[27]袁勤生.现代酶学[M].上海:华东理工大学出版社,2007:81-91.
[28]Ninawe S,Kuhad RC.Bleaching of wheatstraw-rich soda pulp with xylanase from athermoalkalophilic Streptomyces cyaneus SN32[J].Bioresource Technology,2006,97(18):2291-2295.
[29]Taibi Z,Saoudi B,Boudelaa M,et al.Purificationand biochemical characterization of a highlythermostable xylanase from Actinomadura sp.strain Cpt20 isolated from poultry compost[J].Applied Biochemistry and Biotechnology,2012,166(3):663-679.
[30]Zhengchu L,Xiaoyang D,Juzuo Z,et al.Screeningof a xylanase high-producing strain and its rapidseparation and purification[J].Annals ofMicrobiology,2011,61(4):901-906.