重组枯草芽孢杆菌生产角质酶发酵条件优化
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摘要
角质酶是一种多功能酶,属于丝氨酸酯酶,具有称作α/β水解酶折叠的共同结构框架的蛋白质。作为酯酶的一种,角质酶在工业产品和生产工艺中的应用表现出脂肪酶的特性,它既可以参与水解反应也可以参与合成反应。因此其在奶制品工业、洗涤剂工业、化妆品工业、甘油三酯、多聚体、表面活性剂、护肤产品、药物以及农业化学药品等方面都表现出潜在的应用。角质酶在纺织上的应用是近几年兴起的新的研究方向,在染色方面和传统工艺相比能够提高润湿性和水解纤维的能力,缩短处理时间、减少蒸汽和电力消耗以及保护环境等优点。
本论文以一株高积累角质酶的重组枯草芽孢杆菌(Bacillus subtilis WSH 06-07)为出发菌株,以角质酶的高产量、高得率和高生产强度为目标,采用针对发酵过程的外因定性优化(基于微生物反应原理的培养环境优化技术)、内因定性优化(基于代谢特性的分阶段培养技术)等一系列技术对角质酶发酵过程进行优化,研究的主要结果如下:
1.种龄对发酵的影响较大,实验考察了种子的生长情况:9~22 h为种子的对数期,种龄为11 h,将种子接入培养基中有利于菌体的发酵。
2.采用基因工程菌进行发酵,质粒的稳定性和细胞内包涵体对产量影响很大。实验结果表明:在整个发酵过程中,质粒稳定性能维持90%以上。此外,通过聚丙烯酰胺凝胶电泳验证了细胞内没有包涵体,目的蛋白全部在发酵上清液中。
3.摇瓶水平上考察营养条件对B. subtilis WSH 06-07生长和角质酶合成的影响;通过单因素实验和Plackett–Burman实验确定了蔗糖、蛋白胨以及硫酸镁是影响角质酶发酵的主要因素;在此基础上,采用响应面设计方法考察了各营养组分及其浓度的配比关系。实验结果表明:蔗糖和硫酸镁存在交互作用也是影响细胞生长和角质酶合成的主要因素。最终确定角质酶发酵的培养基组分为(g/L):蔗糖32.5,蛋白胨37,酵母膏24,磷酸氢二钠12.54,磷酸二氢钾2.31,硫酸镁4.4。进一步确定较优的环境条件组合为:初始pH 7.5、装液量75 mL/500 mL、接种量5%。
4.在3 L发酵罐中研究了溶氧对角质酶分批发酵的影响。结果表明:在通气量恒定的情况下,不同搅拌转速对菌体干重(DCW)和角质酶产量没有较大影响。因此,在蔗糖浓度为32.5 g/L且通气量控制在1.5 vvm的前提下,将搅拌转速恒定在600 r/min即可满足细胞生长和角质酶合成对溶氧的需求。
5.不同pH控制模式对角质酶分批发酵的影响有较大差异。在发酵过程中控制pH相对于不控制pH角质酶产量有所提高。基于发酵过程中不同pH对菌体比生长速率及比产物合成速率的变化,确定了pH两阶段控制策略,即0~4 h时控制pH 7.5,4 h后将pH调至6.5。通过采用这一优化策略,角质酶酶活有了较大的提高,达170 U/mL,生产强度为16.9 kU/(L·h),比恒定pH 7.5控制模式下分别提高了122.6%和123.2%。
6.实验考察了温度对B. subtilis WSH 06-07生长和产酶的影响。分批培养结果表明:温度在27 oC~40 oC范围内,较高温度对提高细胞生长速度有促进作用,但在40 oC下质粒丢失严重,而较低温度有利于角质酶的合成。在深入分析不同温度下角质酶分批发酵动力学的基础上,提出了温度分阶段控制策略,即发酵前4 h控制为温度37 oC,4 h后切换至30 oC并保持到发酵结束。采用这一最优温度两阶段控制策略,最高酶活和生产强度为312.5 U/(mL)、13.02 KU/(L·h),比37 oC分别提高了83.4%、10.9%。因此温度两阶段控制策略对于提高角质酶产量具有实际效果。
7.实验考察了不同初糖浓度对角质酶分批发酵的影响,实验结果表明:仅通过提高初糖浓度难以实现细胞和角质酶高产量、高得率和高生产强度的有机统一。对此实验考察了分批补料、恒速流加培养方式对B. subtilis WSH 06-07发酵生产角质酶的影响。结果表明:这两种补料培养方式都可以实现细胞和角质酶的高产。综合比较,恒速流加对于B. subtilis WSH 06-07发酵培养来说,无论是细胞得率还是角质酶的高产量、高得率和高生产强度都是较理想的选择。经过31 h的恒速流加培养,DCW达到52.76 g/L,酶活最大达545.87 U/mL。
Cutinases are mutilfunctional enzymes and belong to the family of serine hydrolases containing the so-calledα/βhydrolase fold. As a lipolytic enzyme, cutinase has been presented as a versatile enzyme showing several interesting properties for applications in industrial products and processes. Hydrolytic and synthetic reactions catalyzed by cutinase have potential use in the dairy industry for the hydrolysis of milk fat, in house hold detergents, in the oleochemical industry, in the synthesis of structured triglycerides, polymers and surfactants, in the synthesis of ingredients for personal-care products, and the synthesis of pharmaceuticals and agrochemicals containing one or more chiral centers. The application of cutinase in the textile industry has become a new research direction.Compared with the traditional technique in cotton desizing, cutinase can improve cotton wettability and fabric hydrophilicity in addition to its ability to simplify process, reduce working time, savesteam-water-electricity and protect the environment.
