基于工业化生产的毕赤酵母高效表达木聚糖酶XYL1的研究
|
摘要
木聚糖酶(Xylanase)是一类能专一降解木聚糖为低聚木糖和木糖的水解酶，在制浆造纸、饲料、食品、酿酒等领域具有良好的应用前景，并可为能源短缺、环境污染等相关社会问题提供新的解决方式。木聚糖酶主要来源于枯草芽孢杆菌、类芽孢杆菌、短小芽孢杆菌、链霉菌等原核微生物和黑曲霉、里氏木霉、青霉、等真菌，但上述原始菌株产木聚糖酶的水平较低，难以达到工业化生产要求，从而限制了木聚糖酶在商业上的应用。
有鉴于此，本研究根据毕赤酵母的偏爱密码子人工设计合成了编码链霉菌S38（Streotomyces sp.S38）木聚糖酶XYL1（Xlyanase1）的成熟肽基因，以X33为宿主菌，选用P_(GAP)为启动子，构建组成型表达XYL1的工程菌株X33/pGAPZ A-XYL1。实验表明木聚糖酶XYL1基因成功插入表达载体pGAPZ A中，获得的重组载体pGAPZ A-XYL1，经电转化整合至毕赤酵母X33基因组DNA中。重组工程菌X33/pGAPZ A-XYL1传代稳定，连续传代10代无显著基因丢失，可满足补料分批发酵生产的需要；而连续传代15代的基因丢失率仅为10%。重组工程菌X33/pGAPZ A-XYL1产木聚糖酶活性在摇瓶水平为215±10IU/mL。
发酵液离心收获的上清，经5kDa膜包浓缩置换缓冲液、Q-sephrose FF强阴离子交换层析和Sephadex G-25分子筛联用分离纯化，获得电泳纯度高于90%的重组木聚糖酶XYL1，比活力从796.80IU/mg提高到1328.69IU/mg。肽指纹图谱鉴定结果显示，纯化的XYL1与来源于链霉菌Streptomyces sp. S38的内切β~(-1),4木聚糖酶1（GenBank注册号X98518.1）完全匹配。实验表明重组木聚糖酶XYL1在耐热性方面具有比原酶更好的酶学性质，最适pH为6.0，在pH3.0~9.0时酶活保持稳定；最适反应温度为55℃，且45℃-65℃酶活相对稳定；在60℃处理30min后仍剩余63%的酶活力；重组木聚糖酶XYL1的催化反应机理可以用米氏方程描述，其νmax为5000μmol/min·mg prot，Km为2.0mg/mL。
摇瓶实验比较了甘油、葡萄糖、甲醇这三种碳源对表达的影响，结果表明葡萄糖更适用作发酵的碳源，并确定了发酵初始葡萄糖最适浓度为30g/L。在5L罐中研究了工程菌X33/pGAPZ A-XYL1的连续发酵工艺，结果显示，工程菌发酵第5天进入稳定态：菌体浓度基本稳定在400g/L（WCW）水平，最大菌体产率DCx为8.14g/L.h（WCW）；木聚糖酶XYL1的表达水平稳定在1100IU/mL，木聚糖酶XYL1最大产率DCp达23028IU/L·h。残糖含量在发酵液中维持较低水平，平均为2.56g/L；DO一直稳定在30-40%。连续培养持续了17天，稀释速率D为0.48d~(-1)，产物产率则为补料分批发酵的1.66倍。重组毕赤酵母在发酵过程中的的遗传稳定性较好，未出现明显的基因丢失现象；同时由于料液在反应器中平均停留的时间短，未见明显的蛋白酶降解XYL1的现象。以葡萄糖为限制性底物，通过5L罐的恒化培养，获得相关动力学参数，比生长速率μ与葡萄糖浓度Cs关系符合Monod方程，菌体生长动力学模型μ=0.35Cs（/0.38+Cs），其中μ_(max)=0.35h~(-1)，Ks=0.38g.L~(-1)；比生长速率μ与比产物形成速率的关系呈钟罩型曲线，XYL1生成动力学模型qp=-0.0529μ~2+0.0168μ-0.0003(0.024≤μ≤0.212)；底物消耗动力学模型，q_s=1.6025μ+0.0277；理论最大菌体对葡萄糖得率为YG=0.624g/g，维持系数m=0.0277g/g.h；通过比生长速率μ与菌体生产率DCx的关系研究，推导出最佳稀释速率D_(opt)=0.316h~(-1)，实验预测的最大菌体生产率（DCx）m=7.509g/L·h。对μ与产物生产率DCp的关系进行了研究，μ与产物生产率DCp的关系和μ与qp的关系类似，其峰值出现在μ=0.156h~(-1)。从菌量、产量、菌体得率系数（YX/S）、产物得率系数（YP/S）和比产物形成速率（qp）等动力学参数出发，在50L发酵罐中比较了间歇流加、恒速流加和指数流加等不同流加模式对毕赤酵母工程菌X33/pGAPZ A-XYL1生长和重组XYL1表达的影响，结果显示指数流加是该工程菌高密度发酵的最优的流加模式。在上述指数流加发酵的基础上进一步优化，并采取分段控制工艺进行发酵，最终发酵菌体湿重达415g/L，酶活达2788IU/m L，发酵水平比单纯的指数流加提高了20%。采用分段控制工艺在10m~3生产罐上进行三批次发酵放大试验，平均放罐体积8.152m~3，最高菌湿重达到486g/L，最高酶活3420IU/m L。上述结果表明分段控制工艺放大效果良好，三批次间发酵差异较小，发酵产酶水平比50L罐发酵规模提高了23%，总体工艺稳定。
Xylanase could specific hydrolysis xylan to produce xylo-oligosaccharides and xylose,which showed upstanding application prospect in field of pulping and papermaking, feedindustry, food industry, and Brewing industry, and also provided newly solving strategy toenergy shortage and environmental pollution. Xylanase was widely derived from prokaryoticmicroorganisms including Bacillus subtilis, class of Bacillus, Bacillus pumilus, and fungus suchas Aspergillus niger, Trichodermareesei, Penicillium, Streptomyces. There existed a bottleneckthat the productions of xylanase in parent microorganisms were too low to satisfy therequirement of industry, thus limiting the application of xylanase. According to the Pichiapastoris favored codons, this study designed and artificially synthesized the cDNA fragmentencoding the mature peptide of XYL1(Xlyanase1) from Streotomyces sp.S38. The host X33andthe GAP promoter were selected to construct the constitutive expression strain of XYL1. Therecombinant plasmid pGAPZ A-XYL1was integrated into X33genome DNA byelectroporation. The recombinant strain was transfered of culture stably without obvioussignificant gene loss in10generation, thus satisfied the demand of fed-batch fermentation. Theproduction of XYL1was215±10IU/mLmL in flask fermentation.
The recombianat XYL1was purified through5kDa packge concentration, ion exehnagechromatography Q-Sepharose FF, and molecular sieve Sephadex G-25, with an estimating puritygreater than90%by SDS-PAGE. The specific activity of xylanase was improved from796.80IU/mg to1328.69IU/mg after purification. PMF analysis shown that the purified XYL1matchedto the β~(-1),4endo xylanase1(No. X98518.1) from Streptomyces sp. S38. The recombinant XYL1presented satisfactory enzymatic properties including good thermal stability and wide pHadaptability, which was better than its parental molecule. The optimal pH and temperature forrecombinant XYL1were6.0and55oC, respectively. The catalysis reaction of recombinantXYL1was accorded with the Michaelis-Menton equation, and the Km and Vmax values ofXYL1were2.0mg/mL and5000μmol min~(-1)mg~(-1), respectively.
Glucose was selected as the carbon source for the fermentation of X33/pGAPZ A-XYL1,with an optimal initial concentration of30g/L. The continuous fermentation of X33/pGAPZ A-XYL1was performed in5L fermentor. The fermentation of recombinant XYL1get into stable state in the fifth day,with the steady wet cell weight (WCW) of400g/L and the volumetric cellproductivity (DCx) was8.14g/L.h. The expression level of XYL1was maintained at1100IU/mL, and the volumetric product productivity (DCp) was23028IU/L.h. The concentration ofresidual sugar in fermentation borth was kept constantly at a low level of2.3g/L in thecontinuous fermentation process. The continuous fermentation lasted for17days, with thedilution rate (D)of0.48D~(-1). Finally, the production yield of continuous fermentation was1.66fold of fed-batch fermentation.
The engineering strain X33/pGAPZ A-XYL1displayed genetic stability in thefermentation, as no obvious gene lost was detected. Meanwhile, significant degration of XYL1did not observed in fermentation, resulting from short-term retention of the dosage liquor.Relevant kinetics parameters of fermentation were obtained in the chemostatic culture of X33/pGAPZ A-XYL1in5L fermentor with glucose as the restrictive substrate. The relationshipbetween specific cell growth rate (μ) and substrate concentration (Cs) was according with theMonod equation. The yeast growth kinetics was coincide with the equation, μ=0.35Cs/(0.38+Cs), hence μmax=0.35h~(-1)and Ks=0.38g·L~(-1). The specific cell growth rate (μ) and specificproduct formation rate (qp) was according with the equation qp=-0.0529μ~2+0.0168μ-0.0003(0.024≤μ≤0.212). The substrate consumption kinetics model equation was qs=1.6025μ+0.0277and the theoretical maximum biomass yield coefficient (YG) was0.624g/g. The theoreticaloptimal dilution rate was0.316h~(-1), which was from the study on the relationship of specificgrowth rate and volumetric cell productivity (DCx), and the estimating the maximum volumetriccell productivity (DCx)mwas7.509g/L·h. The relationship of specific growth rate and productformation was also studied, which was similar to the relationship of specific cell growth rate (μ)and specific product formation rate (qp), and the peak value was μ=0.156h~(-1).
We also compaired the influence of different fed-batch mode, including intermissionfed-batch, constant velocity fed-batch and exponential fed-batch in50L fermentor, on thegrowth of Pichia pastoris X33/pGAPZ A-XYL1and the production of recombinant XYL1,which was evaluated by kinetic parameters such as products yield coefficient (Yp/s), biomassyield coefficient(YX/S), specific product formation rate (q_p). Compaired with intermissionfed-batch and constant velocity fed-batch, the exponential fed-batch was the effective fed-batchmode for the high density fermentation of X33/pGAPZ A-XYL1. Based on the exponential fed-batch, we also used the fed-batch controlled in stages strategy in the50-L fermentation.According to the combination fermentation strategy above, the wet cell weight was415gram perliter borth, and the activity of xylanase was2788IU/mL. Finally, three batch of10m~3scalefermentation were carried out, using the fed-batch controlled in stages strategy. Compared to the50L scale fermentation, the yield of XYL1in10m~3scale fermentation was increased by23%.The fermentation process was stable and the highest wet cell weight and xylanase activity in thefermentation borth was486g/L and3420IU/mL respectively.
