重组巴斯德毕赤酵母发酵生产内切几丁质酶的培养条件优化
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摘要
探讨重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件,以期获得最佳的内切几丁质酶活力。以内切几丁质酶活力为指标,通过部分因子试验设计以及响应面法优化确定重组巴斯德毕赤酵母高产内切几丁质酶的最适培养条件。部分因子试验设计筛选的影响重组巴斯德毕赤酵母高产内切几丁质酶的3个关键因子为甲醇、油酸和吐温-80。响应面法优化的上述3个关键因子的最佳浓度分别为0.71%、0.086%和0.31%。重组巴斯德毕赤酵母发酵生产内切几丁质酶的最适培养条件为:酵母膏1%、酵母氮碱(YNB)1.34%、蛋白胨2%、甲醇0.71%、油酸0.086%、吐温-80 0.31%、PTM1 0.8%、pH 6.0。在上述培养条件下,重组巴斯德毕赤酵母产内切几丁质酶的活力高达30.92U/mL。与未优化前相比,酶活力提高了1.44倍。研究结果为内切几丁质酶的产业化生产和应用奠定了良好基础。
The optimal culture conditions of recombinant Pichia pastoris for maximizing endochitinase production were investigated. Using endochitinase activity as target, the optimal culture conditions were determined by fractional factorial design and response surface methodology. Three key factors that had an obvious influence on the activity of endochitinase, screened by fractional factorial design, were methanol, oleic acid and Tween-80. Their optimal concentrations obtained by the response surface methodology with a Box-Behnken design were 0.71%, 0.086% and 0.31%, respectively. The optimal medium for endochitinase production was yeast extract 1%, yeast nitrogen base(YNB) 1.34%, peptone 2%, methanol 0.71%, oleic acid 0.086%, Tween-80 0.31%, and PTM1 0.8%, and the optimal pH was 6.0. Under such a condition, the activity of endochitinase reached 30.92 U/mL, a 1.44-fold increase as compared with that in ordinary unoptimized medium. The results could be regarded as reference for industrial production and use of endochitinase.
The optimal culture conditions of recombinant Pichia pastoris for maximizing endochitinase production were investigated. Using endochitinase activity as target, the optimal culture conditions were determined by fractional factorial design and response surface methodology. Three key factors that had an obvious influence on the activity of endochitinase, screened by fractional factorial design, were methanol, oleic acid and Tween-80. Their optimal concentrations obtained by the response surface methodology with a Box-Behnken design were 0.71%, 0.086% and 0.31%, respectively. The optimal medium for endochitinase production was yeast extract 1%, yeast nitrogen base(YNB) 1.34%, peptone 2%, methanol 0.71%, oleic acid 0.086%, Tween-80 0.31%, and PTM1 0.8%, and the optimal pH was 6.0. Under such a condition, the activity of endochitinase reached 30.92 U/mL, a 1.44-fold increase as compared with that in ordinary unoptimized medium. The results could be regarded as reference for industrial production and use of endochitinase.