In this paper, recombinant Bacillus subtilis WSH 06-07, a strain could accumulate high concentration of cutinase, was selected for cutinase production. According to the principles of fermentation optimization including the optimization of culture conditions based on microbial reactions (outer factor), and the optimization based on metabolic flux analysis (quantitativeness of inner factor), a series of feasible approaches or strategies were carried out to achieve high product concentration, high yield and high productivity of cutinase in the optimization of B. WSH 06-07 cultivation processes, including:
1. Seed aging has a extremely influence in fermentation, and the experiments results were following: the log growth stage was from 9 to 22 h, and the seed aging was 11h, which was beneficial to cutinase fermentation.
2. For recombinant cell, plasmid and cellular insoluble fraction have an important effect on cutinase yield during fermentation. The experiment results showed that plasmid stability was over 90% during the whole fermentation course. Besides, there was no cellular insoluble fraction by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the objection proteins were included in supernant.
3. The required nutrients for cutinase production by B. subtilis WSH 06-07 in shaking flasks were investigated. According to the single-factor experiments and Plackett–Burman design, sucrose, trytone and MgSO4 had a profound influence in cutinase fermentation, base on which, the optimal concentration ratio among them was defined by Response Surface Methodology, and the results were summed that sucrose and MgSO4 had a positive interaction, and their effects on cell growth and cutinase production were the most important elements. Finally, the essential culture medium for cutinase production was determined to be consisted of sucrose 32.5 g/L, trptonse 37 g/L, Na2HPO4 12.54 g/L, KH2PO4 2.31 g/L and MgSO4 4.4 g/L. The environmental conditions of cutinase fermentation were also investigated and an optimal combination was developed: an initial pH of 7.5, a medium content of 75 mL medium in 500 mL flask, and an inoculum size of 5%.
4. The effect of dissolved oxygen (DO) concentration on the batch production of cutinase in a 3 L stirred fermentor by B. subtilis WSH 06-07 was studied. It was found that DCW and cutinase production, especilly for cutinase content, were not greatly influenced by the agitation rate under a fixed aeration rate. When the initial sucrose concentration was 32.5 g/L and the air flow rate was controlled at 1.5 vvm, the DO concentration, under the circumstance of the agitation rate not less than 600 r/min, was sufficient to satisfy the oxygen requirement for better cell growth and cutinae production during the batch fermentation.
5. The modes of pH control for batch cutinase fermentation are extremely different. B. subtilis WSH 06-07 was cultivated by controlling pH, cell growth and cutinase production were improved comparing to without pH control. Based on specific cell growth rate and specific cutinase formation rate under different pH condition, a two stage pH control strategy was developed, in which pH was controlled at 7.5 for the first 4h and then shift to 6.5. By the utilization of this strategy, the yield of cutinase was significantly improved, the maximal cutinase activity and the productivity were 170 U/mL and 16.9 kU/(L·h), which were increased by 122.6% and 123.2%, respectively, compared to that the condition of constant pH 7.5.
6. The effect of temperature, varied from 27 oC to 32 oC, on cell growth and cutinase production the batch fermentation of cultivation was investigated. It was found that cell growth was hastened along with the increase of temperature while lower temperature was more favorable for cutinase formation, however, plasmid lost greatly. Owing to the difference occurred in the optimal temperature for cell growth and cutinase production by thoroughly analyzing the kinetics of batch cutinase production under different temperatures, a two-stage temperature control strategy, in which the temperature was kept at 37 oC during the first 4 h of cultivation, followed by a shift from 37 oC to 30 oC, then maintained at 30 oC until the end of the cultivation, was developed in order to enhance the production of cutinase. Compared with the results under temperature 37 oC control, the two-stage temperature control strategy was confirmed to enhance the ability of cutinase synthesis. As a result, the maximum cutinase production and the productivily were 312.5 U/(mL), 13.02 KU/(L·h), increased by 83.4%、10.9% compareing to at 37 oC, respectively, Therefore, the strategy would have good feasibility in practice.
7. Effects of diverse sucrose concentration on cell growth and cutinaes production were invested by batch fermentation, a sum was found that it was diffuclt to make high product concentration, high yield and high productivity of cutinase harmony by improving sucrose concentration. As to the cultivation of B. subtilis WSH 06-07 by batch or constant fed-batch process in a 3 L stirred fermentor was studied, the resrults shown that it was efficient to realize high cell and high cutinase concentration by two kinds of feeding strateges. However, comhensive comparision of them, constant feeding fermentation was ideal choice to enhance cutinase yield wether cell yield or high product concentration, high yield and high productivity of cutinase. The DCW and cutinase activity were 52.76 g/L, 545.87 U/mL aferte 31 h constant feeding batch cultivation, respectively.