有鉴于此，本研究根据毕赤酵母的偏爱密码子人工设计合成了编码链霉菌S38（Streotomyces sp.S38）木聚糖酶XYL1（Xlyanase1）的成熟肽基因，以X33为宿主菌，选用P_(GAP)为启动子，构建组成型表达XYL1的工程菌株X33/pGAPZ A-XYL1。实验表明木聚糖酶XYL1基因成功插入表达载体pGAPZ A中，获得的重组载体pGAPZ A-XYL1，经电转化整合至毕赤酵母X33基因组DNA中。重组工程菌X33/pGAPZ A-XYL1传代稳定，连续传代10代无显著基因丢失，可满足补料分批发酵生产的需要；而连续传代15代的基因丢失率仅为10%。重组工程菌X33/pGAPZ A-XYL1产木聚糖酶活性在摇瓶水平为215±10IU/mL。
发酵液离心收获的上清，经5kDa膜包浓缩置换缓冲液、Q-sephrose FF强阴离子交换层析和Sephadex G-25分子筛联用分离纯化，获得电泳纯度高于90%的重组木聚糖酶XYL1，比活力从796.80IU/mg提高到1328.69IU/mg。肽指纹图谱鉴定结果显示，纯化的XYL1与来源于链霉菌Streptomyces sp. S38的内切β~(-1),4木聚糖酶1（GenBank注册号X98518.1）完全匹配。实验表明重组木聚糖酶XYL1在耐热性方面具有比原酶更好的酶学性质，最适pH为6.0，在pH3.0~9.0时酶活保持稳定；最适反应温度为55℃，且45℃-65℃酶活相对稳定；在60℃处理30min后仍剩余63%的酶活力；重组木聚糖酶XYL1的催化反应机理可以用米氏方程描述，其νmax为5000μmol/min·mg prot，Km为2.0mg/mL。
摇瓶实验比较了甘油、葡萄糖、甲醇这三种碳源对表达的影响，结果表明葡萄糖更适用作发酵的碳源，并确定了发酵初始葡萄糖最适浓度为30g/L。在5L罐中研究了工程菌X33/pGAPZ A-XYL1的连续发酵工艺，结果显示，工程菌发酵第5天进入稳定态：菌体浓度基本稳定在400g/L（WCW）水平，最大菌体产率DCx为8.14g/L.h（WCW）；木聚糖酶XYL1的表达水平稳定在1100IU/mL，木聚糖酶XYL1最大产率DCp达23028IU/L·h。残糖含量在发酵液中维持较低水平，平均为2.56g/L；DO一直稳定在30-40%。连续培养持续了17天，稀释速率D为0.48d~(-1)，产物产率则为补料分批发酵的1.66倍。重组毕赤酵母在发酵过程中的的遗传稳定性较好，未出现明显的基因丢失现象；同时由于料液在反应器中平均停留的时间短，未见明显的蛋白酶降解XYL1的现象。以葡萄糖为限制性底物，通过5L罐的恒化培养，获得相关动力学参数，比生长速率μ与葡萄糖浓度Cs关系符合Monod方程，菌体生长动力学模型μ=0.35Cs（/0.38+Cs），其中μ_(max)=0.35h~(-1)，Ks=0.38g.L~(-1)；比生长速率μ与比产物形成速率的关系呈钟罩型曲线，XYL1生成动力学模型qp=-0.0529μ~2+0.0168μ-0.0003(0.024≤μ≤0.212)；底物消耗动力学模型，q_s=1.6025μ+0.0277；理论最大菌体对葡萄糖得率为YG=0.624g/g，维持系数m=0.0277g/g.h；通过比生长速率μ与菌体生产率DCx的关系研究，推导出最佳稀释速率D_(opt)=0.316h~(-1)，实验预测的最大菌体生产率（DCx）m=7.509g/L·h。对μ与产物生产率DCp的关系进行了研究，μ与产物生产率DCp的关系和μ与qp的关系类似，其峰值出现在μ=0.156h~(-1)。从菌量、产量、菌体得率系数（YX/S）、产物得率系数（YP/S）和比产物形成速率（qp）等动力学参数出发，在50L发酵罐中比较了间歇流加、恒速流加和指数流加等不同流加模式对毕赤酵母工程菌X33/pGAPZ A-XYL1生长和重组XYL1表达的影响，结果显示指数流加是该工程菌高密度发酵的最优的流加模式。在上述指数流加发酵的基础上进一步优化，并采取分段控制工艺进行发酵，最终发酵菌体湿重达415g/L，酶活达2788IU/m L，发酵水平比单纯的指数流加提高了20%。采用分段控制工艺在10m~3生产罐上进行三批次发酵放大试验，平均放罐体积8.152m~3，最高菌湿重达到486g/L，最高酶活3420IU/m L。上述结果表明分段控制工艺放大效果良好，三批次间发酵差异较小，发酵产酶水平比50L罐发酵规模提高了23%，总体工艺稳定。
Xylanase could specific hydrolysis xylan to produce xylo-oligosaccharides and xylose,which showed upstanding application prospect in field of pulping and papermaking, feedindustry, food industry, and Brewing industry, and also provided newly solving strategy toenergy shortage and environmental pollution. Xylanase was widely derived from prokaryoticmicroorganisms including Bacillus subtilis, class of Bacillus, Bacillus pumilus, and fungus suchas Aspergillus niger, Trichodermareesei, Penicillium, Streptomyces. There existed a bottleneckthat the productions of xylanase in parent microorganisms were too low to satisfy therequirement of industry, thus limiting the application of xylanase. According to the Pichiapastoris favored codons, this study designed and artificially synthesized the cDNA fragmentencoding the mature peptide of XYL1(Xlyanase1) from Streotomyces sp.S38. The host X33andthe GAP promoter were selected to construct the constitutive expression strain of XYL1. Therecombinant plasmid pGAPZ A-XYL1was integrated into X33genome DNA byelectroporation. The recombinant strain was transfered of culture stably without obvioussignificant gene loss in10generation, thus satisfied the demand of fed-batch fermentation. Theproduction of XYL1was215±10IU/mLmL in flask fermentation.
The recombianat XYL1was purified through5kDa packge concentration, ion exehnagechromatography Q-Sepharose FF, and molecular sieve Sephadex G-25, with an estimating puritygreater than90%by SDS-PAGE. The specific activity of xylanase was improved from796.80IU/mg to1328.69IU/mg after purification. PMF analysis shown that the purified XYL1matchedto the β~(-1),4endo xylanase1(No. X98518.1) from Streptomyces sp. S38. The recombinant XYL1presented satisfactory enzymatic properties including good thermal stability and wide pHadaptability, which was better than its parental molecule. The optimal pH and temperature forrecombinant XYL1were6.0and55oC, respectively. The catalysis reaction of recombinantXYL1was accorded with the Michaelis-Menton equation, and the Km and Vmax values ofXYL1were2.0mg/mL and5000μmol min~(-1)mg~(-1), respectively.
Glucose was selected as the carbon source for the fermentation of X33/pGAPZ A-XYL1,with an optimal initial concentration of30g/L. The continuous fermentation of X33/pGAPZ A-XYL1was performed in5L fermentor. The fermentation of recombinant XYL1get into stable state in the fifth day,with the steady wet cell weight (WCW) of400g/L and the volumetric cellproductivity (DCx) was8.14g/L.h. The expression level of XYL1was maintained at1100IU/mL, and the volumetric product productivity (DCp) was23028IU/L.h. The concentration ofresidual sugar in fermentation borth was kept constantly at a low level of2.3g/L in thecontinuous fermentation process. The continuous fermentation lasted for17days, with thedilution rate (D)of0.48D~(-1). Finally, the production yield of continuous fermentation was1.66fold of fed-batch fermentation.
The engineering strain X33/pGAPZ A-XYL1displayed genetic stability in thefermentation, as no obvious gene lost was detected. Meanwhile, significant degration of XYL1did not observed in fermentation, resulting from short-term retention of the dosage liquor.Relevant kinetics parameters of fermentation were obtained in the chemostatic culture of X33/pGAPZ A-XYL1in5L fermentor with glucose as the restrictive substrate. The relationshipbetween specific cell growth rate (μ) and substrate concentration (Cs) was according with theMonod equation. The yeast growth kinetics was coincide with the equation, μ=0.35Cs/(0.38+Cs), hence μmax=0.35h~(-1)and Ks=0.38g·L~(-1). The specific cell growth rate (μ) and specificproduct formation rate (qp) was according with the equation qp=-0.0529μ~2+0.0168μ-0.0003(0.024≤μ≤0.212). The substrate consumption kinetics model equation was qs=1.6025μ+0.0277and the theoretical maximum biomass yield coefficient (YG) was0.624g/g. The theoreticaloptimal dilution rate was0.316h~(-1), which was from the study on the relationship of specificgrowth rate and volumetric cell productivity (DCx), and the estimating the maximum volumetriccell productivity (DCx)mwas7.509g/L·h. The relationship of specific growth rate and productformation was also studied, which was similar to the relationship of specific cell growth rate (μ)and specific product formation rate (qp), and the peak value was μ=0.156h~(-1).
We also compaired the influence of different fed-batch mode, including intermissionfed-batch, constant velocity fed-batch and exponential fed-batch in50L fermentor, on thegrowth of Pichia pastoris X33/pGAPZ A-XYL1and the production of recombinant XYL1,which was evaluated by kinetic parameters such as products yield coefficient (Yp/s), biomassyield coefficient(YX/S), specific product formation rate (q_p). Compaired with intermissionfed-batch and constant velocity fed-batch, the exponential fed-batch was the effective fed-batchmode for the high density fermentation of X33/pGAPZ A-XYL1. Based on the exponential fed-batch, we also used the fed-batch controlled in stages strategy in the50-L fermentation.According to the combination fermentation strategy above, the wet cell weight was415gram perliter borth, and the activity of xylanase was2788IU/mL. Finally, three batch of10m~3scalefermentation were carried out, using the fed-batch controlled in stages strategy. Compared to the50L scale fermentation, the yield of XYL1in10m~3scale fermentation was increased by23%.The fermentation process was stable and the highest wet cell weight and xylanase activity in thefermentation borth was486g/L and3420IU/mL respectively.