引文
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蔡培原,陈祥贵,2008.微生物几丁质酶研究概况及其在食品工程中的应用.生命科学仪器,6(4):32-36
陈少波,喻吴根,2004.几丁质酶研究进展.科技通报,20(5):258-262
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李艳华,梁金钟,范洪臣,2008.响应面法优化γ-聚谷氨酸发酵培养基的研究.食品科技,3:45-48
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王全伟,曲敏,张海玲,徐香玲,2008.菜豆几丁质酶基因Bchi的克隆及其在转基因烟草中的表达.分子植物育种,6(1):53-58
姚婉生,王雪,侯海荣,党立,韩利文,刘可春,2006.几丁寡糖制备的研究进展.山东科学,19(3):28-31
张龙翔,张庭芳,李令媛,2006.生化试验方法和技术.第二版.北京:高等教育出版社.55-59
左爱连,张伟国,2008.利用Design-Expert软件优化丝氨酸羟甲基转移酶产酶培养基.生物技术,18(3):45-49
Carsiolio CN,Benhamous S,Haran S,Cortes C,Gutieerez A,1999.Role of the Trichoderma harzianum endochitinase gene,ech42,in mycoparasition.Applied and Environmental Microbiology,65(3):929-935
Chen SB,Yu WG,2004.Recent progress on chitinase.Bulletin of Science and Technology,20(5):258-262(in Chinese)
Fan YH,Zhang YJ,Yang XY,Pei X,Guo S,2007.Expression of a Beauveria bassiana chitinase(Bbchit1)in Escherichia coli and Pichia pastoris.Protein Expression and Purification,56:93-99
Huang CJ,Chen CY,2005.High-level expression and characterization of two chitinases,ChiCH and ChiCW,of Bacillus cereus 28-9 in Escherichia coli.Biochemical and Biophysical Research Communication,327:8-17
Huang XL,Peng K,Zhou SQ,Huang DY,2010.Study on fermentation conditions for chitinase production of Serratia marcescens.Biotechnology,20(3):64-66(in Chinese)
Li YH,Liang JZ,Fan HC,2008.Optimization ofγ-polyglutamic acid fermentation medium by response surface methodology.Food Science and Technology,3:45-48(in Chinese)
Ping RJ,Sun M,Liu JZ,Wang YJ,Hao JH,Zhang SJ,2008.Optimization of fermentation conditions for marine Bacillus licheniformis MP-2 esterase by response surface methodology.Applied and Environmental Microbiology,14(4):548-552(in Chinese)
Tao Y,Jin H,Long ZF,Zhang L,Ding XQ,2006.Cloning and expression of a chitinase gene from Sanguibacter sp.C4.Acta Genetica Sinica,33(11):1037-1046
Wang HD,Liu WT,Lin BK,Song XH,Lun JS,Hu Z,2009.Isolation of chitinolytic bacteria from the Shantou bay,China and study on their chitinase coding genes.Chinese Journal of Applied and Environmental Biology,15(4):511-514(in Chinese)
Wang QW,Qu M,Zhang HL,Xu XL,2008.Cloning of bean chitinase gene Bchi and its expression in transgenic tobacco.Molecular Plant Breeding,6(1):53-58(in Chinese)
Yao WS,Wang X,Hou HR,Dang L,Han LW,Liu KC,2006.Advances in preparation of chitooligosaccharides.Shandong Science,19(3):28-31(in Chinese)
Yu P,Yan Y,Gu Q,Wang XY,2013.Codon optimisation improves the expression of Trichoderma viride sp.endochitinase in Pichia pastoris.Scientific Reports,3:3043
Zhang LX,Zhang TF,Li LY,2006.Experimental methods and technology of biochemistry.2nd ed.Higher Education Press,Beijing.55-59(in Chinese)
Zuo AL,Zhang WG,2008.Medium optimization of SHMTformation by response surface analysis.Biotechnology,18(3):45-49(in Chinese)
蔡培原,陈祥贵,2008.微生物几丁质酶研究概况及其在食品工程中的应用.生命科学仪器,6(4):32-36
陈少波,喻吴根,2004.几丁质酶研究进展.科技通报,20(5):258-262
黄小龙,彭可,周双清,黄东益,2010.黏质沙雷氏菌产几丁质酶发酵条件的研究.生物技术,20(3):64-66
李艳华,梁金钟,范洪臣,2008.响应面法优化γ-聚谷氨酸发酵培养基的研究.食品科技,3:45-48
平芮巾,孙谧,刘均忠,王跃军,郝建华,张胜军,2008.响应面法优化海洋细菌MP-2酯酶发酵条件.应用与环境生物学报,14(4):548-552
王海东,刘温滔,林伯坤,宋晓慧,伦镜盛,胡忠,2009.汕头海域几丁质酶产生菌的筛选及其几丁质酶编码基因研究.应用与环境生物学报,15(4):511-514
王全伟,曲敏,张海玲,徐香玲,2008.菜豆几丁质酶基因Bchi的克隆及其在转基因烟草中的表达.分子植物育种,6(1):53-58
姚婉生,王雪,侯海荣,党立,韩利文,刘可春,2006.几丁寡糖制备的研究进展.山东科学,19(3):28-31
张龙翔,张庭芳,李令媛,2006.生化试验方法和技术.第二版.北京:高等教育出版社.55-59
左爱连,张伟国,2008.利用Design-Expert软件优化丝氨酸羟甲基转移酶产酶培养基.生物技术,18(3):45-49