本论文以一株高积累角质酶的重组枯草芽孢杆菌(Bacillus subtilis WSH 06-07)为出发菌株,以角质酶的高产量、高得率和高生产强度为目标,采用针对发酵过程的外因定性优化(基于微生物反应原理的培养环境优化技术)、内因定性优化(基于代谢特性的分阶段培养技术)等一系列技术对角质酶发酵过程进行优化,研究的主要结果如下:
1.种龄对发酵的影响较大,实验考察了种子的生长情况:9~22 h为种子的对数期,种龄为11 h,将种子接入培养基中有利于菌体的发酵。
2.采用基因工程菌进行发酵,质粒的稳定性和细胞内包涵体对产量影响很大。实验结果表明:在整个发酵过程中,质粒稳定性能维持90%以上。此外,通过聚丙烯酰胺凝胶电泳验证了细胞内没有包涵体,目的蛋白全部在发酵上清液中。
3.摇瓶水平上考察营养条件对B. subtilis WSH 06-07生长和角质酶合成的影响;通过单因素实验和Plackett–Burman实验确定了蔗糖、蛋白胨以及硫酸镁是影响角质酶发酵的主要因素;在此基础上,采用响应面设计方法考察了各营养组分及其浓度的配比关系。实验结果表明:蔗糖和硫酸镁存在交互作用也是影响细胞生长和角质酶合成的主要因素。最终确定角质酶发酵的培养基组分为(g/L):蔗糖32.5,蛋白胨37,酵母膏24,磷酸氢二钠12.54,磷酸二氢钾2.31,硫酸镁4.4。进一步确定较优的环境条件组合为:初始pH 7.5、装液量75 mL/500 mL、接种量5%。
4.在3 L发酵罐中研究了溶氧对角质酶分批发酵的影响。结果表明:在通气量恒定的情况下,不同搅拌转速对菌体干重(DCW)和角质酶产量没有较大影响。因此,在蔗糖浓度为32.5 g/L且通气量控制在1.5 vvm的前提下,将搅拌转速恒定在600 r/min即可满足细胞生长和角质酶合成对溶氧的需求。
5.不同pH控制模式对角质酶分批发酵的影响有较大差异。在发酵过程中控制pH相对于不控制pH角质酶产量有所提高。基于发酵过程中不同pH对菌体比生长速率及比产物合成速率的变化,确定了pH两阶段控制策略,即0~4 h时控制pH 7.5,4 h后将pH调至6.5。通过采用这一优化策略,角质酶酶活有了较大的提高,达170 U/mL,生产强度为16.9 kU/(L·h),比恒定pH 7.5控制模式下分别提高了122.6%和123.2%。
6.实验考察了温度对B. subtilis WSH 06-07生长和产酶的影响。分批培养结果表明:温度在27 oC~40 oC范围内,较高温度对提高细胞生长速度有促进作用,但在40 oC下质粒丢失严重,而较低温度有利于角质酶的合成。在深入分析不同温度下角质酶分批发酵动力学的基础上,提出了温度分阶段控制策略,即发酵前4 h控制为温度37 oC,4 h后切换至30 oC并保持到发酵结束。采用这一最优温度两阶段控制策略,最高酶活和生产强度为312.5 U/(mL)、13.02 KU/(L·h),比37 oC分别提高了83.4%、10.9%。因此温度两阶段控制策略对于提高角质酶产量具有实际效果。
7.实验考察了不同初糖浓度对角质酶分批发酵的影响,实验结果表明:仅通过提高初糖浓度难以实现细胞和角质酶高产量、高得率和高生产强度的有机统一。对此实验考察了分批补料、恒速流加培养方式对B. subtilis WSH 06-07发酵生产角质酶的影响。结果表明:这两种补料培养方式都可以实现细胞和角质酶的高产。综合比较,恒速流加对于B. subtilis WSH 06-07发酵培养来说,无论是细胞得率还是角质酶的高产量、高得率和高生产强度都是较理想的选择。经过31 h的恒速流加培养,DCW达到52.76 g/L,酶活最大达545.87 U/mL。
Cutinases are mutilfunctional enzymes and belong to the family of serine hydrolases containing the so-calledα/βhydrolase fold. As a lipolytic enzyme, cutinase has been presented as a versatile enzyme showing several interesting properties for applications in industrial products and processes. Hydrolytic and synthetic reactions catalyzed by cutinase have potential use in the dairy industry for the hydrolysis of milk fat, in house hold detergents, in the oleochemical industry, in the synthesis of structured triglycerides, polymers and surfactants, in the synthesis of ingredients for personal-care products, and the synthesis of pharmaceuticals and agrochemicals containing one or more chiral centers. The application of cutinase in the textile industry has become a new research direction.Compared with the traditional technique in cotton desizing, cutinase can improve cotton wettability and fabric hydrophilicity in addition to its ability to simplify process, reduce working time, savesteam-water-electricity and protect the environment.