引文
[1]张嗣良,储炬.多尺度微生物过程优化[M].北京:化学工业出版社,2003:240.
[2]郭志强,顾维智,李佩建,等.木聚糖酶的研究进展[J].营养与日粮,2008,6:18-20.
[3]方洛云,邹晓庭.木聚糖酶基因的分子生物学与基因工程[J].畜禽业,2002,2:2-4.
[4] Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on amino acidsequence similarities [J]. Biochem J.,1993,293:781-788.
[5]连惠芗,汪世华.木聚糖酶的研究与应用[J].武汉工业学院学报,2006,1:42-46.
[6] Maximo C, Ferriera M C, Duarte J. Some Properties of Eucalyptus Kraft Pulp Treated with Xylanase fromAspergillus Niger [J]. World J Microbiol Biotechnol, l998,14:365-367.
[7] Nair SG, Sindhu R, Shashidhar S. Purification and biochemical characterization of two xylanases fromAspergillus sydowii SBS45[J]. Applied Biochemistry and Biotechnology,2008,149(3):229-243.
[8] Hu J, Yu B, Zhang KY, et al. Expression of endo-1,4-beta-xylanase from Trichoderma reesei in Pichiapastoris and functional characterization of the produced enzyme [J]. BMC Biotechnology,2009,9(1):56.
[9] Badhan AK, Chadha BS, Kaur J, et al. Production of multiple xylanolytic and cellulolytic enzymes bythermophilic fungus Myceliophthora sp. IMI387099[J]. Bioresource Technology,2007,98(3):504-510.
[10] OmPraba G, Velmurugan D, Arumugam P, et al. Homology model of a novel thermostable xylanase fromBacillus subtilis-AK1[J]. Journal of Biomolecular Structure and Dynamics,2007,25(3):311-320.
[11]韩双艳,林小琼,张溪,等.枯草芽孢杆菌B2（Bacillus subtilis B2）木聚糖酶在毕赤酵母中的表达[J].现代食品科技,2009,25(8):872-876.
[12] Pason P, Kosugi A, Waeonukul R, et al. Purification and characterization of a multienzyme complexproduced by Paenibacillus curdlanolyticus B-6[J]. Applied Microbiology and Biotechnology,2010,85(3):573-580.
[13]江正兵,宋慧婷,马立新.短小芽孢杆菌木聚糖酶基因在毕赤酵母中的分泌表达及酶学性质研究[J].生物工程学报,2003,1:50-55.
[14]冯佳丽,台喜生,李梅,等.链霉菌产木聚糖酶条件和酶学性质以及重离子诱变对产酶的影响研究.2011,6:205-210.
[15] Zhou JP, Huang HQ, Meng K, et al. Molecular and biochemical characterization of a novel xylanase fromthe symbiotic Sphingobacterium sp.TN19. Applied Microbiology and Biotechnology,2009,85(2):323-333.
[16]付冠华,李端,周晨妍,等.木聚糖酶的研究进展及其应用[J].安徽农业科学,2011,39(35):21566-21568.
[17]刘瑞田,曲音波.木聚糖酶分子的结构区域[J].生物工程进展,1998,18(6):27-28.
[18]刘亮伟,杨海玉,胡瑜,等. F/10木聚糖酶研究进展[J].食品与生物技术学报,2009,28(6):727-732.
[19]刘亮伟,秦天苍,翟继,等. F/10及G/11木聚糖酶家族密码子偏好性分析[J].2008,42(2):223-227.
[20]孙军亭.宇佐美曲霉木聚糖酶基因的改造及表达[D].无锡:江南大学,2009.
[21] Ko EP, Akatsuka H, Moriyama H, et al. Site-directed mutagenesis at aspartate and glutamate residues ofxylanase from Bacillus pumilus [J]. Biochem J,1992,288:117-121.
[22] Poon DK, Webster P, Withers SG, Characterizing the pH-dependent stability and catalytic mechanism ofthe family11xylanase from the alkalophilic Bacillus agaradhaerens [J]. Carbohydr Res,2003,338(5):415-421.
[23] Teplitsky A, Mechaly A, Stojanoff V,et al.Structure determination of the extracellular xylanase fromGeobacillus stearothermophilus by selenomethionyl MAD phasing [J]. Acta Crystallogr D Biol Crystallogr,2004,60(Pt5):836-48.
[24] Chen YL, Tang TY, Cheng KJ. Directed evolution to produce an alkalophilic variant fromaNeocallimastix patriciarumxylanase [J]. Can J Microbiol,2001,47:1088-1094.
[25] McCarthy AA, Morris DD, Bergquist PL, et al. Structure of XynB, a highly thermostable β-1,4-xylanasefrom Dictyoglomus thermophilum Rt46B.1, at1.8resolution[J]. Acta Crystallogr D Biol Crystallogr,2000,56(Pt11):1367-1375.
[26]杨浩萌,姚斌,范云六.木聚糖酶分子结构与重要酶学性质关系的研究进展[J].生物工程学报,2005,21(1):6-11.
[27]程菲菲,赵军旗,石鹏君,等.瓶霉Phialophora sp. P13木聚糖酶基因的克隆及酶学性质分析[J].中国农业科技导报,2012,14(1):85-90.
[28]王坤,罗会颖,石鹏君,等.来源于耐碱真菌Pseudallescheria sp. JSM-2的碱性木聚糖酶基因的克隆、表达及其性质研究[J].生物技术进展.2011,1（1）:61-67.
[29] Verma D, Satyanarayana T. Cloning, expression and applicability of thermo-alkali-stable xylanase ofGeobacillus thermoleovorans in generating xylooligosaccharides from agro-residues [J]. BioresourceTechnology,2012,107:333-338.
[30] Sriyapai T, Somyoonsap P, Matsui K, et al. Cloning of a thermostable xylanase from Actinomadura sp.S14and its expression in Escherichia coli and Pichia pastoris[J]. Journal of Bioscience and Bioengineering.2011,111(5):528-536.
[31] Driss D, Bhiri F, Ghorbel R, et al. Cloning and constitutive expression of His-tagged xylanase GH11from Penicillium occitanis Pol6in Pichia pastoris X33: Purification and characterization[J]. Protein Expr Purif.,2012,83(1):8-14.
[32]张帆,石鹏君,缪礼鸿,等.来源于瓶霉Phialophorasp.G5的酸性木聚糖酶基因的克隆及性质的研究[J].中国农业科技导报,2011,13(3):60-66.
[33]张桂敏,饶犇,叶戋,等.棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析[J].微生物学报,48(6):765-771.
[34]王建设,柏映国,尹俊,等.脂环酸芽孢杆菌Alicyclobacillussp.A15木聚糖酶基因xynA15的克隆表达及性质研究[J].中国农业科技导报,2010,12(6):114-119.
[35] Wang GZ, Luo HY, Wang YR, et al. A novel cold-active xylanase gene from the environmental DNA ofgoat rumen contents: Direct cloning, expression and enzyme characterization[J]. Bioresource Technology,2011,102(3):3330-3336.
[36] Ko CH, Tsai CH, Tu J, et al. Molecular cloning and characterization of a novel thermostable xylanasefrom Paenibacillus campinasensis BL11[J]. Process Biochemistry,2010,45(10):1638-1644.
[37] Qiu ZH, Shi PG, Luo HY, et al. A xylanase with broad pH and temperature adaptability fromStreptomyces megasporus DSM41476, and its potential application in brewing industry[J]. Enzyme andMicrobial Technology,2010,46(6):506-512.
[38] Hwang IT, Lim HK, Song HY, et al. Cloning and characterization of a xylanase, KRICT PX1from thestrain Paenibacillus sp. HPL-001[J]. Biotechnology Advances,2010,28(5):594-601.
[39] Vega E, Farrés GS. Amelia, Beatriz XC, et al. Cloning and expression of a novel, moderatelythermostable xylanase-encoding gene (Cfl xyn11A) from Cellulomonas flavigena[J]. Bioresource Technology,2010,101(14):5539-5545.
[40] Zhou CY, Bai JY, Deng SS, et al. Cloning of a xylanase gene from Aspergillus usamii and its expressionin Escherichia coli [J]. Bioresource Technology,2008,99(4):831-838.
[41] Nádia Skorupa Parachin, Saulo Siqueira, Fabrícia Paula de Faria, et al. Xylanases from Cryptococcusflavus isolate I-11: Enzymatic profile, isolation and heterologous expression of CfXYN1in Saccharomycescerevisiae [J]. Journal of Molecular Catalysis B: Enzymatic,2009,59(1-3):52-57.
[42]何永志,姚斌,王亚茹,等.来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析[J].微生物学报,44（3）:340-344.
[43]周煌凯,龚月生,杨明明,等.耐碱性短小芽孢杆菌木聚糖酶在枯草杆菌中的表达及发酵条件优化[J].饲料工业,2010,31（14）:25-29.
[44]罗建杰,王亚茹,袁铁铮,等.高比活木聚糖酶XYN-W及其突变体在酵母中的高效表达[J].中国农业科技导报,2010,12(5):80-85.
[45] Santosh O, Ramchuran, Bruno Mateus, et al. The methylotrophic yeast Pichia pastoris as a host for theexpression and production of thermostable xylanase from the bacterium Rhodothermus marinus[J]. FEMSYeast Research,2005,5(9):839-850.
[46] He J, Chen DW, Yu B, et al. Optimization of the Trichoderma reesei endo-1,4-beta-xylanase productionby recombinant Pichia pastoris[J]. Biochemical Engineering Journal,2010,52(1):1-6.
[47] Natasha Birijlall, Ayyachamy Manimaran, Kuttanpillai Santhosh Kumar, et al. High level expression of arecombinant xylanase by Pichia pastoris NC38in a5L fermenter and its efficiency in biobleaching of bagassepulp[J]. Bioresource Technology,2011,102(20):9723-9729.
[48] Jia HY, Fan GG, Yan QJ, et al. High-levelexpression of a hyperthermostable Thermotogamaritimaxylanase in Pichia pastoris by codon optimization[J]. Journal of Molecular Catalysis B: Enzymatic,2012,78:72-77.
[49]姚斌.基因工程木聚糖酶XYNB的生产技术[G].生物与酶催化工程产业化促进会技术成果汇编,南京:中国微生物学会酶工程专业委员会,2004:17.
[50]毛爱军,董志杨.高产木聚糖酶工程菌株及其产业化研究[G].生物与酶催化工程产业化促进会技术成果汇编,南京:中国微生物学会酶工程专业委员会,2004:18.
[51]孙宏亮.重组毕赤酵母生产木聚糖酶工艺概述[C].2011饲料酶制剂应用技术国际研讨会暨第二届饲料酶制剂大会,青岛:中国农业科学院饲料研究所《新饲料》杂志社,2011:22-23.
[52] Jeong MY, Kim S, Yun CW, et al. Engineering a de novo internal disulfide bridge to improve the thermalstability of xylanase from Bacillus stearothermophilus No.236[J]. J Biotechnol,2007,127(2):300-309.
[53] George SP, Ahnad A, Rao MB. A novel thermostable xylanase from Thermomonosporasp.: influence ofadditives on thermostability [J]. Bioresour Technol,2001,78(3):221-224.
[54]杨浩萌,王亚茹,伍宁丰,等. N13D、S40E点突变提高木聚糖酶XYNB的热稳定性[J].微生物学通报,2007,34(3):533-536.
[55] Chen Y L, Tang TY, Cheng KJ. Directed evolution to produce an alkalophilic variant from aNeocallimastix patriciarum xylanase [J].Can J Mirobiol,2001,47(12):1088-1094.
[56] Fenel F, Zitting AJ, Kantelinen A. Increased alkali stability in Trichoderma reesei endo-1,4-beta-xylanaseII by site directed mutagenesis [J]. J Biotechnol.2006:121(1):102-107.
[57] Cirino PC, Mayer KM, Daisuke Umeno. Generating Mutant Libraries Using Error-Prone PCR [J].Methods in Molecular Biology,2003,231(1):3-9.
[58] McCullum EO, Williams BA, Zhang J, et al. Random mutagenesis by error-prone PCR [J].2010,634:103-9.
[59] Loo B, Spelberg LJH., Kingma J, et al. Directed Evolution of Epoxide Hydrolase from A radiobactertoward Higher Enantioselectivity by Error-Prone PCR and DNA Shuffling [J]. Chemistry&Biology,2004,11:981-990.
[60] Zhang ZG, Yi ZL, Pei XQ, et al. Improving the thermostability of Geobacillus stearothermophilusxylanase XT6by directed evolution and site-directed mutagenesis [J]. Bioresource Technology,2010,101(23):9272-9278.
[61] Miyazaki K, Takenouchi M, Kondo H, et al. Thermal Stabilization of Bacillus Subtilis Family-11Xylanase by Directed Evolution[J]. J Biol Chem.,2006,281(15):10236-10242.
[62]李清峰,马向东,彭虹旎,等.木聚糖酶耐热性研究进展[J].中国畜牧杂志,200945（11）:57-60.
[63]周晨妍,王武,邬敏辰. Arg引入“Ser/Thr”平面对木聚糖酶XynⅡ热稳定性的影响[J].西北农林科技大学学报(自然科学版),2010,38(1):46-52.
[64] Han C, Yu S, Ouyang J, et al. Enhancing stability of Trichoderma reesei xylanase (XYN II) bysite-directed mutagenesis[J]. Sheng Wu Gong Cheng Xue Bao,2010,26(5):623-629.
[65] Chen XZ, Xu SQ, Zhu MS, et al. Site-directed mutagenesis of an Aspergillus niger xylanase B and itsexpression, purification and enzymatic characterization in Pichia pastoris[J]. Process Biochemistry,2010,45(1):75-80.
[66]吴穗洁,吴文林,刘斌.嗜热酶耐热机制及提高酶热稳定性的研究进展[J].亚热带农业研究,2006,2(4):293-297.
[67]张勇伟,胡虹,刘亮伟,等.木聚糖酶热稳定性化学修饰研究[J].食品与发酵工业,2011,37(5):61-65.
[68] Polizeli ML, Rizzatti AC, Monti R, et al. Xylanases from fungi: properties and industrial applications[J].Appl Microbiol Biotechnol.,2005,67(5):577-91.
[69] Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology, andapplication [J]. Crit Rev Biotechnol.,2002,22(1):33-64.
[70] Beg QK, Kapoor M, Mahajan L, et al. Microbial xylanases and their industrial applications: a review [J].Appl Microbiol Biotechnol.,2001,56(3-4):326-38.
[71]慕娟,问清江,党永,等.木聚糖酶的开发与应用[J].陕西农业科学,2012,1:111-115.
[72] Knob A, Terrasan CRF, Carmona EC. β-Xylosidases from filamentous fungi: an overview[J]. World JMicrobiol Biotechnol,2010,26:389-407.
[73]吴香波,谢益民.木聚糖酶在制浆造纸工业中的应用和研究进展[J].上海造纸,2008,39(5):6-9.
[74]马文,张美云,房桂干.木聚糖酶在制浆造纸中的应用研究进展[J].上海造纸,2008,39(2):9-11.
[75]贺文明.木聚糖酶在美国KP厂的应用[J].国际造纸,2003,22(5):32-34.
[76]尤纪雪,欧阳嘉,沈洁,等.机械力化学效应对麦草浆重组木聚糖酶生物漂白的影响[J].纤维素科学与技术,2010,18(4):25-32.
[77]董毅,陈嘉川,杨桂花,等.木聚糖酶处理改善桉木KP浆质量的研究[J].江苏造纸,2009,3:36-39.
[78]张久政,武书彬,胡惠仁.生物酶用于旧报纸脱墨的研究[J].造纸科学与技术,2003,22(1):14-17.
[79]杨桂花,陈克复,陈嘉川,等.木聚糖酶处理改善速生杨化学机械浆性能的研究[J].林产化学与工业,2009,29(2):105-109.
[80]何承云,林向阳,高雪琴,等.木聚糖酶在馒头制作中的应用研究[J].农产品加工(学刊),2009,6:7-10.
[80]张勤良,王璋,许时婴.中性木聚糖酶在面包制作中的应用[J].食品与发酵工业,2004,30(7):21-25.
[82]李秀婷.微生物木聚糖酶及在食品工业中的应用[J].农业机械学报,2008,39(2):175-179.
[83]郑翔鹏,王国川,林海峰,等.木聚糖酶在啤酒中的应用研究[J].啤酒科技,2008,9:27-36.
[84] Hitzeman RA, Hagie FE, Levine, et al. Expression of a human gene for interferon in yeast [J]. Nature,1981,293:717-722.
[85] Wegner EH.Biochemical conversions by yeast fermentation at high–cell dendities [P]. US. Patent:4414329,1983.
[86] Cregg JM, Cereghino JL,Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. MolBiotechnol,2000,16(1):23-52.
[87] Cunha AE, Clemente JJ, Gomes R, et al. Methanol induction optimization for scFv antibody fragmentproduction in Pichia pastoris [J]. Biotechnol Bioeng.,2004,86(4):458-467.
[88] Ding Y F, Liu, Dai CB. Advances in Pichia pastoris expression system [J]. Chinese Bulletin of LifeSciences,2003,15(1):326-311.
[89]胡世界,罗素兰,张吉贞,等.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6):78-83.
[90] Ohi H, Miura M, Hiramatsu R, et al. The positive and negative cis-acting elements for methanolregulation in the Pichia pastoris AOX2gene [J]. Molecular and General Genetics,1994,243:489-499.
[91]娄瑞娟,罗利龙,张霞,等.巴斯德毕赤酵母表达系统的研究进展和前景展望[J].生物学杂志,2010,27(5):73-76.
[92]王清路,李俏俏,薛金艳,等.巴斯德毕赤酵母表达系统的特点及应用[J].生物技术通讯,2006,17(4):640-643.
[93] Cregg JM, VedvickTS, Raschke WC. Recent advances in the expression of foreign genes in Pichiapastoris [J]. Nature Biotechnology,1993,11:905-910.
[94] Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphatedehydrogenase gene and regulation and use of its promoter[J]. Gene,1997,186(1):37-44.
[95] Menendez J, HernandezL, BanguelaA, et al. Functional production and secretion of theGluconacetobacter diazotrophicus fructose-releasing exo-levanase (LsdB) in Pichia pastoris [J].Enz.Microb.Technol,2004,34(5):446-452.
[96] Dring F, Klapper M, Theis S, et al. Use of the glyceraldehydes-3-phosphate dehydrogenase promoter forproduction of functional mammalian membrane transport proteins in the yeast pichia pastoris [J].Biochem.Biophys.Res.Commun.,199850(2):531－535.
[97] Delroisse JM, Dannau M,Gilsoul JJ, et al.Expression of a synthetic gene encoding a ttriboliumcastaneum carboxylesterase in Pichia pastoris [J]. Protein Expr.Purif.,2005,42(2):286-294.
[98]路蓉.巴斯德毕赤酵母表达系统及其分泌型蛋白表达的研究进展[J].临床和实验医学杂志,2004,3(1):43-47.
[99]耿宏伟,侯红燕,王丕武,等.毕赤酵母表达系统的研究进展[J].食品工程,2011,10:159-162.
[100] Veenhuis M, VanDijken J.P., Harde W. The Significance of Peroxisomes in the Metabolism ofOne-Carbon Compounds in Yeasts [J]. Advances in Microbial Physiology,1983,24:1–82.
[101] Martine M, Zeng S, BergerE.G. The yeast expression system for recombinant glycosyl transferases [J].Glycoconjugate Journal,1999,(16):125-139.
[102] Menendez J, Valdes I, Cabrera N. The ICL1gene of Pichia pastoris, transcriptional regulation and use ofits promoter [J]. Yeast,2003,20(13):1097-10110.
[103] J.R.M. de Almeida, L.M.P. de Moraes, F.A.G. Torres. Molecular characterization of the3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris [J]. Yeast,2005,22:725–737.
[104] Ahn J, Hong J, Lee H, et al. Translation elongation factor1-α gene from Pichia pastoris: molecularcloning, sequence,and use of its promoter[J]. Appl. Genet. Mol. Biotechnol.,2007,74:601-608.
[105] Vassileva A, Chugh DA, Swarninathan S, et al. Effect of copy number on the expression levels ofhepetitis B Surface antigen in the methylotrophic yeast Pichia pastoris [J]. Protein Expr Purif.,2001,21:71.
[106]王龙刚,王宇凯,何成,等.混合补料诱导高拷贝重组毕赤酵母高效表达X-HBsAg [J].中国生物制品学杂志,2010,23(5):521-525.