In this paper, recombinant Bacillus subtilis WSH 06-07, a strain could accumulate high concentration of cutinase, was selected for cutinase production. According to the principles of fermentation optimization including the optimization of culture conditions based on microbial reactions (outer factor), and the optimization based on metabolic flux analysis (quantitativeness of inner factor), a series of feasible approaches or strategies were carried out to achieve high product concentration, high yield and high productivity of cutinase in the optimization of B. WSH 06-07 cultivation processes, including:
1. Seed aging has a extremely influence in fermentation, and the experiments results were following: the log growth stage was from 9 to 22 h, and the seed aging was 11h, which was beneficial to cutinase fermentation.
2. For recombinant cell, plasmid and cellular insoluble fraction have an important effect on cutinase yield during fermentation. The experiment results showed that plasmid stability was over 90% during the whole fermentation course. Besides, there was no cellular insoluble fraction by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the objection proteins were included in supernant.
3. The required nutrients for cutinase production by B. subtilis WSH 06-07 in shaking flasks were investigated. According to the single-factor experiments and Plackett–Burman design, sucrose, trytone and MgSO4 had a profound influence in cutinase fermentation, base on which, the optimal concentration ratio among them was defined by Response Surface Methodology, and the results were summed that sucrose and MgSO4 had a positive interaction, and their effects on cell growth and cutinase production were the most important elements. Finally, the essential culture medium for cutinase production was determined to be consisted of sucrose 32.5 g/L, trptonse 37 g/L, Na2HPO4 12.54 g/L, KH2PO4 2.31 g/L and MgSO4 4.4 g/L. The environmental conditions of cutinase fermentation were also investigated and an optimal combination was developed: an initial pH of 7.5, a medium content of 75 mL medium in 500 mL flask, and an inoculum size of 5%.
4. The effect of dissolved oxygen (DO) concentration on the batch production of cutinase in a 3 L stirred fermentor by B. subtilis WSH 06-07 was studied. It was found that DCW and cutinase production, especilly for cutinase content, were not greatly influenced by the agitation rate under a fixed aeration rate. When the initial sucrose concentration was 32.5 g/L and the air flow rate was controlled at 1.5 vvm, the DO concentration, under the circumstance of the agitation rate not less than 600 r/min, was sufficient to satisfy the oxygen requirement for better cell growth and cutinae production during the batch fermentation.
5. The modes of pH control for batch cutinase fermentation are extremely different. B. subtilis WSH 06-07 was cultivated by controlling pH, cell growth and cutinase production were improved comparing to without pH control. Based on specific cell growth rate and specific cutinase formation rate under different pH condition, a two stage pH control strategy was developed, in which pH was controlled at 7.5 for the first 4h and then shift to 6.5. By the utilization of this strategy, the yield of cutinase was significantly improved, the maximal cutinase activity and the productivity were 170 U/mL and 16.9 kU/(L·h), which were increased by 122.6% and 123.2%, respectively, compared to that the condition of constant pH 7.5.
6. The effect of temperature, varied from 27 oC to 32 oC, on cell growth and cutinase production the batch fermentation of cultivation was investigated. It was found that cell growth was hastened along with the increase of temperature while lower temperature was more favorable for cutinase formation, however, plasmid lost greatly. Owing to the difference occurred in the optimal temperature for cell growth and cutinase production by thoroughly analyzing the kinetics of batch cutinase production under different temperatures, a two-stage temperature control strategy, in which the temperature was kept at 37 oC during the first 4 h of cultivation, followed by a shift from 37 oC to 30 oC, then maintained at 30 oC until the end of the cultivation, was developed in order to enhance the production of cutinase. Compared with the results under temperature 37 oC control, the two-stage temperature control strategy was confirmed to enhance the ability of cutinase synthesis. As a result, the maximum cutinase production and the productivily were 312.5 U/(mL), 13.02 KU/(L·h), increased by 83.4%、10.9% compareing to at 37 oC, respectively, Therefore, the strategy would have good feasibility in practice.
7. Effects of diverse sucrose concentration on cell growth and cutinaes production were invested by batch fermentation, a sum was found that it was diffuclt to make high product concentration, high yield and high productivity of cutinase harmony by improving sucrose concentration. As to the cultivation of B. subtilis WSH 06-07 by batch or constant fed-batch process in a 3 L stirred fermentor was studied, the resrults shown that it was efficient to realize high cell and high cutinase concentration by two kinds of feeding strateges. However, comhensive comparision of them, constant feeding fermentation was ideal choice to enhance cutinase yield wether cell yield or high product concentration, high yield and high productivity of cutinase. The DCW and cutinase activity were 52.76 g/L, 545.87 U/mL aferte 31 h constant feeding batch cultivation, respectively.
引文
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22. Papanikou E, Karamanou S, Economou A. Bacterial protein secretion through the translocase nanomachine [J]. Nature reviews, 2007, 5: 839-851.
23. Desvaux M, Hebraud M. The protein secretion systems in Listeria: inside out bacterial virulence [J]. FEMS microbiology, 2006, 30: 774-805.