[107]井申荣,邹全明,洪愉,等.白细胞介素-10基因多拷贝表达盒的构建及在毕赤酵母中的表达[J].中国生物制品学杂志,2007,2(20):81-86.
[108]谢涛,王红宁.外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展[J].生物技术通讯,2006,3(17):415-417.
[109] Zhao W, Wang JW, Deng RQ, et al. Scale-up fermentation of recombinant Candida rugosa lipaseexpressed in Pichia pastoris using the GAP promoter [J]. J Ind Microbiol Biotechnol,2008,35:189-195.
[110] Soons ZI, Voogt JA, van Straten G, et al. Constant specific growth rate in fed-batch cultivation ofBordetella pertussis using adaptive control [J]. J Biotechnol,2006,125:252-268.
[111] Zhang W, Sinha J, Smith LA, et al. Maximization of production of secreted recombinant proteins inPichia pastoris fed-batch fermentation [J]. Biotechnol Prog.,2005,21:386-393.
[112] Pais-Chanfrau JM, Garcia Y, Licor L, et al. Improving the expression of mini-proinsulin in Pichiapastoris [J]. Biotechnol Lett,2004,26:1269-1272.
[113] Onyeaka H, Nienow AW, Hewitt CJ. Further studies related to the scale-up of high cell densityEscherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when usingaqueous ammonia to control pH [J]. Biotechnol Bioeng,2003,84:474-484.
[114] Goodrick JC, Xu M, Finnegan R, et al. High-level expression and stabilization of recombinant humanchitinase produced in a continuous constitutive Pichia pastoris expression system[J]. Biotechnol Bioeng.,2001,74(6):492-7.
[115] Schilling BM, Goodrick JC, Wan NC. Scale-up of a high cell density continuous culture with Pichiapastoris X-33for the constitutive expression of rh-chitinase [J]. Biotechnol Prog.,2001,17(4):629-33.
[116] Goodrick, Khasa YP, Khushoo A, et al. Kinetic studies of constitutive human granulocyte macrophagecolony stimulating factor hGMCSF expression in continuous culture of Pichia pastoris[J]. J.Biotechnol.Lett.,2007,29:1903-1908.
[117] d’Anjou MC, Daugulis AJ. A rational approach to improving productivity in recombinant Pichia pastorisfermentation[J]. Biotechnol.Bioeng.,2000,2:1–11.
[118] Yamawaki S, Matsumoto T, Ohnishi Y, et al. Production of single-chain variable fragmentantibody (scFv) in fed-batch and continuous culture of Pichia pastoris by two different methanol feedingmethods[J]. J. Biosci. Bioeng.,2007,104:403-407.
[119] Jungo C, Rérat C, Marison IW, et al. Quantitative characterization of the regulation of the synthesis ofalcohol oxidase and of the expression of recombinant avidin in a Pichia pastoris Mut+strain[J]. EnzymeMicrob.,2006,39:936-944.
[120] Zhang W, Liu CP, Inan M, et al. Optimization of cell density and dilution rate in Pichia pastoriscontinuous fermentations for production of recombinant proteins[J]. J. Ind. Microbiol. Biotechnol.,2004,31:330–334.
[121] Nakano A, Lee C Y, Yoshida A, et al. Effects of methanol feeding methods on chimeric α-amylaseexpression continuous culture of Pichia pastoris[J]. J. Biosci. Bioeng.,2006,101:227–231.
[122] Khasa Y.P., Khushoo A., Srivastava L., et al. Kinetic studies of constitutive humangranulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture ofPichia pastoris [J]. Biotechnol. Lett.,2007,29:1903–1908.
[123] Maurer M., Kühleitner M., Gasser B., et al. Versatile modeling and optimization of fed-batch processesfor the production of secreted heterologous proteins with Pichia pastoris[J]. Microb. Cell act,2006,5:37.
[124] Kansoh A.L. and Gammal A.. Xylanolytic activities of Streptomyces sp, taxonomy production, partialpurification and utilization of agricultural wastes [J]. Acta Microbiol. Immunol. Hung.,2001,48:39–52.
[125] Lenartovicz V, Marques De Souza, et al. Temperature effect in the production of multiple xylanases byAspergillus fumigates [J]. J Basic Microbiol.,2002,42:388-95.
[126] Xu Z, Bai Y, Sun W. et al. Screening and fermentation conditions of a bacterial strain over–producingxylanase[J]. Wei Sheng Wu Xue Bao.,2000,40:440–3.
[127] Haq IU, Tasneem M, Raana K, et al. Optimization of Cultural Conditions for the Production of Xylanaseby Chemically Mutated Strain of Aspergillus niger GCBCX-20[J]. Int. J. Agri. Biol.,2004,6(6):1115–1118.
[128]禹慧明,林勇,徐有良,等.木聚糖酶高产菌株选育[J].工业微生物,2002,31(1):43-45.
[129]李市场,吴敏,姚建铭,等.黑曲霉木聚糖酶及其在饲料和制备低聚木糖等方面的应用[G].生物与酶催化工程产业化促进会技术成果汇编,南京:中国微生物学会酶工程专业委员会,2004:20.
[130]王晓丹,郭丽琼,赵力超,等.木聚糖酶酶活性测定方法及酶活性单位定义[J].食品与发酵工业,2009,7(09):34-36.
[131] Georis J, Giannottaa F, Buyl ED, et al. Purification and properties of three endo-b-1,4-xylanasesproduced by Streptomyces sp. strain S38which differ in their ability to enhance the bleaching of kraft pulps[J].Enzyme and Microbial Technology,2000,26:178–186.
[132] Peng Y, Bu W, Kang LY. Methylotrophic yeast system [J]. Biotechnol Information,2000,1:38-41.
[133] Invitrogen corporation.Pichia Expression Kit (A Manual of Methods for Expression of RecombinantProteins in Pichia pastoris)[M]. www.invitrogen.com,2002.
[134]孙君社.酶与酶工程及其应用[M].北京:化学工业出版社,2006:15-18.
[135]郭勇,郑穗平.酶学[M].广州:华南理工大学出版社,2005,83-92.
[136] Kimura T, Ito J, Kawano A, et al. Purification, characterization, and molecular cloning of acidophilicxylanase from penicillium sp.40[J]. Biosci Biotechnol Biochem.,2000,64(6):1230-1237.
[137] Saraswat V, Bisaria VS, Purification, Characterization and Substrate Specificities of XylanaseIsoenzymes from Melanocarpus albomyces IIS68[J]. Bioscience,Biotechnology, and Biochemistry,2000,64(6):1173-1180.
[138]张红莲,姚斌,王亚茄,等.具有高比活性的新木聚糖酶XYNB的酶学性质研究及其编码基因的克隆和表达[J].2003,48,4:364-368.
[139] Esteves F D L, Gouders T, Josette L B, et al. Improving the alkalophilic performances of the Xyl1xylanase from Streptomyces sp. S38: Structural comparison and mutational analysis[J]. Protein Sci.,2005,14:292-302.
[140] Zhang AL, Zhang TY, Luo JX, et al. Constitutive expression of human angiostatin in Pichia pastoris byhigh-density cell culture[J]. J Ind Microbiol Biotechnol.,2007,34(2):117-22.
[141] Suzuki H, Kishimoto M, Kamoshita Y., et al. On-line control of feeding of medium components toattain high cell density[J]. Bioprocess and Biosystems Engineering,2000,22(5):433-440.
[142] Curvers S, Linnemann J, Klauser T, et al. Recombinant protein production with Pichia pastoris incontinuous fermentation-kinetic analysis of growth and product formation[J]. Eng Life Sci,2002,8:229-235.
[143] Boze H, Laborde C, Chemardin P, et al. High-level secretory production of recombinant porcinefollicle-stimulating hormone by Pichia pastoris[J]. Process Biochem,2001,36:907-913.
[144] Brierley RA, Bussineau C, Kosson R, et al. Fermentation development of recombinant Pichia pastorisexpressing the heterologous gene: bovine lysozyme[J]. Ann N Y Acad Sci,1990,589:350-362.
[145] Curvers S, Brixius P, Klauser T, et al. Human chymotrypsinogen B production with Pichia pastoris byintegrated development of fermentation and downstream processing[J]. Fermentation. Biotechnol Prog,2001,17:495-502.
[146] Gasser B, Maurer M, Gach J, et al. Engineering of Pichia pastoris for improved production of antibodyfragments[J]. Biotechnol Bioeng,2006,94:353-361.
[147] Jenzsch M, Gnoth S, Beck M, et al. A Open-loop control of the biomass concentration within the growthphase of recombinant protein production processes[J].Journal of Biotechnology,2006,127:84-94.
[148] Enfors S-O, Jahic M, Rozkov A, et al. Physiological responses to mixing in large scale bioreactors[J].Journal of Biotechnology,2001,85:175-185.
[149] Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehydes-3-phosphatedehydrogenase gene and regulation and use of its promoter[J]. Gene,1997,186:37–44
[150]戚以政,汪叔雄.生物反应动力学与反应器[M].北京:化学工业出版社,2007:160-170.
[151]俞俊棠.新编生物工艺学（下册）[M].北京:化学工业出版社,2003:68-75.
[152]梁世中.生物工程与设备[M].北京:中国轻工业出版社,2005:26-27,148-158.
[153] d'Anjou MC, Daugulis AJ. A rational approach to improving productivity in recombinant Pichia pastorisfermentation [J]. Biotechnol Bioeng.,2001,72(1):1-11.
[154] Zhang W, Sinha J, Smith LA, et al. Maximization of production of secreted recombinant proteins inPichia pastoris fed-batch fermentation [J]. Biotechnol Prog.,2005,21:386-393.
[155] Jenzsch M, Gnoth S, Beck M, et al. A Open-loop control of the biomass concentration within the growthphase of recombinant protein production processes[J]. Journal of Biotechnology,2006,127:84-94.
[156] Jenzsch M, Gnoth S, Kleinschmidt M, et al. Improving the batch-to-batch reproducibility in microbialcultures during recombinant protein production by guiding the process along a predefined total biomassprofile[J]. Bioprocess Biosyst Eng.,2006,29:315-321.
[157] Wei Zhao, Jinwen Wang, Riqiang Deng, et al. Scale-up fermentation of recombinant Candida rugosalipase expressed in Pichia pastoris using the GAP promoter[J]. J Ind Microbiol Biotechnol,2008,35:189–195.
[2]郭志强,顾维智,李佩建,等.木聚糖酶的研究进展[J].营养与日粮,2008,6:18-20.