24. Pukatzki S, Ma A T, Sturtevant D, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103: 1528-1533.
25. Skillman K M, Barnard T J, Peterson J H, et al. Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter [J]. Molecular microbiology, 2005, 58: 945-958.
26. Binnewies T T, Bendtsen J D, Hallin P F, et al. Genome Update: Protein secretion systems in 225 bacterial genomes [J]. Microbiology (Reading, England), 2005, 151: 1013-1016.
27. Doyle S M, Braswell E H, Teschke C M. SecA folds via a dimeric intermediate [J].Biochemistry, 2000, 39: 11667-11676.
28. Jilaveanu L B, Oliver D. SecA dimer cross-linked at its subunit interface is functional for protein translocation [J]. Journal of bacteriology, 2006, 188: 335-338.
29. Robson A, Booth A E, Gold V A, et al. A large conformational change couples the ATP binding site of SecA to the SecY protein channel [J]. Journal of molecular biology, 2007, 374: 965-976.
30. Dautin N, Bernstein H D. Protein secretion in gram-negative bacteria via the autotransporter pathway [J]. Annual review of microbiology, 2007, 61: 89-112.
31. Ling Lin F, Zi Rong X, Wei Fen L, et al. Protein secretion pathways in Bacillus subtilis implication for optimization of heterologous protein secretion[J]. Biotechnology advances, 2007, 25: 1-12.
32. Kolattududy K [P]. US pat, 5545547, 1996-08-13.
33. Tadayuki S [P]. J P pat, 08092023, 1996-09-04.
34. Kim Y H, Ahn J Y, Moon S H, et al. Biodegradation and detoxification of organophosphate insecticide malathion by Fusarium oxysporum f. sp. pisi cutinase [J]. Chemosphere, 2005, 60: 1349-1355.
35. Johan S. Laundry detergent and/or fabric care compositions comprising a modified transferase [P]. US pat, 6410498, 2002-06.
36. Fett W F G. H, Moreau R A, Osman S F, et al. Cutinase production by Streptomyces. spp [J]. Curr Microbiol, 1992, 25: 165-171.
37. Shimao M. Biodegradation of plastics [J]. Current opinion in biotechnology, 2001, 12: 242-247.
38. Murphy C A J A C. Fusarium polycaprolactone depolymerase is cutinase [J]. Applied and environmental microbiology, 1996, 62 (2): 456-460.
39. Fernando R R, Teles J M S C. Enzymatic degreasing of a solid waste from the leather industry by lipases [J]. Biotechnology letters, 2001, 23: 1159-1163.
40. Degani O, Gepstein S, Dosoretz C G. Potential use of cutinase in enzymatic scouring of cotton fiber cuticle [J]. Applied biochemistry and biotechnology, 2002, 102: 277-289.
41. Ofir D S G. Potential use of cutinase in enzymatic scouring of cotton fiber cuticle [J]. Applied biochemistry and biotechnology, 2002, 102: 277-289.
42. Andersen B K. Method of treating polyester fabrics. [P]. US pat: 1999, 11: 5997584.
43. Kellis J, James T. Enzymatic modification of the surface of a polyester fiber [P]. US pat, 6254645, 2001, 07.
44.毕凤珍,李寅,陈坚等. Thermobifida fusca产角质酶摇瓶发酵条件研究[J].应用与环境生物学报, 2005, 11(5): 608-610.
45.张守亮,华兆折,陈坚等Thermobi fida f usca WSH03211突变株合成角质酶的发酵条件[J].食品与生物技术学报, 2006, 25(5): 44-49.
46. Pedersen S, Nesgaard L, Baptista R P, et al. pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS [J]. Biopolymers, 2006, 83: 619-629.
47. Baptista R P, Santos A M, Fedorov A, et al. Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles [J]. Journal of biotechnology, 2003, 102: 241-249.
48. Egmond M R, De, V J. Fusarium solani pisi cutinase [J]. Biochimie, 2000, 82: 1015-1021.
49. Griswold K E, Mahmood N A, Iverson B L. Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm [J]. Protein expression and purification, 2003, 27: 134-142.
50 Calado C R, Ferreira B S, Fonseca M M, et al. Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain [J]. Journal of biotechnology, 2004, 109: 147-158.
51. Calado C R, Hamilton G E, Cabral J M, et al. Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae [J]. Bioseparation, 2001, 10: 87-97.
52. Parker S K, Curtin K M, Vasil M L. Purification and characterization of mycobacterial phospholipase A: an activity associated with mycobacterial cutinase [J]. Journal of bacteriology, 2007, 189: 4153-4160.
53. Ahn J Y, Kim Y H, Min J, et al. Accelerated degradation of dipentyl phthalate by Fusarium oxysporum f. sp. pisi cutinase and toxicity evaluation of its degradation products using bioluminescent bacteria [J]. Current microbiology, 2006, 52: 340-344.
54. Araujo R, Silva C, Oneill A, et al. Tailoring cutinase activity towards polyethylene terephthalate and polyamide 6,6 fibers [J]. Journal of biotechnology, 2007, 128: 849-857.