[3]方洛云,邹晓庭.木聚糖酶基因的分子生物学与基因工程[J].畜禽业,2002,2:2-4.
[4] Henrissat B, Bairoch A. New families in the classification of glycosyl hydrolases based on amino acidsequence similarities [J]. Biochem J.,1993,293:781-788.
[5]连惠芗,汪世华.木聚糖酶的研究与应用[J].武汉工业学院学报,2006,1:42-46.
[6] Maximo C, Ferriera M C, Duarte J. Some Properties of Eucalyptus Kraft Pulp Treated with Xylanase fromAspergillus Niger [J]. World J Microbiol Biotechnol, l998,14:365-367.
[7] Nair SG, Sindhu R, Shashidhar S. Purification and biochemical characterization of two xylanases fromAspergillus sydowii SBS45[J]. Applied Biochemistry and Biotechnology,2008,149(3):229-243.
[8] Hu J, Yu B, Zhang KY, et al. Expression of endo-1,4-beta-xylanase from Trichoderma reesei in Pichiapastoris and functional characterization of the produced enzyme [J]. BMC Biotechnology,2009,9(1):56.
[9] Badhan AK, Chadha BS, Kaur J, et al. Production of multiple xylanolytic and cellulolytic enzymes bythermophilic fungus Myceliophthora sp. IMI387099[J]. Bioresource Technology,2007,98(3):504-510.
[10] OmPraba G, Velmurugan D, Arumugam P, et al. Homology model of a novel thermostable xylanase fromBacillus subtilis-AK1[J]. Journal of Biomolecular Structure and Dynamics,2007,25(3):311-320.
[11]韩双艳,林小琼,张溪,等.枯草芽孢杆菌B2（Bacillus subtilis B2）木聚糖酶在毕赤酵母中的表达[J].现代食品科技,2009,25(8):872-876.
[12] Pason P, Kosugi A, Waeonukul R, et al. Purification and characterization of a multienzyme complexproduced by Paenibacillus curdlanolyticus B-6[J]. Applied Microbiology and Biotechnology,2010,85(3):573-580.
[13]江正兵,宋慧婷,马立新.短小芽孢杆菌木聚糖酶基因在毕赤酵母中的分泌表达及酶学性质研究[J].生物工程学报,2003,1:50-55.
[14]冯佳丽,台喜生,李梅,等.链霉菌产木聚糖酶条件和酶学性质以及重离子诱变对产酶的影响研究.2011,6:205-210.
[15] Zhou JP, Huang HQ, Meng K, et al. Molecular and biochemical characterization of a novel xylanase fromthe symbiotic Sphingobacterium sp.TN19. Applied Microbiology and Biotechnology,2009,85(2):323-333.
[16]付冠华,李端,周晨妍,等.木聚糖酶的研究进展及其应用[J].安徽农业科学,2011,39(35):21566-21568.
[17]刘瑞田,曲音波.木聚糖酶分子的结构区域[J].生物工程进展,1998,18(6):27-28.
[18]刘亮伟,杨海玉,胡瑜,等. F/10木聚糖酶研究进展[J].食品与生物技术学报,2009,28(6):727-732.
[19]刘亮伟,秦天苍,翟继,等. F/10及G/11木聚糖酶家族密码子偏好性分析[J].2008,42(2):223-227.
[20]孙军亭.宇佐美曲霉木聚糖酶基因的改造及表达[D].无锡:江南大学,2009.
[21] Ko EP, Akatsuka H, Moriyama H, et al. Site-directed mutagenesis at aspartate and glutamate residues ofxylanase from Bacillus pumilus [J]. Biochem J,1992,288:117-121.
[22] Poon DK, Webster P, Withers SG, Characterizing the pH-dependent stability and catalytic mechanism ofthe family11xylanase from the alkalophilic Bacillus agaradhaerens [J]. Carbohydr Res,2003,338(5):415-421.
[23] Teplitsky A, Mechaly A, Stojanoff V,et al.Structure determination of the extracellular xylanase fromGeobacillus stearothermophilus by selenomethionyl MAD phasing [J]. Acta Crystallogr D Biol Crystallogr,2004,60(Pt5):836-48.
[24] Chen YL, Tang TY, Cheng KJ. Directed evolution to produce an alkalophilic variant fromaNeocallimastix patriciarumxylanase [J]. Can J Microbiol,2001,47:1088-1094.
[25] McCarthy AA, Morris DD, Bergquist PL, et al. Structure of XynB, a highly thermostable β-1,4-xylanasefrom Dictyoglomus thermophilum Rt46B.1, at1.8resolution[J]. Acta Crystallogr D Biol Crystallogr,2000,56(Pt11):1367-1375.
[26]杨浩萌,姚斌,范云六.木聚糖酶分子结构与重要酶学性质关系的研究进展[J].生物工程学报,2005,21(1):6-11.
[27]程菲菲,赵军旗,石鹏君,等.瓶霉Phialophora sp. P13木聚糖酶基因的克隆及酶学性质分析[J].中国农业科技导报,2012,14(1):85-90.
[28]王坤,罗会颖,石鹏君,等.来源于耐碱真菌Pseudallescheria sp. JSM-2的碱性木聚糖酶基因的克隆、表达及其性质研究[J].生物技术进展.2011,1（1）:61-67.
[29] Verma D, Satyanarayana T. Cloning, expression and applicability of thermo-alkali-stable xylanase ofGeobacillus thermoleovorans in generating xylooligosaccharides from agro-residues [J]. BioresourceTechnology,2012,107:333-338.
[30] Sriyapai T, Somyoonsap P, Matsui K, et al. Cloning of a thermostable xylanase from Actinomadura sp.S14and its expression in Escherichia coli and Pichia pastoris[J]. Journal of Bioscience and Bioengineering.2011,111(5):528-536.
[31] Driss D, Bhiri F, Ghorbel R, et al. Cloning and constitutive expression of His-tagged xylanase GH11from Penicillium occitanis Pol6in Pichia pastoris X33: Purification and characterization[J]. Protein Expr Purif.,2012,83(1):8-14.
[32]张帆,石鹏君,缪礼鸿,等.来源于瓶霉Phialophorasp.G5的酸性木聚糖酶基因的克隆及性质的研究[J].中国农业科技导报,2011,13(3):60-66.
[33]张桂敏,饶犇,叶戋,等.棉花黄萎病真菌Verticillium dahliae木聚糖酶基因的克隆、表达和酶学性质分析[J].微生物学报,48(6):765-771.
[34]王建设,柏映国,尹俊,等.脂环酸芽孢杆菌Alicyclobacillussp.A15木聚糖酶基因xynA15的克隆表达及性质研究[J].中国农业科技导报,2010,12(6):114-119.
[35] Wang GZ, Luo HY, Wang YR, et al. A novel cold-active xylanase gene from the environmental DNA ofgoat rumen contents: Direct cloning, expression and enzyme characterization[J]. Bioresource Technology,2011,102(3):3330-3336.
[36] Ko CH, Tsai CH, Tu J, et al. Molecular cloning and characterization of a novel thermostable xylanasefrom Paenibacillus campinasensis BL11[J]. Process Biochemistry,2010,45(10):1638-1644.
[37] Qiu ZH, Shi PG, Luo HY, et al. A xylanase with broad pH and temperature adaptability fromStreptomyces megasporus DSM41476, and its potential application in brewing industry[J]. Enzyme andMicrobial Technology,2010,46(6):506-512.
[38] Hwang IT, Lim HK, Song HY, et al. Cloning and characterization of a xylanase, KRICT PX1from thestrain Paenibacillus sp. HPL-001[J]. Biotechnology Advances,2010,28(5):594-601.
[39] Vega E, Farrés GS. Amelia, Beatriz XC, et al. Cloning and expression of a novel, moderatelythermostable xylanase-encoding gene (Cfl xyn11A) from Cellulomonas flavigena[J]. Bioresource Technology,2010,101(14):5539-5545.
[40] Zhou CY, Bai JY, Deng SS, et al. Cloning of a xylanase gene from Aspergillus usamii and its expressionin Escherichia coli [J]. Bioresource Technology,2008,99(4):831-838.
[41] Nádia Skorupa Parachin, Saulo Siqueira, Fabrícia Paula de Faria, et al. Xylanases from Cryptococcusflavus isolate I-11: Enzymatic profile, isolation and heterologous expression of CfXYN1in Saccharomycescerevisiae [J]. Journal of Molecular Catalysis B: Enzymatic,2009,59(1-3):52-57.
[42]何永志,姚斌,王亚茹,等.来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析[J].微生物学报,44（3）:340-344.
[43]周煌凯,龚月生,杨明明,等.耐碱性短小芽孢杆菌木聚糖酶在枯草杆菌中的表达及发酵条件优化[J].饲料工业,2010,31（14）:25-29.
[44]罗建杰,王亚茹,袁铁铮,等.高比活木聚糖酶XYN-W及其突变体在酵母中的高效表达[J].中国农业科技导报,2010,12(5):80-85.
[45] Santosh O, Ramchuran, Bruno Mateus, et al. The methylotrophic yeast Pichia pastoris as a host for theexpression and production of thermostable xylanase from the bacterium Rhodothermus marinus[J]. FEMSYeast Research,2005,5(9):839-850.
[46] He J, Chen DW, Yu B, et al. Optimization of the Trichoderma reesei endo-1,4-beta-xylanase productionby recombinant Pichia pastoris[J]. Biochemical Engineering Journal,2010,52(1):1-6.
[47] Natasha Birijlall, Ayyachamy Manimaran, Kuttanpillai Santhosh Kumar, et al. High level expression of arecombinant xylanase by Pichia pastoris NC38in a5L fermenter and its efficiency in biobleaching of bagassepulp[J]. Bioresource Technology,2011,102(20):9723-9729.
[48] Jia HY, Fan GG, Yan QJ, et al. High-levelexpression of a hyperthermostable Thermotogamaritimaxylanase in Pichia pastoris by codon optimization[J]. Journal of Molecular Catalysis B: Enzymatic,2012,78:72-77.
[49]姚斌.基因工程木聚糖酶XYNB的生产技术[G].生物与酶催化工程产业化促进会技术成果汇编,南京:中国微生物学会酶工程专业委员会,2004:17.
[50]毛爱军,董志杨.高产木聚糖酶工程菌株及其产业化研究[G].生物与酶催化工程产业化促进会技术成果汇编,南京:中国微生物学会酶工程专业委员会,2004:18.