55. Goncalves A M, Serro A P, Aires-Barros M R, et al. Effects of ionic surfactants used in reversed micelles on cutinase activity and stability [J]. Biochimica et biophysica acta, 2000, 1480: 92-106.
56. Bandmann N, Collet E, Leijen J, et al. Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems [J]. Journal of biotechnology, 2000, 79: 161-172.
57. Costa M J, Cunha M T, Cabral J M, et al. Scale-up of recombinant cutinase recoveryby whole broth extraction with PEG-phosphate aqueous two-phase [J]. Bioseparation, 2000, 9: 231-238.
58. Cunha M T, Costa M J, Calado C R, et al. Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins [J]. Journal of biotechnology, 2003, 100: 55-64.
59. Fernandes S, Johansson G, Hatti-Kaul R. Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate [J]. Biotechnology and bioengineering, 2001, 73: 465-475.
60. Fernandes S, Mattiasson B, Hatti-Kaul R. Recovery of recombinant cutinase using detergent foam [J]. Biotechnology progress, 2002, 18: 116-123.
61. Kepka C, Collet E, Roos F, et al. Two-step recovery process for tryptophan tagged cutinase: interfacing aqueous two-phase extraction and hydrophobic interaction chromatography [J]. Journal of chromatography, 2005, 1075: 33-41.
62. Kwon Y, Han Z, Karatan E, et al. Antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers [J]. Analytical chemistry, 2004, 76: 5713-5720.
63. Lin T S, Kolattukudy P E. Induction of a biopolyester hydrolase (cutinase) by low levels of cutin monomers in Fusarium solani f.sp. pisi [J]. Journal of bacteriology, 1978, 133: 942-951.
64. Fragaa G M L. Production of cutinase by Fusarium oxysporum in solid-state fermentation using agro-industrial residues [J]. Journal of Biotechnology, 2007, 131: 11-16.
65. Gabriela M L F. Production of cutinase by Fusarium oxysporum in solid-state fermentation using agro-industrial residues [J]. Journal of Biotechnology, 2007, 12: 211-241.
66. Du G. C, Zhang S L, Hua Z Z, et al. Enhanced cutinase production with Thermobifida fusca by two-stage pH control strategy [J]. Biotechnology journal, 2007, 2: 365-369.
67. Calado C R, Almeida C, Cabral J M, et al. Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain [J]. Journal of bioscience and bioengineering, 2003, 96: 141-148.
68. Ferreira B S, Calado C R, Van Keulen F, et al. Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects [J]. Applied microbiology and biotechnology, 2003, 61: 69-76.
69. Ahuja S K, Ferreira G M, Moreira A R. Application of Plackett-Burman design andresponse surface methodology to achieve exponential growth for aggregated shipworm bacterium [J]. Biotechnology and bioengineering, 2004, 85: 666-675.
70. Maarten R, Egmond J D V. Fusarium solani pisi cutinase [J]. Biochimie, 2000, 82: 67-69.
71.宁正祥主编.食品成分分析手册[M].北京:中国轻工业出版社, 1998.
72. Wu Q L, Chen T, Gan Y, et al. Optimization of riboflavin production by recombinant Bacillus subtilis RH 44 using statistical designs [J] Applied microbiology and biotechnology, 2007, 76: 783-794.
73. Institute S. SAS User’s Guide, SAS Institute Inc Cary, NC, 2003.
74. Gong H R. Simple and effective system for use with response surface methodology [J] Cereal Science Today 1972, 17: 309-334.
75. Joglekar A M M A T. Product Excellence through design of experiments [J]. Cereal Foods World, 1987, 32: 857-868.
76.俞俊堂,唐孝宣.生物工艺学[M].上海:华东理工大学出版社, 1997, 75-107.
77.梅乐和,姚善泾,林东强.生化生产工艺学[M].北京:科学出版社, 1999, 186-201.
78.李寅,陈坚.发酵过程优化原理与实践[M].北京:化学工业出版社, 2002, 24-28.
79.熊宗贵.发酵工艺原理[M].北京:中国医药科技出版社, 1999.
80.李友荣,储炬.现代工业发酵调控学[M].北京:化学工业出版社, 2002, 230-301.
81. Calado C R, Monteiro S M S, Cabral J M S, et al. Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiae [J]. Biosci. Bioeng, 2002, 93: 354-359.
82.高海军,李寅,陈坚.高细胞密度发酵技术[M].化学工业出版社, 2006, 198-199.
83. Ning S A. The effect of carbon dioxide gas on the growth of Candida utilis yeasts during continuous cultivation chemostat [J]. Mikrobiologiia, 1976, 45(1): 67-72.
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22. Papanikou E, Karamanou S, Economou A. Bacterial protein secretion through the translocase nanomachine [J]. Nature reviews, 2007, 5: 839-851.
23. Desvaux M, Hebraud M. The protein secretion systems in Listeria: inside out bacterial virulence [J]. FEMS microbiology, 2006, 30: 774-805.
24. Pukatzki S, Ma A T, Sturtevant D, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103: 1528-1533.
25. Skillman K M, Barnard T J, Peterson J H, et al. Efficient secretion of a folded protein domain by a monomeric bacterial autotransporter [J]. Molecular microbiology, 2005, 58: 945-958.
26. Binnewies T T, Bendtsen J D, Hallin P F, et al. Genome Update: Protein secretion systems in 225 bacterial genomes [J]. Microbiology (Reading, England), 2005, 151: 1013-1016.
27. Doyle S M, Braswell E H, Teschke C M. SecA folds via a dimeric intermediate [J].Biochemistry, 2000, 39: 11667-11676.
28. Jilaveanu L B, Oliver D. SecA dimer cross-linked at its subunit interface is functional for protein translocation [J]. Journal of bacteriology, 2006, 188: 335-338.
29. Robson A, Booth A E, Gold V A, et al. A large conformational change couples the ATP binding site of SecA to the SecY protein channel [J]. Journal of molecular biology, 2007, 374: 965-976.
30. Dautin N, Bernstein H D. Protein secretion in gram-negative bacteria via the autotransporter pathway [J]. Annual review of microbiology, 2007, 61: 89-112.
31. Ling Lin F, Zi Rong X, Wei Fen L, et al. Protein secretion pathways in Bacillus subtilis implication for optimization of heterologous protein secretion[J]. Biotechnology advances, 2007, 25: 1-12.
32. Kolattududy K [P]. US pat, 5545547, 1996-08-13.
33. Tadayuki S [P]. J P pat, 08092023, 1996-09-04.
34. Kim Y H, Ahn J Y, Moon S H, et al. Biodegradation and detoxification of organophosphate insecticide malathion by Fusarium oxysporum f. sp. pisi cutinase [J]. Chemosphere, 2005, 60: 1349-1355.
35. Johan S. Laundry detergent and/or fabric care compositions comprising a modified transferase [P]. US pat, 6410498, 2002-06.
36. Fett W F G. H, Moreau R A, Osman S F, et al. Cutinase production by Streptomyces. spp [J]. Curr Microbiol, 1992, 25: 165-171.
37. Shimao M. Biodegradation of plastics [J]. Current opinion in biotechnology, 2001, 12: 242-247.
38. Murphy C A J A C. Fusarium polycaprolactone depolymerase is cutinase [J]. Applied and environmental microbiology, 1996, 62 (2): 456-460.
39. Fernando R R, Teles J M S C. Enzymatic degreasing of a solid waste from the leather industry by lipases [J]. Biotechnology letters, 2001, 23: 1159-1163.
40. Degani O, Gepstein S, Dosoretz C G. Potential use of cutinase in enzymatic scouring of cotton fiber cuticle [J]. Applied biochemistry and biotechnology, 2002, 102: 277-289.
41. Ofir D S G. Potential use of cutinase in enzymatic scouring of cotton fiber cuticle [J]. Applied biochemistry and biotechnology, 2002, 102: 277-289.
42. Andersen B K. Method of treating polyester fabrics. [P]. US pat: 1999, 11: 5997584.
43. Kellis J, James T. Enzymatic modification of the surface of a polyester fiber [P]. US pat, 6254645, 2001, 07.
44.毕凤珍,李寅,陈坚等. Thermobifida fusca产角质酶摇瓶发酵条件研究[J].应用与环境生物学报, 2005, 11(5): 608-610.
45.张守亮,华兆折,陈坚等Thermobi fida f usca WSH03211突变株合成角质酶的发酵条件[J].食品与生物技术学报, 2006, 25(5): 44-49.
46. Pedersen S, Nesgaard L, Baptista R P, et al. pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS [J]. Biopolymers, 2006, 83: 619-629.
47. Baptista R P, Santos A M, Fedorov A, et al. Activity, conformation and dynamics of cutinase adsorbed on poly(methyl methacrylate) latex particles [J]. Journal of biotechnology, 2003, 102: 241-249.
48. Egmond M R, De, V J. Fusarium solani pisi cutinase [J]. Biochimie, 2000, 82: 1015-1021.
49. Griswold K E, Mahmood N A, Iverson B L. Effects of codon usage versus putative 5'-mRNA structure on the expression of Fusarium solani cutinase in the Escherichia coli cytoplasm [J]. Protein expression and purification, 2003, 27: 134-142.
50 Calado C R, Ferreira B S, Fonseca M M, et al. Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain [J]. Journal of biotechnology, 2004, 109: 147-158.
51. Calado C R, Hamilton G E, Cabral J M, et al. Direct product sequestration of a recombinant cutinase from batch fermentations of Saccharomyces cerevisiae [J]. Bioseparation, 2001, 10: 87-97.
52. Parker S K, Curtin K M, Vasil M L. Purification and characterization of mycobacterial phospholipase A: an activity associated with mycobacterial cutinase [J]. Journal of bacteriology, 2007, 189: 4153-4160.
53. Ahn J Y, Kim Y H, Min J, et al. Accelerated degradation of dipentyl phthalate by Fusarium oxysporum f. sp. pisi cutinase and toxicity evaluation of its degradation products using bioluminescent bacteria [J]. Current microbiology, 2006, 52: 340-344.