[51]孙宏亮.重组毕赤酵母生产木聚糖酶工艺概述[C].2011饲料酶制剂应用技术国际研讨会暨第二届饲料酶制剂大会,青岛:中国农业科学院饲料研究所《新饲料》杂志社,2011:22-23.
[52] Jeong MY, Kim S, Yun CW, et al. Engineering a de novo internal disulfide bridge to improve the thermalstability of xylanase from Bacillus stearothermophilus No.236[J]. J Biotechnol,2007,127(2):300-309.
[53] George SP, Ahnad A, Rao MB. A novel thermostable xylanase from Thermomonosporasp.: influence ofadditives on thermostability [J]. Bioresour Technol,2001,78(3):221-224.
[54]杨浩萌,王亚茹,伍宁丰,等. N13D、S40E点突变提高木聚糖酶XYNB的热稳定性[J].微生物学通报,2007,34(3):533-536.
[55] Chen Y L, Tang TY, Cheng KJ. Directed evolution to produce an alkalophilic variant from aNeocallimastix patriciarum xylanase [J].Can J Mirobiol,2001,47(12):1088-1094.
[56] Fenel F, Zitting AJ, Kantelinen A. Increased alkali stability in Trichoderma reesei endo-1,4-beta-xylanaseII by site directed mutagenesis [J]. J Biotechnol.2006:121(1):102-107.
[57] Cirino PC, Mayer KM, Daisuke Umeno. Generating Mutant Libraries Using Error-Prone PCR [J].Methods in Molecular Biology,2003,231(1):3-9.
[58] McCullum EO, Williams BA, Zhang J, et al. Random mutagenesis by error-prone PCR [J].2010,634:103-9.
[59] Loo B, Spelberg LJH., Kingma J, et al. Directed Evolution of Epoxide Hydrolase from A radiobactertoward Higher Enantioselectivity by Error-Prone PCR and DNA Shuffling [J]. Chemistry&Biology,2004,11:981-990.
[60] Zhang ZG, Yi ZL, Pei XQ, et al. Improving the thermostability of Geobacillus stearothermophilusxylanase XT6by directed evolution and site-directed mutagenesis [J]. Bioresource Technology,2010,101(23):9272-9278.
[61] Miyazaki K, Takenouchi M, Kondo H, et al. Thermal Stabilization of Bacillus Subtilis Family-11Xylanase by Directed Evolution[J]. J Biol Chem.,2006,281(15):10236-10242.
[62]李清峰,马向东,彭虹旎,等.木聚糖酶耐热性研究进展[J].中国畜牧杂志,200945（11）:57-60.
[63]周晨妍,王武,邬敏辰. Arg引入“Ser/Thr”平面对木聚糖酶XynⅡ热稳定性的影响[J].西北农林科技大学学报(自然科学版),2010,38(1):46-52.
[64] Han C, Yu S, Ouyang J, et al. Enhancing stability of Trichoderma reesei xylanase (XYN II) bysite-directed mutagenesis[J]. Sheng Wu Gong Cheng Xue Bao,2010,26(5):623-629.
[65] Chen XZ, Xu SQ, Zhu MS, et al. Site-directed mutagenesis of an Aspergillus niger xylanase B and itsexpression, purification and enzymatic characterization in Pichia pastoris[J]. Process Biochemistry,2010,45(1):75-80.
[66]吴穗洁,吴文林,刘斌.嗜热酶耐热机制及提高酶热稳定性的研究进展[J].亚热带农业研究,2006,2(4):293-297.
[67]张勇伟,胡虹,刘亮伟,等.木聚糖酶热稳定性化学修饰研究[J].食品与发酵工业,2011,37(5):61-65.
[68] Polizeli ML, Rizzatti AC, Monti R, et al. Xylanases from fungi: properties and industrial applications[J].Appl Microbiol Biotechnol.,2005,67(5):577-91.
[69] Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology, andapplication [J]. Crit Rev Biotechnol.,2002,22(1):33-64.
[70] Beg QK, Kapoor M, Mahajan L, et al. Microbial xylanases and their industrial applications: a review [J].Appl Microbiol Biotechnol.,2001,56(3-4):326-38.
[71]慕娟,问清江,党永,等.木聚糖酶的开发与应用[J].陕西农业科学,2012,1:111-115.
[72] Knob A, Terrasan CRF, Carmona EC. β-Xylosidases from filamentous fungi: an overview[J]. World JMicrobiol Biotechnol,2010,26:389-407.
[73]吴香波,谢益民.木聚糖酶在制浆造纸工业中的应用和研究进展[J].上海造纸,2008,39(5):6-9.
[74]马文,张美云,房桂干.木聚糖酶在制浆造纸中的应用研究进展[J].上海造纸,2008,39(2):9-11.
[75]贺文明.木聚糖酶在美国KP厂的应用[J].国际造纸,2003,22(5):32-34.
[76]尤纪雪,欧阳嘉,沈洁,等.机械力化学效应对麦草浆重组木聚糖酶生物漂白的影响[J].纤维素科学与技术,2010,18(4):25-32.
[77]董毅,陈嘉川,杨桂花,等.木聚糖酶处理改善桉木KP浆质量的研究[J].江苏造纸,2009,3:36-39.
[78]张久政,武书彬,胡惠仁.生物酶用于旧报纸脱墨的研究[J].造纸科学与技术,2003,22(1):14-17.
[79]杨桂花,陈克复,陈嘉川,等.木聚糖酶处理改善速生杨化学机械浆性能的研究[J].林产化学与工业,2009,29(2):105-109.
[80]何承云,林向阳,高雪琴,等.木聚糖酶在馒头制作中的应用研究[J].农产品加工(学刊),2009,6:7-10.
[80]张勤良,王璋,许时婴.中性木聚糖酶在面包制作中的应用[J].食品与发酵工业,2004,30(7):21-25.
[82]李秀婷.微生物木聚糖酶及在食品工业中的应用[J].农业机械学报,2008,39(2):175-179.
[83]郑翔鹏,王国川,林海峰,等.木聚糖酶在啤酒中的应用研究[J].啤酒科技,2008,9:27-36.
[84] Hitzeman RA, Hagie FE, Levine, et al. Expression of a human gene for interferon in yeast [J]. Nature,1981,293:717-722.
[85] Wegner EH.Biochemical conversions by yeast fermentation at high–cell dendities [P]. US. Patent:4414329,1983.
[86] Cregg JM, Cereghino JL,Shi J, et al. Recombinant protein expression in Pichia pastoris [J]. MolBiotechnol,2000,16(1):23-52.
[87] Cunha AE, Clemente JJ, Gomes R, et al. Methanol induction optimization for scFv antibody fragmentproduction in Pichia pastoris [J]. Biotechnol Bioeng.,2004,86(4):458-467.
[88] Ding Y F, Liu, Dai CB. Advances in Pichia pastoris expression system [J]. Chinese Bulletin of LifeSciences,2003,15(1):326-311.
[89]胡世界,罗素兰,张吉贞,等.巴斯德毕赤酵母表达系统及其高水平表达策略[J].生物技术,2007,17(6):78-83.
[90] Ohi H, Miura M, Hiramatsu R, et al. The positive and negative cis-acting elements for methanolregulation in the Pichia pastoris AOX2gene [J]. Molecular and General Genetics,1994,243:489-499.
[91]娄瑞娟,罗利龙,张霞,等.巴斯德毕赤酵母表达系统的研究进展和前景展望[J].生物学杂志,2010,27(5):73-76.
[92]王清路,李俏俏,薛金艳,等.巴斯德毕赤酵母表达系统的特点及应用[J].生物技术通讯,2006,17(4):640-643.
[93] Cregg JM, VedvickTS, Raschke WC. Recent advances in the expression of foreign genes in Pichiapastoris [J]. Nature Biotechnology,1993,11:905-910.
[94] Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehyde-3-phosphatedehydrogenase gene and regulation and use of its promoter[J]. Gene,1997,186(1):37-44.
[95] Menendez J, HernandezL, BanguelaA, et al. Functional production and secretion of theGluconacetobacter diazotrophicus fructose-releasing exo-levanase (LsdB) in Pichia pastoris [J].Enz.Microb.Technol,2004,34(5):446-452.
[96] Dring F, Klapper M, Theis S, et al. Use of the glyceraldehydes-3-phosphate dehydrogenase promoter forproduction of functional mammalian membrane transport proteins in the yeast pichia pastoris [J].Biochem.Biophys.Res.Commun.,199850(2):531－535.
[97] Delroisse JM, Dannau M,Gilsoul JJ, et al.Expression of a synthetic gene encoding a ttriboliumcastaneum carboxylesterase in Pichia pastoris [J]. Protein Expr.Purif.,2005,42(2):286-294.
[98]路蓉.巴斯德毕赤酵母表达系统及其分泌型蛋白表达的研究进展[J].临床和实验医学杂志,2004,3(1):43-47.
[99]耿宏伟,侯红燕,王丕武,等.毕赤酵母表达系统的研究进展[J].食品工程,2011,10:159-162.
[100] Veenhuis M, VanDijken J.P., Harde W. The Significance of Peroxisomes in the Metabolism ofOne-Carbon Compounds in Yeasts [J]. Advances in Microbial Physiology,1983,24:1–82.
[101] Martine M, Zeng S, BergerE.G. The yeast expression system for recombinant glycosyl transferases [J].Glycoconjugate Journal,1999,(16):125-139.
[102] Menendez J, Valdes I, Cabrera N. The ICL1gene of Pichia pastoris, transcriptional regulation and use ofits promoter [J]. Yeast,2003,20(13):1097-10110.
[103] J.R.M. de Almeida, L.M.P. de Moraes, F.A.G. Torres. Molecular characterization of the3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris [J]. Yeast,2005,22:725–737.
[104] Ahn J, Hong J, Lee H, et al. Translation elongation factor1-α gene from Pichia pastoris: molecularcloning, sequence,and use of its promoter[J]. Appl. Genet. Mol. Biotechnol.,2007,74:601-608.
[105] Vassileva A, Chugh DA, Swarninathan S, et al. Effect of copy number on the expression levels ofhepetitis B Surface antigen in the methylotrophic yeast Pichia pastoris [J]. Protein Expr Purif.,2001,21:71.
[106]王龙刚,王宇凯,何成,等.混合补料诱导高拷贝重组毕赤酵母高效表达X-HBsAg [J].中国生物制品学杂志,2010,23(5):521-525.
[107]井申荣,邹全明,洪愉,等.白细胞介素-10基因多拷贝表达盒的构建及在毕赤酵母中的表达[J].中国生物制品学杂志,2007,2(20):81-86.