54. Araujo R, Silva C, Oneill A, et al. Tailoring cutinase activity towards polyethylene terephthalate and polyamide 6,6 fibers [J]. Journal of biotechnology, 2007, 128: 849-857.
55. Goncalves A M, Serro A P, Aires-Barros M R, et al. Effects of ionic surfactants used in reversed micelles on cutinase activity and stability [J]. Biochimica et biophysica acta, 2000, 1480: 92-106.
56. Bandmann N, Collet E, Leijen J, et al. Genetic engineering of the Fusarium solani pisi lipase cutinase for enhanced partitioning in PEG-phosphate aqueous two-phase systems [J]. Journal of biotechnology, 2000, 79: 161-172.
57. Costa M J, Cunha M T, Cabral J M, et al. Scale-up of recombinant cutinase recoveryby whole broth extraction with PEG-phosphate aqueous two-phase [J]. Bioseparation, 2000, 9: 231-238.
58. Cunha M T, Costa M J, Calado C R, et al. Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins [J]. Journal of biotechnology, 2003, 100: 55-64.
59. Fernandes S, Johansson G, Hatti-Kaul R. Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate [J]. Biotechnology and bioengineering, 2001, 73: 465-475.
60. Fernandes S, Mattiasson B, Hatti-Kaul R. Recovery of recombinant cutinase using detergent foam [J]. Biotechnology progress, 2002, 18: 116-123.
61. Kepka C, Collet E, Roos F, et al. Two-step recovery process for tryptophan tagged cutinase: interfacing aqueous two-phase extraction and hydrophobic interaction chromatography [J]. Journal of chromatography, 2005, 1075: 33-41.
62. Kwon Y, Han Z, Karatan E, et al. Antibody arrays prepared by cutinase-mediated immobilization on self-assembled monolayers [J]. Analytical chemistry, 2004, 76: 5713-5720.
63. Lin T S, Kolattukudy P E. Induction of a biopolyester hydrolase (cutinase) by low levels of cutin monomers in Fusarium solani f.sp. pisi [J]. Journal of bacteriology, 1978, 133: 942-951.
64. Fragaa G M L. Production of cutinase by Fusarium oxysporum in solid-state fermentation using agro-industrial residues [J]. Journal of Biotechnology, 2007, 131: 11-16.
65. Gabriela M L F. Production of cutinase by Fusarium oxysporum in solid-state fermentation using agro-industrial residues [J]. Journal of Biotechnology, 2007, 12: 211-241.
66. Du G. C, Zhang S L, Hua Z Z, et al. Enhanced cutinase production with Thermobifida fusca by two-stage pH control strategy [J]. Biotechnology journal, 2007, 2: 365-369.
67. Calado C R, Almeida C, Cabral J M, et al. Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain [J]. Journal of bioscience and bioengineering, 2003, 96: 141-148.
68. Ferreira B S, Calado C R, Van Keulen F, et al. Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects [J]. Applied microbiology and biotechnology, 2003, 61: 69-76.
69. Ahuja S K, Ferreira G M, Moreira A R. Application of Plackett-Burman design andresponse surface methodology to achieve exponential growth for aggregated shipworm bacterium [J]. Biotechnology and bioengineering, 2004, 85: 666-675.
70. Maarten R, Egmond J D V. Fusarium solani pisi cutinase [J]. Biochimie, 2000, 82: 67-69.
71.宁正祥主编.食品成分分析手册[M].北京:中国轻工业出版社, 1998.
72. Wu Q L, Chen T, Gan Y, et al. Optimization of riboflavin production by recombinant Bacillus subtilis RH 44 using statistical designs [J] Applied microbiology and biotechnology, 2007, 76: 783-794.
73. Institute S. SAS User’s Guide, SAS Institute Inc Cary, NC, 2003.
74. Gong H R. Simple and effective system for use with response surface methodology [J] Cereal Science Today 1972, 17: 309-334.
75. Joglekar A M M A T. Product Excellence through design of experiments [J]. Cereal Foods World, 1987, 32: 857-868.
76.俞俊堂,唐孝宣.生物工艺学[M].上海:华东理工大学出版社, 1997, 75-107.
77.梅乐和,姚善泾,林东强.生化生产工艺学[M].北京:科学出版社, 1999, 186-201.
78.李寅,陈坚.发酵过程优化原理与实践[M].北京:化学工业出版社, 2002, 24-28.
79.熊宗贵.发酵工艺原理[M].北京:中国医药科技出版社, 1999.
80.李友荣,储炬.现代工业发酵调控学[M].北京:化学工业出版社, 2002, 230-301.
81. Calado C R, Monteiro S M S, Cabral J M S, et al. Effect of pre-fermentation on the production of cutinase by a recombinant Saccharomyces cerevisiae [J]. Biosci. Bioeng, 2002, 93: 354-359.
82.高海军,李寅,陈坚.高细胞密度发酵技术[M].化学工业出版社, 2006, 198-199.
83. Ning S A. The effect of carbon dioxide gas on the growth of Candida utilis yeasts during continuous cultivation chemostat [J]. Mikrobiologiia, 1976, 45(1): 67-72.