[108]谢涛,王红宁.外源基因在巴斯德毕赤酵母中多拷贝整合的研究进展[J].生物技术通讯,2006,3(17):415-417.
[109] Zhao W, Wang JW, Deng RQ, et al. Scale-up fermentation of recombinant Candida rugosa lipaseexpressed in Pichia pastoris using the GAP promoter [J]. J Ind Microbiol Biotechnol,2008,35:189-195.
[110] Soons ZI, Voogt JA, van Straten G, et al. Constant specific growth rate in fed-batch cultivation ofBordetella pertussis using adaptive control [J]. J Biotechnol,2006,125:252-268.
[111] Zhang W, Sinha J, Smith LA, et al. Maximization of production of secreted recombinant proteins inPichia pastoris fed-batch fermentation [J]. Biotechnol Prog.,2005,21:386-393.
[112] Pais-Chanfrau JM, Garcia Y, Licor L, et al. Improving the expression of mini-proinsulin in Pichiapastoris [J]. Biotechnol Lett,2004,26:1269-1272.
[113] Onyeaka H, Nienow AW, Hewitt CJ. Further studies related to the scale-up of high cell densityEscherichia coli fed-batch fermentations: the additional effect of a changing microenvironment when usingaqueous ammonia to control pH [J]. Biotechnol Bioeng,2003,84:474-484.
[114] Goodrick JC, Xu M, Finnegan R, et al. High-level expression and stabilization of recombinant humanchitinase produced in a continuous constitutive Pichia pastoris expression system[J]. Biotechnol Bioeng.,2001,74(6):492-7.
[115] Schilling BM, Goodrick JC, Wan NC. Scale-up of a high cell density continuous culture with Pichiapastoris X-33for the constitutive expression of rh-chitinase [J]. Biotechnol Prog.,2001,17(4):629-33.
[116] Goodrick, Khasa YP, Khushoo A, et al. Kinetic studies of constitutive human granulocyte macrophagecolony stimulating factor hGMCSF expression in continuous culture of Pichia pastoris[J]. J.Biotechnol.Lett.,2007,29:1903-1908.
[117] d’Anjou MC, Daugulis AJ. A rational approach to improving productivity in recombinant Pichia pastorisfermentation[J]. Biotechnol.Bioeng.,2000,2:1–11.
[118] Yamawaki S, Matsumoto T, Ohnishi Y, et al. Production of single-chain variable fragmentantibody (scFv) in fed-batch and continuous culture of Pichia pastoris by two different methanol feedingmethods[J]. J. Biosci. Bioeng.,2007,104:403-407.
[119] Jungo C, Rérat C, Marison IW, et al. Quantitative characterization of the regulation of the synthesis ofalcohol oxidase and of the expression of recombinant avidin in a Pichia pastoris Mut+strain[J]. EnzymeMicrob.,2006,39:936-944.
[120] Zhang W, Liu CP, Inan M, et al. Optimization of cell density and dilution rate in Pichia pastoriscontinuous fermentations for production of recombinant proteins[J]. J. Ind. Microbiol. Biotechnol.,2004,31:330–334.
[121] Nakano A, Lee C Y, Yoshida A, et al. Effects of methanol feeding methods on chimeric α-amylaseexpression continuous culture of Pichia pastoris[J]. J. Biosci. Bioeng.,2006,101:227–231.
[122] Khasa Y.P., Khushoo A., Srivastava L., et al. Kinetic studies of constitutive humangranulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture ofPichia pastoris [J]. Biotechnol. Lett.,2007,29:1903–1908.
[123] Maurer M., Kühleitner M., Gasser B., et al. Versatile modeling and optimization of fed-batch processesfor the production of secreted heterologous proteins with Pichia pastoris[J]. Microb. Cell act,2006,5:37.
[124] Kansoh A.L. and Gammal A.. Xylanolytic activities of Streptomyces sp, taxonomy production, partialpurification and utilization of agricultural wastes [J]. Acta Microbiol. Immunol. Hung.,2001,48:39–52.
[125] Lenartovicz V, Marques De Souza, et al. Temperature effect in the production of multiple xylanases byAspergillus fumigates [J]. J Basic Microbiol.,2002,42:388-95.
[126] Xu Z, Bai Y, Sun W. et al. Screening and fermentation conditions of a bacterial strain over–producingxylanase[J]. Wei Sheng Wu Xue Bao.,2000,40:440–3.
[127] Haq IU, Tasneem M, Raana K, et al. Optimization of Cultural Conditions for the Production of Xylanaseby Chemically Mutated Strain of Aspergillus niger GCBCX-20[J]. Int. J. Agri. Biol.,2004,6(6):1115–1118.
[128]禹慧明,林勇,徐有良,等.木聚糖酶高产菌株选育[J].工业微生物,2002,31(1):43-45.
[129]李市场,吴敏,姚建铭,等.黑曲霉木聚糖酶及其在饲料和制备低聚木糖等方面的应用[G].生物与酶催化工程产业化促进会技术成果汇编,南京:中国微生物学会酶工程专业委员会,2004:20.
[130]王晓丹,郭丽琼,赵力超,等.木聚糖酶酶活性测定方法及酶活性单位定义[J].食品与发酵工业,2009,7(09):34-36.
[131] Georis J, Giannottaa F, Buyl ED, et al. Purification and properties of three endo-b-1,4-xylanasesproduced by Streptomyces sp. strain S38which differ in their ability to enhance the bleaching of kraft pulps[J].Enzyme and Microbial Technology,2000,26:178–186.
[132] Peng Y, Bu W, Kang LY. Methylotrophic yeast system [J]. Biotechnol Information,2000,1:38-41.
[133] Invitrogen corporation.Pichia Expression Kit (A Manual of Methods for Expression of RecombinantProteins in Pichia pastoris)[M]. www.invitrogen.com,2002.
[134]孙君社.酶与酶工程及其应用[M].北京:化学工业出版社,2006:15-18.
[135]郭勇,郑穗平.酶学[M].广州:华南理工大学出版社,2005,83-92.
[136] Kimura T, Ito J, Kawano A, et al. Purification, characterization, and molecular cloning of acidophilicxylanase from penicillium sp.40[J]. Biosci Biotechnol Biochem.,2000,64(6):1230-1237.
[137] Saraswat V, Bisaria VS, Purification, Characterization and Substrate Specificities of XylanaseIsoenzymes from Melanocarpus albomyces IIS68[J]. Bioscience,Biotechnology, and Biochemistry,2000,64(6):1173-1180.
[138]张红莲,姚斌,王亚茄,等.具有高比活性的新木聚糖酶XYNB的酶学性质研究及其编码基因的克隆和表达[J].2003,48,4:364-368.
[139] Esteves F D L, Gouders T, Josette L B, et al. Improving the alkalophilic performances of the Xyl1xylanase from Streptomyces sp. S38: Structural comparison and mutational analysis[J]. Protein Sci.,2005,14:292-302.
[140] Zhang AL, Zhang TY, Luo JX, et al. Constitutive expression of human angiostatin in Pichia pastoris byhigh-density cell culture[J]. J Ind Microbiol Biotechnol.,2007,34(2):117-22.
[141] Suzuki H, Kishimoto M, Kamoshita Y., et al. On-line control of feeding of medium components toattain high cell density[J]. Bioprocess and Biosystems Engineering,2000,22(5):433-440.
[142] Curvers S, Linnemann J, Klauser T, et al. Recombinant protein production with Pichia pastoris incontinuous fermentation-kinetic analysis of growth and product formation[J]. Eng Life Sci,2002,8:229-235.
[143] Boze H, Laborde C, Chemardin P, et al. High-level secretory production of recombinant porcinefollicle-stimulating hormone by Pichia pastoris[J]. Process Biochem,2001,36:907-913.
[144] Brierley RA, Bussineau C, Kosson R, et al. Fermentation development of recombinant Pichia pastorisexpressing the heterologous gene: bovine lysozyme[J]. Ann N Y Acad Sci,1990,589:350-362.
[145] Curvers S, Brixius P, Klauser T, et al. Human chymotrypsinogen B production with Pichia pastoris byintegrated development of fermentation and downstream processing[J]. Fermentation. Biotechnol Prog,2001,17:495-502.
[146] Gasser B, Maurer M, Gach J, et al. Engineering of Pichia pastoris for improved production of antibodyfragments[J]. Biotechnol Bioeng,2006,94:353-361.
[147] Jenzsch M, Gnoth S, Beck M, et al. A Open-loop control of the biomass concentration within the growthphase of recombinant protein production processes[J].Journal of Biotechnology,2006,127:84-94.
[148] Enfors S-O, Jahic M, Rozkov A, et al. Physiological responses to mixing in large scale bioreactors[J].Journal of Biotechnology,2001,85:175-185.
[149] Waterham HR, Digan ME, Koutz PJ, et al. Isolation of the Pichia pastoris glyceraldehydes-3-phosphatedehydrogenase gene and regulation and use of its promoter[J]. Gene,1997,186:37–44
[150]戚以政,汪叔雄.生物反应动力学与反应器[M].北京:化学工业出版社,2007:160-170.
[151]俞俊棠.新编生物工艺学（下册）[M].北京:化学工业出版社,2003:68-75.
[152]梁世中.生物工程与设备[M].北京:中国轻工业出版社,2005:26-27,148-158.
[153] d'Anjou MC, Daugulis AJ. A rational approach to improving productivity in recombinant Pichia pastorisfermentation [J]. Biotechnol Bioeng.,2001,72(1):1-11.
[154] Zhang W, Sinha J, Smith LA, et al. Maximization of production of secreted recombinant proteins inPichia pastoris fed-batch fermentation [J]. Biotechnol Prog.,2005,21:386-393.
[155] Jenzsch M, Gnoth S, Beck M, et al. A Open-loop control of the biomass concentration within the growthphase of recombinant protein production processes[J]. Journal of Biotechnology,2006,127:84-94.
[156] Jenzsch M, Gnoth S, Kleinschmidt M, et al. Improving the batch-to-batch reproducibility in microbialcultures during recombinant protein production by guiding the process along a predefined total biomassprofile[J]. Bioprocess Biosyst Eng.,2006,29:315-321.
[157] Wei Zhao, Jinwen Wang, Riqiang Deng, et al. Scale-up fermentation of recombinant Candida rugosalipase expressed in Pichia pastoris using the GAP promoter[J]. J Ind Microbiol Biotechnol,2008,35:189–195.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |