Streptomyces sp.M-Z18发酵生产ε-聚赖氨酸的碳源供给策略与过程调控研究
|
摘要
ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是微生物通过非核糖体合成方式催化25~35个L-赖氨酸单体,以α-COOH和ε-NH_2相互缩合的方式而形成的一种同型L-赖氨酸链状聚合物,分子量一般为2500~4500 Da。由于其具有抑菌性广、水溶性强、热稳定性好、pH值使用范围广以及安全性高等优点,目前主要作为食品防腐剂应用在食品防腐和保鲜领域,已经在日本形成数十亿日元市场。同时,作为一种阳离子型生物聚合物,ε-PL还广泛应用于生物材料和药物载体研究。因此,研究开发ε-PL发酵技术以提高ε-PL发酵水平,实现低成本、高效率ε-PL发酵生产,为我国建立ε-PL产业提供技术支撑。
本论文利用一株ε-PL高产菌Streptomyces sp. M-Z18,提出了工业甘油、甘油和葡萄糖混合物、前体L-赖氨酸作为发酵底物的供给策略,通过发酵过程优化与调控技术,显著提高了ε-PL发酵水平;考察了甘油和葡萄糖作碳源引起ε-PL合成的差异;研究了Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL的机制;建立了转化前体L-赖氨酸耦合发酵生产ε-PL的工艺。具体研究内容如下:
(1)在确定工业甘油作为发酵碳源的前提下,筛选出相匹配的牛肉浸膏、(NH4)2SO_4、KH_2PO_4、K_2HPO_4、MgSO_4·7H_2O和FeSO_4·7H_2O作为Streptomyces sp. M-Z18合成ε-PL的营养成分;借助Plackett-Burman设计确定了甘油、硫酸铵和K_2HPO_4是影响ε-PL合成的关键营养成分;利用响应面分析构建出最优营养组合为:甘油60 g/L,(NH4)2SO_4 5 g/L,牛肉浸膏10 g/L,KH_2PO_4 4 g/L,MgSO_4·7H_2O 0.8 g/L,FeSO_4·7H_2O 0.05 g/L。利用该优化培养基自然发酵生产ε-PL(pH值不控制),摇瓶ε-PL产量达到2.27 g/L,菌体干重达到7.75 g/L;5 L发酵罐分批发酵ε-PL产量为3.5 g/L,是出发培养基(M3G培养基)的3倍。
(2)在研究pH值对ε-PL发酵过程影响的基础上,以最大ε-PL比合成速率为调控目标,建立了一种利用甘油为碳源的两阶段pH值控制策略发酵生产ε-PL工艺。该工艺使得ε-PL分批发酵产量和产率达到9.13 g/L和4.76 g/L/d,较最优单一pH值控制发酵(pH3.5)分别提高16.6%和52.1%。结合甘油和硫酸铵流加技术,ε-PL发酵产量达到30.11 g/L,ε-PL产率为4.18 g/L/d,转化率达到13.2%。根据葡萄糖和甘油发酵生产ε-PL的互补性优点,提出了甘油-葡萄糖双碳源发酵生产ε-PL工艺。研究发现,Streptomyces sp. M-Z18不仅能够同步消耗甘油和葡萄糖用于菌体生长和ε-PL合成,并且显著缩短了发酵时间,提高了ε-PL合成速率。当葡萄糖和甘油混合比例为30/30(w/w)时,发酵速率比任何单一碳源都显著提高:较葡萄糖快25.4%,较甘油快32.8%。甘油-葡萄糖双碳源(30/30,w/w)补料-分批发酵使得ε-PL发酵产量达到35.14 g/L,ε-PL产率为4.85 g/L/d,转化率为12.1%。
(3)在考察甘油和葡萄糖对Streptomyces sp. M-Z18合成ε-PL影响时,发现甘油和葡萄糖引起了ε-PL合成的显著差异。通过对两种碳源下ε-PL发酵过程关键酶活性变化考察,发现磷酸烯醇式丙酮酸羧化酶(PEPC)在以葡萄糖为碳源的发酵过程中表现出较高活性,而天门冬氨酸激酶(ASPK)和ε-PL合成酶(Pls)在以甘油为碳源的发酵过程中活性较高;两种碳源下ε-PL发酵过程代谢通量分析表明:以甘油为碳源减小了杂氨基酸合成和菌体合成通量,增大了磷酸戊糖途径、TCA循环回补途径、天门冬氨酸族氨基酸合成途径和L-赖氨酸合成等途径的代谢流量,而TCA循环代谢通量基本保持不变,这说明甘油作碳源能够使得更多的碳代谢流流向ε-PL的合成途径,减少了代谢副产物的生成,提高了产物转化率;考察不同还原度碳源(葡萄糖酸、葡萄糖和山梨醇)对菌体生长和ε-PL合成影响,发现碳源还原度越接近前体L-赖氨酸还原度越有利于ε-PL合成。在上述实验结论的基础上,提出了甘油优于葡萄糖合成ε-PL的可能机制在于:①甘油作为小分子多元醇通过水取代机理稳定了酶的空间构象而提高了ε-PL合成途径中天门冬氨酸激酶和ε-PL合成酶活性,从而增大了ε-PL合成途径代谢通量,提高了ε-PL产量;②甘油与L-赖氨酸还原度相同,实现了底物和产物前体的氧化还原平衡,减少了代谢副产物生成,提高了产物转化率;③甘油比葡萄糖为ε-PL合成提供了更多的能量辅因子ATP。
(4)利用两阶段培养方法考察前体L-赖氨酸对ε-PL合成影响时,发现低浓度(1~2 g/L)L-赖氨酸能够显著促进ε-PL合成。利用同位素标记方法(L-(U-13C)赖氨酸)和核磁共振分析技术(NMR)研究L-赖氨酸转化机制时,发现L-赖氨酸是作为整体直接参与ε-PL合成,且细胞转化前体L-赖氨酸比例为40%左右,该比例不会随着前体L-赖氨酸浓度的增加而提高。在对甘油、pH值、L-赖氨酸和细胞膜通透性等因素的考察基础上,建立了Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL体系并强化了该转化过程。通过对转化过程的研究,发现转化体系合成ε-PL来源于两条途径:①转化外源L-赖氨酸合成ε-PL途径;②转化甘油形成内源L-赖氨酸合成ε-PL途径。
(5)在考察pH值对5 L发酵罐规模Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL过程影响的基础上,建立了5 L发酵罐规模补料-分批转化体系,使得ε-PL合成能力达到15 g/L。前体L-赖氨酸添加方式对ε-PL发酵过程影响研究结果表明,在发酵后期添加1 g/L L-赖氨酸有利于实现发酵与前体L-赖氨酸转化的同步进行。结合甘油单一碳源和甘油-葡萄糖双碳源发酵生产ε-PL工艺,初步建立转化前体L-赖氨酸耦合发酵生产ε-PL工艺。两种ε-PL生产工艺分别实现ε-PL发酵产量达到33.76 g/L和37.6 g/L。
ε-poly-L-lysine (ε-PL), consists of 25~35 L-lysine residues with linkages betweenα-carboxyl groups andε-amino groups, is a homopolymer produced by microbial nonribosomal peptide synthetases (NRPSs).ε-PL shows strong antimicrobial activity against a wide spectrum of microorganisms (including bacteria and fungi), water soluble, thermalstability, wide used range of pH and safety. It is mainly used as a food preservative in several countries, especially in Japan. At present,ε-PL has been formed billions of yen in Japan market. Therefore, it is important to develop an efficientε-PL fermentation strategy for its industrial manufacture in China.
In this dissertation, a highε-PL producing strain, Streptomyces sp. M-Z18 was used as a model to demonstrate the effect of glycerol and mixed with glucose as carbon sources onε-PL production by process optimization and regulation. Meanwhile, the differences between glycerol and glucose onε-PL production were investigated. Based on the well understanding of mechanisms in theε-PL formation from precursor L-lysine, whole-cell biotransformation method forε-PL production directly from L-lysine was established. The main results were described as follows:
(1) In order to improveε-PL production of Streptomyces sp. M-Z18, the effects of nutritional conditions (carbon sources, nitrogen sources, phosphate salts and metal ions) onε-PL production and cell growth were investigated in shaking flask. The results of one-time-one-factor showed that glycerol, beef extract, (NH_4)2SO_4, KH_2PO_4, K2HPO_4, MgSO_4·7H_2O and FeSO_4·7H_2O were the optimal nutritions forε-PL production and cell growth; Plackett-Burman design was determined glycerol, (NH_4)2SO_4 and K2HPO_4 were the key nutritions forε-PL production. The optimized conditions were determined by using response surface methodology as follows: 60 g/L glycerol,5 g/L (NH_4)2SO_4,10 g/L beef extract,4 g/L KH_2PO_4,0.8 g/L MgSO_4·7H_2O,0.05 g/L FeSO_4·7H_2O. Under the optimized fermentation conditions,ε-PL production and DCW were achieved at 2.27 g/L and 7.75 g/L, respectively, in shake-flask fermentation. Furthermore, the batch fermentation results showed that the production ofε-PL was yielded 3.5 g/L after 96 h in 5 L fermenter under pH uncontrolled strategy, it was enhanced 3-fold than M3G medium.
(2) Based on the effect of pH onε-PL production, this dissertation developed a novel two-stage pH control strategy under the direction by the highest specificε-PL formation rate. By applying this strategy, the maximalε-PL concentration and productivity had reached at 9.13 g/L and 4.76 g/L/day, respectively, it is higher by 16.6% and 52.1% than the optimal one-stage pH control process (pH3.5). Combined with glycerol and (NH_4)2SO_4 feeding strategy, fed-batch fermentation was performed. After 173 h fermentation, theε-PL concentration, productivity and yield reached at 30.11 g/L, 4.18 g/L/day and 13.2%, respectively. Furthermore, due to the complementary advantages of glucose and glycerol forε-PL fermentation, the effect of glucose-glycerol mixed carbon sources onε-PL fermentation were investigated. The results of experiment showed that glycerol and glucose simultaneously consumed by Streptomyces sp. M-Z18 for cell growth andε-PL synthesis. In addition, glycerol-glucose fermentation could significantly reduce the fermentation time and improve theε-PL productivity much. When the ratio of glycerol to glucose at 30/30 (w/w), the batch fermentation time was shorten than single carbon source fermentation by 25.4% (glucose) and 32.8% (glycerol). Finally, fed-batch fermentation with glucose and glycerol as a mixed carbon source (30/30,w/w) achieved maximumε-PL concentration, productivity and yield of 35.14 g/L, 4.85 g/L/d and 12.1%, respectively.
(3) When glycerol and glucose were used as carbon sources forε-PL production, it is found that glycerol and glucose make significant differences onε-PL synthesis. To explain these differences detailed, key enzymes activities, metabolic flux analysis (MFA) and reduction degree of carbon sources were investigated. Results from the key enzymes evaluation showed that phosphoenolpyruvate carboxylase activity in the glucose medium was higher than glycerol, however, the activities of aspartate kinase andε-PL synthase in glycerol was superior than glucose. MFA showed that glycerol as carbon source was reduced the flux of amino acids synthesis (except L-lysine) and cell growth, increased the flux of pentose phosphate pathway, TCA cycle anaplerotic reaction, aspartic acid family amino acid biosynthesis and L-lysine pathway. However, the flux of TCA cycle remained unchanged compared with glucose as carbon source. It was indicated that glycerol as carbon source improved the flux of target metabolic and reduced the by-product. The effect of different reduction degrees of carbon sources (gluconic acid, glucose and sorbitol) on cell growth andε-PL synthesis showed that reduction degree played an important role inε-PL production. Based on the above experimental results, the possible mechanisms on glycerol superior than glucose as carbon source forε-PL production were proposed as follows:①glycerol as the polyol molecules replaced water for supporting the spatial structure of the enzymes and thereby improved the aspartate kinase andε-PL synthase activities. Finally, it had increased the flux ofε-PL synthesis pathway and enhancement ofε-PL production;②glycerol and L-lysine have the same degree of reduction, so it could reduce the metabolic by-products generated to keep redox balance and improve the yield ofε-PL;③glycerol have more reduction degree than glucose, so it could provide more ATP forε-PL synthesis.
(4) To investigate the effects of precursor L-lysine addition concentration onε-PL synthesis, two-stage culture method was performed and found that low concentrations of L-lysine could significantly promote the production ofε-PL. To reveal the relationship between L-lysine and enhancement ofε-PL production, isotope labeling method (L-(U-13C) lysine) and nuclear magnetic resonance (NMR) were used and found that 40% L-lysine as a whole directly involved in theε-PL synthesis. Moreover, this ratio is not improved when external L-lysine concentration is increased. When the effects of L-lysine, glycerol, pH and cell membrane permeability on Streptomyces sp. M-Z18 whole-cell biotransformation process, the system of whole-cell conversion of L-lysine toε-PL were established. Based on the above experimental results,ε-PL synthesis in the system derived from two ways:①conversion of exogenous L-lysine;②conversion of glycerol to endogenous L-lysine.
(5) In order to develop of fed-batch whole-cell biotransformation system in 5 L fermentor, the effect of pH on the process of whole-cell biotransformation was investigated and the highestε-PL production by 15 g/L was achieved at the optimal culture conditions. We have investigated the effect of the ways of added L-lysine onε-PL production and found that addition 1 g/L L-lysine at the late of fermentation was benefit for coupled fermentation with biotransformation. Based on the two-stage pH control strategy and glycerol-glucose mixed carbon source fermentation strategy, we developed two types of coupled fermentation with biotransformation strategies forε-PL production and achievedε-PL production of 33.76 g/L and 37.6 g/L, respectively.
本论文利用一株ε-PL高产菌Streptomyces sp. M-Z18,提出了工业甘油、甘油和葡萄糖混合物、前体L-赖氨酸作为发酵底物的供给策略,通过发酵过程优化与调控技术,显著提高了ε-PL发酵水平;考察了甘油和葡萄糖作碳源引起ε-PL合成的差异;研究了Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL的机制;建立了转化前体L-赖氨酸耦合发酵生产ε-PL的工艺。具体研究内容如下:
(1)在确定工业甘油作为发酵碳源的前提下,筛选出相匹配的牛肉浸膏、(NH4)2SO_4、KH_2PO_4、K_2HPO_4、MgSO_4·7H_2O和FeSO_4·7H_2O作为Streptomyces sp. M-Z18合成ε-PL的营养成分;借助Plackett-Burman设计确定了甘油、硫酸铵和K_2HPO_4是影响ε-PL合成的关键营养成分;利用响应面分析构建出最优营养组合为:甘油60 g/L,(NH4)2SO_4 5 g/L,牛肉浸膏10 g/L,KH_2PO_4 4 g/L,MgSO_4·7H_2O 0.8 g/L,FeSO_4·7H_2O 0.05 g/L。利用该优化培养基自然发酵生产ε-PL(pH值不控制),摇瓶ε-PL产量达到2.27 g/L,菌体干重达到7.75 g/L;5 L发酵罐分批发酵ε-PL产量为3.5 g/L,是出发培养基(M3G培养基)的3倍。
(2)在研究pH值对ε-PL发酵过程影响的基础上,以最大ε-PL比合成速率为调控目标,建立了一种利用甘油为碳源的两阶段pH值控制策略发酵生产ε-PL工艺。该工艺使得ε-PL分批发酵产量和产率达到9.13 g/L和4.76 g/L/d,较最优单一pH值控制发酵(pH3.5)分别提高16.6%和52.1%。结合甘油和硫酸铵流加技术,ε-PL发酵产量达到30.11 g/L,ε-PL产率为4.18 g/L/d,转化率达到13.2%。根据葡萄糖和甘油发酵生产ε-PL的互补性优点,提出了甘油-葡萄糖双碳源发酵生产ε-PL工艺。研究发现,Streptomyces sp. M-Z18不仅能够同步消耗甘油和葡萄糖用于菌体生长和ε-PL合成,并且显著缩短了发酵时间,提高了ε-PL合成速率。当葡萄糖和甘油混合比例为30/30(w/w)时,发酵速率比任何单一碳源都显著提高:较葡萄糖快25.4%,较甘油快32.8%。甘油-葡萄糖双碳源(30/30,w/w)补料-分批发酵使得ε-PL发酵产量达到35.14 g/L,ε-PL产率为4.85 g/L/d,转化率为12.1%。
(3)在考察甘油和葡萄糖对Streptomyces sp. M-Z18合成ε-PL影响时,发现甘油和葡萄糖引起了ε-PL合成的显著差异。通过对两种碳源下ε-PL发酵过程关键酶活性变化考察,发现磷酸烯醇式丙酮酸羧化酶(PEPC)在以葡萄糖为碳源的发酵过程中表现出较高活性,而天门冬氨酸激酶(ASPK)和ε-PL合成酶(Pls)在以甘油为碳源的发酵过程中活性较高;两种碳源下ε-PL发酵过程代谢通量分析表明:以甘油为碳源减小了杂氨基酸合成和菌体合成通量,增大了磷酸戊糖途径、TCA循环回补途径、天门冬氨酸族氨基酸合成途径和L-赖氨酸合成等途径的代谢流量,而TCA循环代谢通量基本保持不变,这说明甘油作碳源能够使得更多的碳代谢流流向ε-PL的合成途径,减少了代谢副产物的生成,提高了产物转化率;考察不同还原度碳源(葡萄糖酸、葡萄糖和山梨醇)对菌体生长和ε-PL合成影响,发现碳源还原度越接近前体L-赖氨酸还原度越有利于ε-PL合成。在上述实验结论的基础上,提出了甘油优于葡萄糖合成ε-PL的可能机制在于:①甘油作为小分子多元醇通过水取代机理稳定了酶的空间构象而提高了ε-PL合成途径中天门冬氨酸激酶和ε-PL合成酶活性,从而增大了ε-PL合成途径代谢通量,提高了ε-PL产量;②甘油与L-赖氨酸还原度相同,实现了底物和产物前体的氧化还原平衡,减少了代谢副产物生成,提高了产物转化率;③甘油比葡萄糖为ε-PL合成提供了更多的能量辅因子ATP。
(4)利用两阶段培养方法考察前体L-赖氨酸对ε-PL合成影响时,发现低浓度(1~2 g/L)L-赖氨酸能够显著促进ε-PL合成。利用同位素标记方法(L-(U-13C)赖氨酸)和核磁共振分析技术(NMR)研究L-赖氨酸转化机制时,发现L-赖氨酸是作为整体直接参与ε-PL合成,且细胞转化前体L-赖氨酸比例为40%左右,该比例不会随着前体L-赖氨酸浓度的增加而提高。在对甘油、pH值、L-赖氨酸和细胞膜通透性等因素的考察基础上,建立了Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL体系并强化了该转化过程。通过对转化过程的研究,发现转化体系合成ε-PL来源于两条途径:①转化外源L-赖氨酸合成ε-PL途径;②转化甘油形成内源L-赖氨酸合成ε-PL途径。
(5)在考察pH值对5 L发酵罐规模Streptomyces sp. M-Z18转化前体L-赖氨酸合成ε-PL过程影响的基础上,建立了5 L发酵罐规模补料-分批转化体系,使得ε-PL合成能力达到15 g/L。前体L-赖氨酸添加方式对ε-PL发酵过程影响研究结果表明,在发酵后期添加1 g/L L-赖氨酸有利于实现发酵与前体L-赖氨酸转化的同步进行。结合甘油单一碳源和甘油-葡萄糖双碳源发酵生产ε-PL工艺,初步建立转化前体L-赖氨酸耦合发酵生产ε-PL工艺。两种ε-PL生产工艺分别实现ε-PL发酵产量达到33.76 g/L和37.6 g/L。
ε-poly-L-lysine (ε-PL), consists of 25~35 L-lysine residues with linkages betweenα-carboxyl groups andε-amino groups, is a homopolymer produced by microbial nonribosomal peptide synthetases (NRPSs).ε-PL shows strong antimicrobial activity against a wide spectrum of microorganisms (including bacteria and fungi), water soluble, thermalstability, wide used range of pH and safety. It is mainly used as a food preservative in several countries, especially in Japan. At present,ε-PL has been formed billions of yen in Japan market. Therefore, it is important to develop an efficientε-PL fermentation strategy for its industrial manufacture in China.
In this dissertation, a highε-PL producing strain, Streptomyces sp. M-Z18 was used as a model to demonstrate the effect of glycerol and mixed with glucose as carbon sources onε-PL production by process optimization and regulation. Meanwhile, the differences between glycerol and glucose onε-PL production were investigated. Based on the well understanding of mechanisms in theε-PL formation from precursor L-lysine, whole-cell biotransformation method forε-PL production directly from L-lysine was established. The main results were described as follows:
(1) In order to improveε-PL production of Streptomyces sp. M-Z18, the effects of nutritional conditions (carbon sources, nitrogen sources, phosphate salts and metal ions) onε-PL production and cell growth were investigated in shaking flask. The results of one-time-one-factor showed that glycerol, beef extract, (NH_4)2SO_4, KH_2PO_4, K2HPO_4, MgSO_4·7H_2O and FeSO_4·7H_2O were the optimal nutritions forε-PL production and cell growth; Plackett-Burman design was determined glycerol, (NH_4)2SO_4 and K2HPO_4 were the key nutritions forε-PL production. The optimized conditions were determined by using response surface methodology as follows: 60 g/L glycerol,5 g/L (NH_4)2SO_4,10 g/L beef extract,4 g/L KH_2PO_4,0.8 g/L MgSO_4·7H_2O,0.05 g/L FeSO_4·7H_2O. Under the optimized fermentation conditions,ε-PL production and DCW were achieved at 2.27 g/L and 7.75 g/L, respectively, in shake-flask fermentation. Furthermore, the batch fermentation results showed that the production ofε-PL was yielded 3.5 g/L after 96 h in 5 L fermenter under pH uncontrolled strategy, it was enhanced 3-fold than M3G medium.
(2) Based on the effect of pH onε-PL production, this dissertation developed a novel two-stage pH control strategy under the direction by the highest specificε-PL formation rate. By applying this strategy, the maximalε-PL concentration and productivity had reached at 9.13 g/L and 4.76 g/L/day, respectively, it is higher by 16.6% and 52.1% than the optimal one-stage pH control process (pH3.5). Combined with glycerol and (NH_4)2SO_4 feeding strategy, fed-batch fermentation was performed. After 173 h fermentation, theε-PL concentration, productivity and yield reached at 30.11 g/L, 4.18 g/L/day and 13.2%, respectively. Furthermore, due to the complementary advantages of glucose and glycerol forε-PL fermentation, the effect of glucose-glycerol mixed carbon sources onε-PL fermentation were investigated. The results of experiment showed that glycerol and glucose simultaneously consumed by Streptomyces sp. M-Z18 for cell growth andε-PL synthesis. In addition, glycerol-glucose fermentation could significantly reduce the fermentation time and improve theε-PL productivity much. When the ratio of glycerol to glucose at 30/30 (w/w), the batch fermentation time was shorten than single carbon source fermentation by 25.4% (glucose) and 32.8% (glycerol). Finally, fed-batch fermentation with glucose and glycerol as a mixed carbon source (30/30,w/w) achieved maximumε-PL concentration, productivity and yield of 35.14 g/L, 4.85 g/L/d and 12.1%, respectively.
(3) When glycerol and glucose were used as carbon sources forε-PL production, it is found that glycerol and glucose make significant differences onε-PL synthesis. To explain these differences detailed, key enzymes activities, metabolic flux analysis (MFA) and reduction degree of carbon sources were investigated. Results from the key enzymes evaluation showed that phosphoenolpyruvate carboxylase activity in the glucose medium was higher than glycerol, however, the activities of aspartate kinase andε-PL synthase in glycerol was superior than glucose. MFA showed that glycerol as carbon source was reduced the flux of amino acids synthesis (except L-lysine) and cell growth, increased the flux of pentose phosphate pathway, TCA cycle anaplerotic reaction, aspartic acid family amino acid biosynthesis and L-lysine pathway. However, the flux of TCA cycle remained unchanged compared with glucose as carbon source. It was indicated that glycerol as carbon source improved the flux of target metabolic and reduced the by-product. The effect of different reduction degrees of carbon sources (gluconic acid, glucose and sorbitol) on cell growth andε-PL synthesis showed that reduction degree played an important role inε-PL production. Based on the above experimental results, the possible mechanisms on glycerol superior than glucose as carbon source forε-PL production were proposed as follows:①glycerol as the polyol molecules replaced water for supporting the spatial structure of the enzymes and thereby improved the aspartate kinase andε-PL synthase activities. Finally, it had increased the flux ofε-PL synthesis pathway and enhancement ofε-PL production;②glycerol and L-lysine have the same degree of reduction, so it could reduce the metabolic by-products generated to keep redox balance and improve the yield ofε-PL;③glycerol have more reduction degree than glucose, so it could provide more ATP forε-PL synthesis.
(4) To investigate the effects of precursor L-lysine addition concentration onε-PL synthesis, two-stage culture method was performed and found that low concentrations of L-lysine could significantly promote the production ofε-PL. To reveal the relationship between L-lysine and enhancement ofε-PL production, isotope labeling method (L-(U-13C) lysine) and nuclear magnetic resonance (NMR) were used and found that 40% L-lysine as a whole directly involved in theε-PL synthesis. Moreover, this ratio is not improved when external L-lysine concentration is increased. When the effects of L-lysine, glycerol, pH and cell membrane permeability on Streptomyces sp. M-Z18 whole-cell biotransformation process, the system of whole-cell conversion of L-lysine toε-PL were established. Based on the above experimental results,ε-PL synthesis in the system derived from two ways:①conversion of exogenous L-lysine;②conversion of glycerol to endogenous L-lysine.
(5) In order to develop of fed-batch whole-cell biotransformation system in 5 L fermentor, the effect of pH on the process of whole-cell biotransformation was investigated and the highestε-PL production by 15 g/L was achieved at the optimal culture conditions. We have investigated the effect of the ways of added L-lysine onε-PL production and found that addition 1 g/L L-lysine at the late of fermentation was benefit for coupled fermentation with biotransformation. Based on the two-stage pH control strategy and glycerol-glucose mixed carbon source fermentation strategy, we developed two types of coupled fermentation with biotransformation strategies forε-PL production and achievedε-PL production of 33.76 g/L and 37.6 g/L, respectively.
引文
[1] Shima S, Sakai H. Polylysine produced by Streptomyces [J]. Agric Biol Chem, 1977, 41:1807-1809.
[2]施庆珊,陈仪本,欧阳友生.ε-聚赖氨酸的微生物合成与降解[J].生物技术, 2004, 14:77-79.
[3] Hiraki J, Ichikawa T, Ninomiya S, et al. Use of ADME studies to confirm the safety ofε-polylysine as a preservative in food [J]. Regul Toxicol Pharm, 2003,37:328-340.
[4] Shima S, Oshima S, Sakai H. Biosynthesis ofε-poly-L-lysine by washed mycelium of Streptomyces albulus No.346 [J]. Nippon Nogeikagaku Kaishi, 1983, 57:221-226 (in Japanese).
[5] Nishikawa M, Ogawa K. Distribution of microbes producing antimicrobialε-poly-L- lysine polymers in soil micro?ora determined by a novel method [J]. Appl Environ Microbiol, 2002, 68: 3575-3581.
[6]朱宏阳,徐虹,吴群等.ε-聚赖氨酸生产菌株的筛选和鉴定[J].微生物学通报, 2005, 32:127-130.
[7] Ouyang J, Xu H, Li S, et al. Production ofε-poly-L-lysine by newly isolated Kitasatospora sp. PL6-3 [J]. Biotechnol J. 2006, 1(12):1459-1463
[8]李树,陈旭升,廖莉娟,等.ε-聚赖氨酸产生菌的筛选方法改进[J].食品与生物技术学报, 2010,29(2):282-287
[9] Li S, Tang L, Chen XS, et al. Isolation and characterization of a novelε-poly-L-lysine producing strain:Streptomyces griseofuscus [J]. J Ind Microbiol Biotechnol, 2011, 38: 557-563
[10]贾士儒,许春英,谭之磊,等.ε-聚赖氨酸产生菌TUST-2的分离鉴定[J].微生物学报, 2010, 50:191-196
[11] Shih IL, Shen MH, Van YT. Microbial synthesis of poly(ε-lysine) and its various applications [J]. Bioresour Technol, 2006,97:1148-1159.
[12] Hiraki J, Hatakeyama M, Morita H, et al. Improvedε-poly-L-lysine production of an S-(2-aminoethyl)-L-cysteine resistant mutant of Streptomyces albulus [J]. Seibutu Kougaku Kaishi, 1998, 76:487-493.
[13]陈玮玮,朱宏阳,徐虹.ε-聚赖氨酸高产菌株选育及分批发酵的研究[J].工业微生物, 2007, 37:28-30.
[14]丛茂林,许鹏,谭之磊,等.氮离子注入法筛选ε-聚赖氨酸高产菌株[J].现代食品科技, 2009, 25:491-493.
[15]岩田敏治,白石慎治,岩泽由美子,等.大量产生ε-聚-L-赖氨酸的菌株和生产方法[P].中国专利, CN1260004A, 1997-4-23.
[16] Shih IL, Shen MH. Application of response surface methodology to optimize production of poly-ε-lysine by Streptomyces albulus IFO 14147 [J]. Enzyme Microb Technol, 2006, 39: 15-21
[17] Saimura M, Takehara M, Mizukami S, et al. Biosynthesis of nearly monodispersed poly(ε-L-lysine) in Streptomyces species [J]. Biotechnol Lett, 2008, 30:377-385.
[18] Yamanaka K, Maruyama C, Takagi H, et al. epsilon-poly-l-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase [J]. Nat Chem Biol, 2008, 4:766-772.
[19] Kobayashi K, Nishikawa M. Promotion ofε-poly-L-lysine production by iron in Kitasatospora Kifunense [J]. World J Microbiol Biotechnol, 2007, 23:1033-1036.
[20] Wang GL, Jia SR, Wang T, et al. Effect of ferrous ion onε-poly-L-lysine biosynthesis by Streptomyces diastatochromogenes CGMCC3145 [J]. Curr Microbiol, 2011, 62:1062- 1067.
[21] Kahar P, Iwata T, Hiraki J, et al. Enhancement ofε-polylysine production by Streptomyces albulus strain 410 using pH control [J]. J Biosci Bioeng, 2001, 91:190-194.
[22] Kahar P, Kobayashi K, Iwata T, et al. Production ofε-polylysine in an airlift bioreactor (ABR) [J]. J Biosci Bioeng, 2002, 93:274-280.
[23] Shih IL, Shen MH. Optimization of cell growth and poly(ε-lysine) production in batch and fed-batch cultures by Streptomyces albulus IFO 14147 [J]. Process Biochem, 2006, 41:1644-1649.
[24] Jia SR, Wang GL, Sun YF, et al. Improvement ofε-poly-L-lysine production by Streptomyces albulus TUST2 employing a feeding strategy [C]. In: The 3rd International Conference on Bioinformatics and Biomedical Engineering (iCBBE 2009). 2009, Beijing, China. 1-4. doi:10.1109/ICBBE.2009.5162940
[25] Hiraki J, Suzuki E. Process for producingε-poly-L-lysine with immobilized Streptomyces albulus [P]. US. Patent. US 5900363.1999-05-04
[26] Zhang Y, Feng XH, Xu H, et al.ε-Poly-L-lysine production by immobilized cells of Kitasatospora sp. MY 5-36 in repeated fed-batch cultures [J]. Bioresour Technol, 2010,101:5523-5527.
[27]徐虹,张扬,冯小海,等.一种吸附固定化发酵生产ε-聚赖氨酸的工艺[P].中国专利, CN 101509021A, 2009-08-19.
[28] Shima S, Matsuoka H, Iwamoto T, et al. Antimicrobial action of epsilon-poly-l-lysine [J]. J Antibiot, 1984,37:1449-1455.
[29] Liu SG, Wu QP, Zhang JM, et al. Production ofε-poly-L-lysine by Streptomyces sp. using resin-based, in situ product removal [J]. Biotechnol Lett, 2011,33:1581-1585.
[30]贾士儒,莫治文,谭之磊,等.一种提高ε-聚-L-赖氨酸产量的新方法[P].中国专利, CN101671703A, 2010-03-17.
[31] Takehara M, Aihara Y, Kawai S, et al. Strain producing low molecular weightε-poly-L-lysine and production of low molecular weightε-poly-l-lysine using the strain [J]. Japan patent. 017159.2001.
[32] Nishikawa M, Ogawa K. Inhibition ofε-poly-l-lysine biosynthesis in Streptomyctaceae bacteria by short-chain polyols [J]. Appl Environ Microbiol, 2006,72:2306-2312.
[33] Nishikawa M. Molecular mass control using polyanionic cyclodextrin derivatives for the epsilon-poly-l-lysine biosynthesis by Streptomyces [J]. Enzyme Microb Technol, 2009,45:295-298
[34] Takehara M, Hibino A , Saimura M, et al. High-yield production of short chain length poly(ε-L-lysine)consisting of 5-20 residues by Streptomyces aureofaciens, and its antimicrobial activity [J]. Biotechnol Lett, 2010, 32:1299-1303.
[35] Kito M, Takimoto R, Yoshida T, et al. Purification and characterization of anε-poly-L- lysine-degrading enzyme from anε-poly-L-lysine-producing strain of Streptomyces albulus [J]. Arch Microbiol, 2002, 178:325-330.
[36] Kito M, Onji Y, Yoshida T, et al. Occurrence ofε-poly-L-lysine-degrading enzyme inε- poly-L-lysine-tolerant Sphingobacterium multivorum OJ10: purification and characterization [J]. FEMS Microbiol Lett, 2002, 207:147-151.
[37] Takimoto R, Kito M, Yoshda T, et al. Characterization of microbial enzymes catalyzing the degradation ofε-poly-L-lysine [C]. In: Proceedings of Annual Meeting of the Society for Bioscience Biotechnology and Biochemistry. Osaka: Japan, 2002, 297-301.
[38] Hamano Y, Yoshida T, Kito M, et al. Biological function of the pld gene product that degradesε-poly-L-lysine in Streptomyces albulus [J]. Appl Microbiol Biotechnol, 2006, 72:173-181.
[39] Feng XH, Xu H, Xu XY, et al. Purification and some properties ofε-poly-L-lysine- degrading enzyme from Kitasatospora sp. CCTCC M205012 [J]. Process Biochem, 2008, 43:667-672.
[40]谭之磊,贾士儒,赵颖,等.淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质[J].高等学校化学学报, 2009,30: 2404-2408.
[41] Hamano Y, Nicchu I, Shimizu T, et al.ε-Poly-L-lysine producer, Streptomyces albulus,has feedback-inhibition resistant aspartokinase [J]. Appl Microbiol Biotechnol, 2007, 76:873-882
[42] Kawai T, Kubota T, Hiraki J, et al. Biosynthesis ofε-poly-L-lysine in a cell-free system of Streptomyces albulus [J]. Biochem Biophys Res Commun, 2003, 311:635-640.
[43] Yamanaka K, Kito N, Imokawa Y, et al. Mechanism ofε-Poly-L-Lysine production and accumulation revealed by identification and analysis of anε-Poly-L-Lysine-degrading enzyme [J]. Appl Environ Microbiol, 2010, 76:5669-5675.
[44] Hirohara H, Saimura M, Takehara M, et al. Substantially mono-dispersed poly(ε-L- lysine)s frequently occurred in newly isolated strains of Streptomyces sp. [J]. Appl Microbiol Biotechnol, 2007, 76:1009-1016.
[45] Hamano Y, Nicchu I, Hoshino Y, et al. Development of genedelivery systems for theε-poly-L-lysine producer, Streptomyces albulus [J]. J Biosci Bioeng, 2005, 99:636-641.
[46] Yamanaka K, Kito N, Kita A, et al. Development of a recombinantε-poly-L-lysine synthetase expression system to perform mutational analysis [J]. J Biosci Bioeng, 2011, 111:646-649.
[47]郭明,胡昌华.生物转化—从全细胞催化到代谢工程[J].中国生物工程杂志, 2010, 30:110-115.
[48]蔡真,李寅.工业生物技术专刊序言[J].生物工程学报, 2011, 27:971-975.
[49] Schoemaker HE, Mink D, Wubbolts MG. Dispelling the myths-Biocatalysis in industrial synthsis [J]. Science, 2003, 299:1694-1697.
[50]刘惠.酵母生物转化生产S-腺苷-L-蛋氨酸辅产谷胱甘肽机理及工艺的研究[D]:[博士学位论文].杭州:浙江大学, 2002.
[51] Shimoni E, Ravid U, Shoham Y. Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin [J]. J Biotechnol, 2000, 78:1-9.
[52]张琪,王秀伶,王世英,等.牛瘤胃分离菌株静息细胞培养体系生物转化黄豆苷原[J].生物工程学报, 2010, 26: 35-41.
[53]徐莉,袁生,陈婷.恶臭假单胞菌NA-1菌体培养转化和静息细胞转化联合工艺生产6-羟基烟酸研究[J].微生物学报, 2006, 46:63-67.
[54] Zhu YH, Li JH, Liu L, et al. Production ofα-ketoisocaproate via free-whole-cell biotransformation by Rhodococcus opacus DSM 43250 with L-leucine as the substrate [J]. Enzyme Microb Technol, 2011, 49:321-325.
[55]白凤武.无载体固定化细胞的研究进展[J].生物工程进展2000, 20:32-36.
[56] Hattori T, Furusaka C. Chemical activities of E. coli adsorbed on a resin [J]. J Biochem, 1960, 48:831-837.
[57]崔建涛,李建新,王育红,等.细胞固定化技术的研究进展[J].农产品加工·学刊, 2007, 88:24-26.
[58]李冀新,张超,高虹.固定化细胞技术应用研究进展[J].化学与生物工程, 2006, 23:5-7.
[59]付雯,张晓勇,周金燕,等.固定化枯草芽孢杆菌发酵生产捷安肽素[J].应用与环境生物学报, 2009, 15:230-234.
[60] Kar S, Mandal A, Das Mohapatra PK, et al. Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads [J]. J Ind Microbiol Biotechnol, 2008, 35:245-249.
[61]向智男,宁正祥.植物性天然防腐剂及其在食品中的应用[J].中国食品添加剂, 2004, 3:79-82.
[62]储炬,李友荣.现代工业发酵调控学[M].北京:化学工业出版社, 2002, 230-235.
[63] Kennedy M, Krouse D. Strategies for improving fermentation medium performance: a review [J]. J Ind Microbiol Biotechnol, 1999, 23:456-475.
[64] Shima S, Sakai H. Poly-L-lysine produced by Streptomyces. Part II. Taxonomy and fermentation studies [J]. Agric Biol Chem, 1981, 45:2497-2502.
[65] Prapulla SG, Jacob Z, Chand N, et al. Maximization of lipid production by rhodotorula gracilris CFR-1 using response surface methodology [J]. Biotechnol Bioeng, 1992, 40:965-970
[66] Bajaj IB, Lele SS, Singhal RS. A statistical approach to optimization of fermentative production of poly(γ-glutamic acid) from Bacillus licheniformis NCIM 2324 [J]. Bioresour Technol, 2008, 100:826-832.
[67] Majumder A, Singh A, Goyal A. Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization [J]. Carbohydr Polym, 2009, 75:150-156.
[68] Willke TH, Vorlop KD. Industrial bioconversion of renewable resources as an alternative to conventional chemistry [J]. Appl Microbiol Biotechnol, 2004, 66:131-134.
[69] Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering [J]. Biotechnol Bioeng, 2006, 94:821-829.
[70] http://www.16ds.com/products/232. 2011-08-24
[71] Raj SM, Rathnasingh C, Jo JE, et al. Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain [J]. Process Biochem, 2008, 43:1440-1446.
[72] Petrov K, Petrova P. High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31 [J]. Appl Microbiol Biotechnol, 2009, 84:659-665.
[73] Menzel K, Zeng AP, Deckwer WD. High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae [J]. Enzym Microb Technol, 1997, 20:82-86.
[74] Mothes G, Schnorpfeil C, Ackermann JU. Production of PHB from crude glycerol [J]. Eng Life Sci, 2007, 7:475-479.
[75] Lee PC, Lee SY, Chang HN. Kinetic study on succinic acid and acetic acid formation during continuous cultures of Anaerobiospirillum succiniciproducens grown on glycerol [J]. Bioprocess Biosyst Eng, 2010, 33:465-471.
[76] da Silva GP, Mack M, Contiero J. Glycerol: A promising and abundant carbon source for industrial microbiology [J]. Biotechnol Adv, 2009, 27, 30-39.
[77]张超,张东荣,贺魏,等.一种简便的ε-聚赖氨酸产生菌的筛选方法[J].山东大学学报(医学版), 2006, 44:1104-1107.
[78]陈旭升.ε-聚赖氨酸高产菌株选育与发酵过程优化[D]: [硕士论文].无锡:江南大学, 2008.
[79] Itzhaki RF. Colorimetric method for estimating polylysine and polyarginine [J]. Anal Biochem, 1972, 50:569-574.
[80] Bankar SB, Singhal RS. Optimization of poly-ε-lysine production by Streptomyces noursei NRRL 5126 [J]. Bioresour Technol, 2010, 101: 8370-8375.
[81] Wittmann C, Becker J. The L-lysine story: from metabolic pathways to industrial production [J]. Microbiol Monogr, 2007, 5:39-70.
[82] Plackett RL, Burman JP. The design of optimum multifactorial experiments [J]. Biometrica, 1944, 33:305-325.
[83] Guo WQ, Ren NQ, Wang XJ, et al. Optimization of culture conditions for hydrogen production by Ethanoligenens harbinense B49 using response surface methodology [J]. Bioresour Technol, 2009, 100:1192-1196.
[84]江曙,朱丽,黄为一.磷酸盐对阿扎霉素B生物合成的调节[J].中国抗生素杂志, 2005, 30:73-75.
[85]焦瑞身.微生物工程[M].北京:化学工业出版社, 2005, 25.
[86]陈坚,刘立明,堵国成.发酵过程优化原理与技术[M].北京:化学工业出版社, 2009, 3-7.
[87] Xiu LZ, Chen X, Sun YQ, et al. Stoichiometric analysis and experimental investigation of glycerol-glucose co-fermentation in Klebsiella pneumoniae under microaerobicconditions [J]. Biochem Eng J, 2007,33: 42-52.
[88]谢志鹏,徐志南,郑建明,等.靛酚蓝反应测定发酵液中的氨态氮[J].浙江大学学报(工学版), 2005, 39:437-444.
[89] Kito M, Takimoto R, Yoshida T, et al. Purification and characterization of anε-poly-L-lysine-degrading enzyme from anε-poly-L-lysine-producing strain of Streptomyces albulus [J]. Arch Microbiol, 2002, 178:325-330.
[90] Meijnen JP, Verhoef S, Briedjlal AA, et al. Improved p-hydroxybenzoate production by engineered Pseudomonas putida S12 by using a mixed-substrate feeding strategy [J]. Appl Microbiol Biotechnol, 2011, 90:885-893.
[91] Saint-Amans S, Girbal L, Andrade J, et al. Regulation of carbon and electron flow in clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures [J]. J Bacteriol, 2001,183:1748-1754.
[92] de Noronha PP, Nielsen J, Bazin MJ. Pathway kinetics and metabolic control analysis of a high-yielding strain of Penicillum chrysogenum during fed batch cultivation [J]. Biotech Bioeng, 1996,51:168-176
[93] Christensen B, Gombert AK, Nielsen J. Analysis of flux estimates based on 13C-labelling experiments [J]. Eur J Biochem, 2002, 269:2795-2800.
[94] Klapa MI, Aon JC, Stephanopoulos G. Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry [J]. Eur J Biochem, 2003, 270: 3525-3542.
[95] Schuster S, Dandekar T, Fell DA. Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering [J]. Trends Biotechnol, 1999,17:53-60.
[96] Wolfgang W. 13C metabolic flux analysis [J]. Metab Eng, 2001,3:195-206.
[97] Lee K, Berthiaume F, Stephanopouls GN, et al. Metabolic flux analysis a powerful tool for monitoring tissue function [J]. Tissue Eng, 1999,5:347-368.
[98] Stephanopoulos GN, Aristidou AA, Nielsen J. Metabolic engineering [M], Academic Press, New York, 1998.
[99] Hirohara H, Takehara M, Saimura M, et al. Biosynthesis of poly(ε-L-lysine)s in two newly isolated strains of Streptomyces sp. [J]. Appl Microbiol Biotechnol, 2006, 73:321-331.
[100]Delaunay S, Daran-Lapujade P, Engasser JM, et al. Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum [J]. J Ind Microbiol Biotechnol, 2004, 31:183-188.
[101]Mori M, Shiio I. Purification and some properties of phosphoenolpyruvate carboxylasefrom Brevibacterium ?avum and its aspartate-overproducing mutant [J]. J Biochem, 1985, 97:1119-1128.
[102]Borodina I, Schǒller C, Eliasson A, et al. Metabolic network analysis of streptomyces tenebrarius, a streptomyces species with an active entner-doudoroff pathway[J]. Appl Environ Microbiol, 2005, 71:2294-2302.
[103]Kirkpatrick JR, Doolin LE, Godfrey OW. Lysine biosynthesis in streptomyces lipmanii: implications in antibiotic biosynthesis [J]. Antimicrob Agents Chemother, 1973, 4:542-550.
[104]Kosuge T, Hoshino T. Lysine is synthesized through theα-aminoadipate pathway in Thermus thermophilus [J]. FEMS Microbiol Lett, 1998, 169:361-367.
[105]Cummins CS, Harris H. Studies on the cell wall composition and taxonomy of Actinomycetales and related groups [J]. J Gen Microbiol, 1958, 18:173-189.
[106]Ingraham JL, Maal?e O, Neidhardt FC. Growth of the Bacterial Cell [M]. Sinauer, Sunderland, MA, 1983.
[107]Daae EB, Ison AP. Classification and sensitivity analysis of a proposed primary metabolic reaction network for Streptomyces lividans [J]. Metab Eng ,1999, 1:153-165.
[108]Li J, Yang Y, Chu J. Quantitative metabolic ?ux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chrysogenum fed with soybean oil [J]. Bioprocess Biosyst Eng, 2010, 33:1119-1129.
[109]Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem, 1976, 72:248-254.
[110]Henderson JW, Ricker RD, Bidlingmeyer BA, et al. Rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids. USA (Agilent App Note 5980-1193E): Agilent Technologies, 2000.
[111]Van Briesen JM. Evaluation of methods to predict bacterial yield using thermodynamics [J]. Biodegradation, 2002, 13: 171-190.
[112]Liu Y, Zhang YG, Zhang RB,et al. Glycerol/Glucose co-fermentation: one more proficient process to producepropionic acid by Propionibacterium acidipropionic [J]. Curr Microbiol, 2011, 62:152-158.
[113]Kim YS, Lee JH, Kim NH, et al. Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered Escherichia coli [J]. Appl Microbiol Biotechnol, 2011, 90:489-497.
[114] Sola Penna A, Meyer Fernandes JR. Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than othersugars? [J]. Arch Biochem Biophys, 1998, 360:10-14.
[115]诸葛健,王正祥.工业微生物实验技术手册[M].中国轻工业出版社, 1994, 520-521
[116]Zhao W, Yang RJ. Protective effect of sorbitol on enzymes exposed to microsecond pulsed electric field [J]. J Phys Chem B, 2008, 112:14018-14025.
[117]Suwannakham S. Metabolic engineering for enhanced propionic acid fermentation by Propionibacterium acidipropionici[D]. The Ohio State University, 2005.
[118]Coral J, Karp SG, Porto de Souza, et al. Batch fermentation model of propionic acid production by Propionibacterium acidipropionici in different carbon sources [J]. Appl Biochem Biotechnol, 2008, 151:333-341.
[119]Lewis PV, Yang ST. Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate [J]. Appl Microbiol Biotechnol, 1992, 37:437-442.
[120]San KY, Bennett GN, Berrios-Rivera SJ, et al. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli [J]. Metab Eng, 2002, 4:182-192.
[121]Saanchez AM, Bennett GN, San KY. Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium [J]. J Biotechnol, 2005, 117:395-405.
[122]Alfafara CG, Kanda A, Shioi T, et al. Effect of amino acids on glutathione production by Saccharomyces cerevsiae [J]. Appl Microbiol Biotechnol, 1992, 36:538-540.
[123]Larroche C, Besson I, Gros JB. High pyrazine production by Bacillus subtilis in solid substrate fermentation on ground soybeans [J]. Process Biochem, 1999,34:667-674.
[124]Thorne CB, Gomez CG, Noyes HE, et al. Production of glutamyl polypeptide by Bacillus subtilis [J]. J Bacteriol, 1954, 68:307-315.
[125]Yao J, Xu H, Shi NN, et al. Analysis of carbon metabolism and improvement ofγ-Polyglutamic acid production from Bacillus subtilis NX-2 [J]. Appl Biochem Biotechnol, 2010, 160:2332-2341.
[126]Alfalfa CG, Miura K, Shimizu H, et a1. Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae [J]. Appl Microbiol Biotechnol, 1992, 37:141-146.
[127]de Carvalho CCCR. Enzymatic and whole cell catalysis: Finding new strategies for old processe [J]. Biotechnol Adv, 2011, 29:75-83.
[128]Hama S, Yamaji H, Kaieda M, et al. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production [J]. Biochem Eng J, 2004, 21:155-160.
[129]Liu Y, Hama H, Fujita Y, et al. Production of S-lactoylglutathione by high activity wholecell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols [J]. Biotechnol Bioeng, 1999, 64:54-60.
[130]van der Werf MJ, Hartmans S, van den Tweel WJJ. Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by triton X-100 for efficient production of D-matate [J]. Appl Microbiol Biotechnol, 1995, 43:590-594.
[131]Silveira MM, Jonas R. The biotechnological production of sorbitol [J]. Appl Microbiol Biotechnol, 2002, 59:400-409.
[132]Upadhya R, Nagajyothi, Bhat SG. Stabilization of D-amino caid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation ofα-ketoacids [J]. Biotechnol Bioeng, 2000, 68:430-436.
[133]Cavonas M, Torroglosa T, Kleber H, et al. Effect of salt stress on crotonobetaine and D-carnitine biotransformation of L-carnitine by resting cells of Escherichia coli [J]. J Basic Microbiol, 2003, 43:259-268.
[134]Matsumoto T, Takahash S, Kaieda M, et al. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production [J]. Appl Microbiol Biotechnol, 2001, 57:515-520.
[135]Breedveld MW, Zevenhuizen LP, Zehnder AJB. Synthesis of cyclicβ-1,2-glucans by Rhizobium leguminosarum Biovartrifolii TA-1: factors influencing excretion [J]. J Bacteriol, 1992, 174:6336-6342.
[136]Isoai A, Kimura H, Reichert A, et al. Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysis prepared with recombinant cells [J]. Biotechnol Bioeng, 2002, 80:22-32.
[137]Ni Y, Chen R. Lipoprotein mutation accelerates substrate-permeability limited toluene dioxygenase- catalyzed reaction [J]. Biotechnol Prog, 2005, 21:799-805.
[138]Liang GB, Liao X, Du GC, et al. Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis [J]. J Appl Microbiol, 2008, 105:1432-1440.
[139]Sonntag K, Eggeling K, De Graaf AA, et al. Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum quantification by 13C- and 1H-NMR spectroscopy [J]. Eur J Biochem, 1993, 213:1325-1331.
[140]Kunioka M, Goto A. Biosynthesis of poly(γ-glutamic acid) from L-glutamic acid, citric acid and ammonium sulfate in Bacillus subtilis IFO3335 [J]. Appl Microbiol Biotechnol, 1995, 44:501-506.
[141]Maeda S, Kunimotob KK, Sasaki C, et al. Characterization of microbial poly (ε-L- lysine) by FT-IR, Raman and solid state 13C NMR spectroscopies [J]. J Mol Struct, 2003,655:149-155.
[142]杨革,王宁,张超.全细胞催化合成医用纺织新材料聚γ-谷氨酸[J].过程工程学报, 2010, 10:777-780.
[143]Chen RR. Permeability issues in whole-cell bioprocesses and cellular membrane engineering [J]. Appl Microbiol Biotechnol, 2007, 74:730-738.
[144]刘志钰,张东旭,堵国成,等. Paecilomyces sp. S152胞外漆酶的合成调控[J].生物技术学报, 2010, 29:952-958.
[145]Liang GB, Du GC, Chen J. A novel strategy of enhanced glutathione production in high cell density cultivation of Candida utilis—Cysteine addition combined with dissolved oxygen controlling [J]. Enzyme Microb Technol, 2008, 42:284-289.
[146]Wei GY, Wang DH, Chen J. Overproduction of glutathione by L-cysteine addition and a temperature-shift strategy [J]. Biotechnol Bioproc E, 2008, 13:347-353.
[147]Yoon SH, Do JH, Lee SY, et al. Production of poly-γ-glutamic acid by fed-batch culture of Bacillus Licheniformis [J]. Biotechnol Lett, 2000, 22:585-588.
[2]施庆珊,陈仪本,欧阳友生.ε-聚赖氨酸的微生物合成与降解[J].生物技术, 2004, 14:77-79.
[3] Hiraki J, Ichikawa T, Ninomiya S, et al. Use of ADME studies to confirm the safety ofε-polylysine as a preservative in food [J]. Regul Toxicol Pharm, 2003,37:328-340.
[4] Shima S, Oshima S, Sakai H. Biosynthesis ofε-poly-L-lysine by washed mycelium of Streptomyces albulus No.346 [J]. Nippon Nogeikagaku Kaishi, 1983, 57:221-226 (in Japanese).
[5] Nishikawa M, Ogawa K. Distribution of microbes producing antimicrobialε-poly-L- lysine polymers in soil micro?ora determined by a novel method [J]. Appl Environ Microbiol, 2002, 68: 3575-3581.
[6]朱宏阳,徐虹,吴群等.ε-聚赖氨酸生产菌株的筛选和鉴定[J].微生物学通报, 2005, 32:127-130.
[7] Ouyang J, Xu H, Li S, et al. Production ofε-poly-L-lysine by newly isolated Kitasatospora sp. PL6-3 [J]. Biotechnol J. 2006, 1(12):1459-1463
[8]李树,陈旭升,廖莉娟,等.ε-聚赖氨酸产生菌的筛选方法改进[J].食品与生物技术学报, 2010,29(2):282-287
[9] Li S, Tang L, Chen XS, et al. Isolation and characterization of a novelε-poly-L-lysine producing strain:Streptomyces griseofuscus [J]. J Ind Microbiol Biotechnol, 2011, 38: 557-563
[10]贾士儒,许春英,谭之磊,等.ε-聚赖氨酸产生菌TUST-2的分离鉴定[J].微生物学报, 2010, 50:191-196
[11] Shih IL, Shen MH, Van YT. Microbial synthesis of poly(ε-lysine) and its various applications [J]. Bioresour Technol, 2006,97:1148-1159.
[12] Hiraki J, Hatakeyama M, Morita H, et al. Improvedε-poly-L-lysine production of an S-(2-aminoethyl)-L-cysteine resistant mutant of Streptomyces albulus [J]. Seibutu Kougaku Kaishi, 1998, 76:487-493.
[13]陈玮玮,朱宏阳,徐虹.ε-聚赖氨酸高产菌株选育及分批发酵的研究[J].工业微生物, 2007, 37:28-30.
[14]丛茂林,许鹏,谭之磊,等.氮离子注入法筛选ε-聚赖氨酸高产菌株[J].现代食品科技, 2009, 25:491-493.
[15]岩田敏治,白石慎治,岩泽由美子,等.大量产生ε-聚-L-赖氨酸的菌株和生产方法[P].中国专利, CN1260004A, 1997-4-23.
[16] Shih IL, Shen MH. Application of response surface methodology to optimize production of poly-ε-lysine by Streptomyces albulus IFO 14147 [J]. Enzyme Microb Technol, 2006, 39: 15-21
[17] Saimura M, Takehara M, Mizukami S, et al. Biosynthesis of nearly monodispersed poly(ε-L-lysine) in Streptomyces species [J]. Biotechnol Lett, 2008, 30:377-385.
[18] Yamanaka K, Maruyama C, Takagi H, et al. epsilon-poly-l-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase [J]. Nat Chem Biol, 2008, 4:766-772.
[19] Kobayashi K, Nishikawa M. Promotion ofε-poly-L-lysine production by iron in Kitasatospora Kifunense [J]. World J Microbiol Biotechnol, 2007, 23:1033-1036.
[20] Wang GL, Jia SR, Wang T, et al. Effect of ferrous ion onε-poly-L-lysine biosynthesis by Streptomyces diastatochromogenes CGMCC3145 [J]. Curr Microbiol, 2011, 62:1062- 1067.
[21] Kahar P, Iwata T, Hiraki J, et al. Enhancement ofε-polylysine production by Streptomyces albulus strain 410 using pH control [J]. J Biosci Bioeng, 2001, 91:190-194.
[22] Kahar P, Kobayashi K, Iwata T, et al. Production ofε-polylysine in an airlift bioreactor (ABR) [J]. J Biosci Bioeng, 2002, 93:274-280.
[23] Shih IL, Shen MH. Optimization of cell growth and poly(ε-lysine) production in batch and fed-batch cultures by Streptomyces albulus IFO 14147 [J]. Process Biochem, 2006, 41:1644-1649.
[24] Jia SR, Wang GL, Sun YF, et al. Improvement ofε-poly-L-lysine production by Streptomyces albulus TUST2 employing a feeding strategy [C]. In: The 3rd International Conference on Bioinformatics and Biomedical Engineering (iCBBE 2009). 2009, Beijing, China. 1-4. doi:10.1109/ICBBE.2009.5162940
[25] Hiraki J, Suzuki E. Process for producingε-poly-L-lysine with immobilized Streptomyces albulus [P]. US. Patent. US 5900363.1999-05-04
[26] Zhang Y, Feng XH, Xu H, et al.ε-Poly-L-lysine production by immobilized cells of Kitasatospora sp. MY 5-36 in repeated fed-batch cultures [J]. Bioresour Technol, 2010,101:5523-5527.
[27]徐虹,张扬,冯小海,等.一种吸附固定化发酵生产ε-聚赖氨酸的工艺[P].中国专利, CN 101509021A, 2009-08-19.
[28] Shima S, Matsuoka H, Iwamoto T, et al. Antimicrobial action of epsilon-poly-l-lysine [J]. J Antibiot, 1984,37:1449-1455.
[29] Liu SG, Wu QP, Zhang JM, et al. Production ofε-poly-L-lysine by Streptomyces sp. using resin-based, in situ product removal [J]. Biotechnol Lett, 2011,33:1581-1585.
[30]贾士儒,莫治文,谭之磊,等.一种提高ε-聚-L-赖氨酸产量的新方法[P].中国专利, CN101671703A, 2010-03-17.
[31] Takehara M, Aihara Y, Kawai S, et al. Strain producing low molecular weightε-poly-L-lysine and production of low molecular weightε-poly-l-lysine using the strain [J]. Japan patent. 017159.2001.
[32] Nishikawa M, Ogawa K. Inhibition ofε-poly-l-lysine biosynthesis in Streptomyctaceae bacteria by short-chain polyols [J]. Appl Environ Microbiol, 2006,72:2306-2312.
[33] Nishikawa M. Molecular mass control using polyanionic cyclodextrin derivatives for the epsilon-poly-l-lysine biosynthesis by Streptomyces [J]. Enzyme Microb Technol, 2009,45:295-298
[34] Takehara M, Hibino A , Saimura M, et al. High-yield production of short chain length poly(ε-L-lysine)consisting of 5-20 residues by Streptomyces aureofaciens, and its antimicrobial activity [J]. Biotechnol Lett, 2010, 32:1299-1303.
[35] Kito M, Takimoto R, Yoshida T, et al. Purification and characterization of anε-poly-L- lysine-degrading enzyme from anε-poly-L-lysine-producing strain of Streptomyces albulus [J]. Arch Microbiol, 2002, 178:325-330.
[36] Kito M, Onji Y, Yoshida T, et al. Occurrence ofε-poly-L-lysine-degrading enzyme inε- poly-L-lysine-tolerant Sphingobacterium multivorum OJ10: purification and characterization [J]. FEMS Microbiol Lett, 2002, 207:147-151.
[37] Takimoto R, Kito M, Yoshda T, et al. Characterization of microbial enzymes catalyzing the degradation ofε-poly-L-lysine [C]. In: Proceedings of Annual Meeting of the Society for Bioscience Biotechnology and Biochemistry. Osaka: Japan, 2002, 297-301.
[38] Hamano Y, Yoshida T, Kito M, et al. Biological function of the pld gene product that degradesε-poly-L-lysine in Streptomyces albulus [J]. Appl Microbiol Biotechnol, 2006, 72:173-181.
[39] Feng XH, Xu H, Xu XY, et al. Purification and some properties ofε-poly-L-lysine- degrading enzyme from Kitasatospora sp. CCTCC M205012 [J]. Process Biochem, 2008, 43:667-672.
[40]谭之磊,贾士儒,赵颖,等.淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质[J].高等学校化学学报, 2009,30: 2404-2408.
[41] Hamano Y, Nicchu I, Shimizu T, et al.ε-Poly-L-lysine producer, Streptomyces albulus,has feedback-inhibition resistant aspartokinase [J]. Appl Microbiol Biotechnol, 2007, 76:873-882
[42] Kawai T, Kubota T, Hiraki J, et al. Biosynthesis ofε-poly-L-lysine in a cell-free system of Streptomyces albulus [J]. Biochem Biophys Res Commun, 2003, 311:635-640.
[43] Yamanaka K, Kito N, Imokawa Y, et al. Mechanism ofε-Poly-L-Lysine production and accumulation revealed by identification and analysis of anε-Poly-L-Lysine-degrading enzyme [J]. Appl Environ Microbiol, 2010, 76:5669-5675.
[44] Hirohara H, Saimura M, Takehara M, et al. Substantially mono-dispersed poly(ε-L- lysine)s frequently occurred in newly isolated strains of Streptomyces sp. [J]. Appl Microbiol Biotechnol, 2007, 76:1009-1016.
[45] Hamano Y, Nicchu I, Hoshino Y, et al. Development of genedelivery systems for theε-poly-L-lysine producer, Streptomyces albulus [J]. J Biosci Bioeng, 2005, 99:636-641.
[46] Yamanaka K, Kito N, Kita A, et al. Development of a recombinantε-poly-L-lysine synthetase expression system to perform mutational analysis [J]. J Biosci Bioeng, 2011, 111:646-649.
[47]郭明,胡昌华.生物转化—从全细胞催化到代谢工程[J].中国生物工程杂志, 2010, 30:110-115.
[48]蔡真,李寅.工业生物技术专刊序言[J].生物工程学报, 2011, 27:971-975.
[49] Schoemaker HE, Mink D, Wubbolts MG. Dispelling the myths-Biocatalysis in industrial synthsis [J]. Science, 2003, 299:1694-1697.
[50]刘惠.酵母生物转化生产S-腺苷-L-蛋氨酸辅产谷胱甘肽机理及工艺的研究[D]:[博士学位论文].杭州:浙江大学, 2002.
[51] Shimoni E, Ravid U, Shoham Y. Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin [J]. J Biotechnol, 2000, 78:1-9.
[52]张琪,王秀伶,王世英,等.牛瘤胃分离菌株静息细胞培养体系生物转化黄豆苷原[J].生物工程学报, 2010, 26: 35-41.
[53]徐莉,袁生,陈婷.恶臭假单胞菌NA-1菌体培养转化和静息细胞转化联合工艺生产6-羟基烟酸研究[J].微生物学报, 2006, 46:63-67.
[54] Zhu YH, Li JH, Liu L, et al. Production ofα-ketoisocaproate via free-whole-cell biotransformation by Rhodococcus opacus DSM 43250 with L-leucine as the substrate [J]. Enzyme Microb Technol, 2011, 49:321-325.
[55]白凤武.无载体固定化细胞的研究进展[J].生物工程进展2000, 20:32-36.
[56] Hattori T, Furusaka C. Chemical activities of E. coli adsorbed on a resin [J]. J Biochem, 1960, 48:831-837.
[57]崔建涛,李建新,王育红,等.细胞固定化技术的研究进展[J].农产品加工·学刊, 2007, 88:24-26.
[58]李冀新,张超,高虹.固定化细胞技术应用研究进展[J].化学与生物工程, 2006, 23:5-7.
[59]付雯,张晓勇,周金燕,等.固定化枯草芽孢杆菌发酵生产捷安肽素[J].应用与环境生物学报, 2009, 15:230-234.
[60] Kar S, Mandal A, Das Mohapatra PK, et al. Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads [J]. J Ind Microbiol Biotechnol, 2008, 35:245-249.
[61]向智男,宁正祥.植物性天然防腐剂及其在食品中的应用[J].中国食品添加剂, 2004, 3:79-82.
[62]储炬,李友荣.现代工业发酵调控学[M].北京:化学工业出版社, 2002, 230-235.
[63] Kennedy M, Krouse D. Strategies for improving fermentation medium performance: a review [J]. J Ind Microbiol Biotechnol, 1999, 23:456-475.
[64] Shima S, Sakai H. Poly-L-lysine produced by Streptomyces. Part II. Taxonomy and fermentation studies [J]. Agric Biol Chem, 1981, 45:2497-2502.
[65] Prapulla SG, Jacob Z, Chand N, et al. Maximization of lipid production by rhodotorula gracilris CFR-1 using response surface methodology [J]. Biotechnol Bioeng, 1992, 40:965-970
[66] Bajaj IB, Lele SS, Singhal RS. A statistical approach to optimization of fermentative production of poly(γ-glutamic acid) from Bacillus licheniformis NCIM 2324 [J]. Bioresour Technol, 2008, 100:826-832.
[67] Majumder A, Singh A, Goyal A. Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization [J]. Carbohydr Polym, 2009, 75:150-156.
[68] Willke TH, Vorlop KD. Industrial bioconversion of renewable resources as an alternative to conventional chemistry [J]. Appl Microbiol Biotechnol, 2004, 66:131-134.
[69] Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering [J]. Biotechnol Bioeng, 2006, 94:821-829.
[70] http://www.16ds.com/products/232. 2011-08-24
[71] Raj SM, Rathnasingh C, Jo JE, et al. Production of 3-hydroxypropionic acid from glycerol by a novel recombinant Escherichia coli BL21 strain [J]. Process Biochem, 2008, 43:1440-1446.
[72] Petrov K, Petrova P. High production of 2,3-butanediol from glycerol by Klebsiella pneumoniae G31 [J]. Appl Microbiol Biotechnol, 2009, 84:659-665.
[73] Menzel K, Zeng AP, Deckwer WD. High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae [J]. Enzym Microb Technol, 1997, 20:82-86.
[74] Mothes G, Schnorpfeil C, Ackermann JU. Production of PHB from crude glycerol [J]. Eng Life Sci, 2007, 7:475-479.
[75] Lee PC, Lee SY, Chang HN. Kinetic study on succinic acid and acetic acid formation during continuous cultures of Anaerobiospirillum succiniciproducens grown on glycerol [J]. Bioprocess Biosyst Eng, 2010, 33:465-471.
[76] da Silva GP, Mack M, Contiero J. Glycerol: A promising and abundant carbon source for industrial microbiology [J]. Biotechnol Adv, 2009, 27, 30-39.
[77]张超,张东荣,贺魏,等.一种简便的ε-聚赖氨酸产生菌的筛选方法[J].山东大学学报(医学版), 2006, 44:1104-1107.
[78]陈旭升.ε-聚赖氨酸高产菌株选育与发酵过程优化[D]: [硕士论文].无锡:江南大学, 2008.
[79] Itzhaki RF. Colorimetric method for estimating polylysine and polyarginine [J]. Anal Biochem, 1972, 50:569-574.
[80] Bankar SB, Singhal RS. Optimization of poly-ε-lysine production by Streptomyces noursei NRRL 5126 [J]. Bioresour Technol, 2010, 101: 8370-8375.
[81] Wittmann C, Becker J. The L-lysine story: from metabolic pathways to industrial production [J]. Microbiol Monogr, 2007, 5:39-70.
[82] Plackett RL, Burman JP. The design of optimum multifactorial experiments [J]. Biometrica, 1944, 33:305-325.
[83] Guo WQ, Ren NQ, Wang XJ, et al. Optimization of culture conditions for hydrogen production by Ethanoligenens harbinense B49 using response surface methodology [J]. Bioresour Technol, 2009, 100:1192-1196.
[84]江曙,朱丽,黄为一.磷酸盐对阿扎霉素B生物合成的调节[J].中国抗生素杂志, 2005, 30:73-75.
[85]焦瑞身.微生物工程[M].北京:化学工业出版社, 2005, 25.
[86]陈坚,刘立明,堵国成.发酵过程优化原理与技术[M].北京:化学工业出版社, 2009, 3-7.
[87] Xiu LZ, Chen X, Sun YQ, et al. Stoichiometric analysis and experimental investigation of glycerol-glucose co-fermentation in Klebsiella pneumoniae under microaerobicconditions [J]. Biochem Eng J, 2007,33: 42-52.
[88]谢志鹏,徐志南,郑建明,等.靛酚蓝反应测定发酵液中的氨态氮[J].浙江大学学报(工学版), 2005, 39:437-444.
[89] Kito M, Takimoto R, Yoshida T, et al. Purification and characterization of anε-poly-L-lysine-degrading enzyme from anε-poly-L-lysine-producing strain of Streptomyces albulus [J]. Arch Microbiol, 2002, 178:325-330.
[90] Meijnen JP, Verhoef S, Briedjlal AA, et al. Improved p-hydroxybenzoate production by engineered Pseudomonas putida S12 by using a mixed-substrate feeding strategy [J]. Appl Microbiol Biotechnol, 2011, 90:885-893.
[91] Saint-Amans S, Girbal L, Andrade J, et al. Regulation of carbon and electron flow in clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures [J]. J Bacteriol, 2001,183:1748-1754.
[92] de Noronha PP, Nielsen J, Bazin MJ. Pathway kinetics and metabolic control analysis of a high-yielding strain of Penicillum chrysogenum during fed batch cultivation [J]. Biotech Bioeng, 1996,51:168-176
[93] Christensen B, Gombert AK, Nielsen J. Analysis of flux estimates based on 13C-labelling experiments [J]. Eur J Biochem, 2002, 269:2795-2800.
[94] Klapa MI, Aon JC, Stephanopoulos G. Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry [J]. Eur J Biochem, 2003, 270: 3525-3542.
[95] Schuster S, Dandekar T, Fell DA. Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering [J]. Trends Biotechnol, 1999,17:53-60.
[96] Wolfgang W. 13C metabolic flux analysis [J]. Metab Eng, 2001,3:195-206.
[97] Lee K, Berthiaume F, Stephanopouls GN, et al. Metabolic flux analysis a powerful tool for monitoring tissue function [J]. Tissue Eng, 1999,5:347-368.
[98] Stephanopoulos GN, Aristidou AA, Nielsen J. Metabolic engineering [M], Academic Press, New York, 1998.
[99] Hirohara H, Takehara M, Saimura M, et al. Biosynthesis of poly(ε-L-lysine)s in two newly isolated strains of Streptomyces sp. [J]. Appl Microbiol Biotechnol, 2006, 73:321-331.
[100]Delaunay S, Daran-Lapujade P, Engasser JM, et al. Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum [J]. J Ind Microbiol Biotechnol, 2004, 31:183-188.
[101]Mori M, Shiio I. Purification and some properties of phosphoenolpyruvate carboxylasefrom Brevibacterium ?avum and its aspartate-overproducing mutant [J]. J Biochem, 1985, 97:1119-1128.
[102]Borodina I, Schǒller C, Eliasson A, et al. Metabolic network analysis of streptomyces tenebrarius, a streptomyces species with an active entner-doudoroff pathway[J]. Appl Environ Microbiol, 2005, 71:2294-2302.
[103]Kirkpatrick JR, Doolin LE, Godfrey OW. Lysine biosynthesis in streptomyces lipmanii: implications in antibiotic biosynthesis [J]. Antimicrob Agents Chemother, 1973, 4:542-550.
[104]Kosuge T, Hoshino T. Lysine is synthesized through theα-aminoadipate pathway in Thermus thermophilus [J]. FEMS Microbiol Lett, 1998, 169:361-367.
[105]Cummins CS, Harris H. Studies on the cell wall composition and taxonomy of Actinomycetales and related groups [J]. J Gen Microbiol, 1958, 18:173-189.
[106]Ingraham JL, Maal?e O, Neidhardt FC. Growth of the Bacterial Cell [M]. Sinauer, Sunderland, MA, 1983.
[107]Daae EB, Ison AP. Classification and sensitivity analysis of a proposed primary metabolic reaction network for Streptomyces lividans [J]. Metab Eng ,1999, 1:153-165.
[108]Li J, Yang Y, Chu J. Quantitative metabolic ?ux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chrysogenum fed with soybean oil [J]. Bioprocess Biosyst Eng, 2010, 33:1119-1129.
[109]Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding [J]. Anal Biochem, 1976, 72:248-254.
[110]Henderson JW, Ricker RD, Bidlingmeyer BA, et al. Rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids. USA (Agilent App Note 5980-1193E): Agilent Technologies, 2000.
[111]Van Briesen JM. Evaluation of methods to predict bacterial yield using thermodynamics [J]. Biodegradation, 2002, 13: 171-190.
[112]Liu Y, Zhang YG, Zhang RB,et al. Glycerol/Glucose co-fermentation: one more proficient process to producepropionic acid by Propionibacterium acidipropionic [J]. Curr Microbiol, 2011, 62:152-158.
[113]Kim YS, Lee JH, Kim NH, et al. Increase of lycopene production by supplementing auxiliary carbon sources in metabolically engineered Escherichia coli [J]. Appl Microbiol Biotechnol, 2011, 90:489-497.
[114] Sola Penna A, Meyer Fernandes JR. Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than othersugars? [J]. Arch Biochem Biophys, 1998, 360:10-14.
[115]诸葛健,王正祥.工业微生物实验技术手册[M].中国轻工业出版社, 1994, 520-521
[116]Zhao W, Yang RJ. Protective effect of sorbitol on enzymes exposed to microsecond pulsed electric field [J]. J Phys Chem B, 2008, 112:14018-14025.
[117]Suwannakham S. Metabolic engineering for enhanced propionic acid fermentation by Propionibacterium acidipropionici[D]. The Ohio State University, 2005.
[118]Coral J, Karp SG, Porto de Souza, et al. Batch fermentation model of propionic acid production by Propionibacterium acidipropionici in different carbon sources [J]. Appl Biochem Biotechnol, 2008, 151:333-341.
[119]Lewis PV, Yang ST. Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate [J]. Appl Microbiol Biotechnol, 1992, 37:437-442.
[120]San KY, Bennett GN, Berrios-Rivera SJ, et al. Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli [J]. Metab Eng, 2002, 4:182-192.
[121]Saanchez AM, Bennett GN, San KY. Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium [J]. J Biotechnol, 2005, 117:395-405.
[122]Alfafara CG, Kanda A, Shioi T, et al. Effect of amino acids on glutathione production by Saccharomyces cerevsiae [J]. Appl Microbiol Biotechnol, 1992, 36:538-540.
[123]Larroche C, Besson I, Gros JB. High pyrazine production by Bacillus subtilis in solid substrate fermentation on ground soybeans [J]. Process Biochem, 1999,34:667-674.
[124]Thorne CB, Gomez CG, Noyes HE, et al. Production of glutamyl polypeptide by Bacillus subtilis [J]. J Bacteriol, 1954, 68:307-315.
[125]Yao J, Xu H, Shi NN, et al. Analysis of carbon metabolism and improvement ofγ-Polyglutamic acid production from Bacillus subtilis NX-2 [J]. Appl Biochem Biotechnol, 2010, 160:2332-2341.
[126]Alfalfa CG, Miura K, Shimizu H, et a1. Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae [J]. Appl Microbiol Biotechnol, 1992, 37:141-146.
[127]de Carvalho CCCR. Enzymatic and whole cell catalysis: Finding new strategies for old processe [J]. Biotechnol Adv, 2011, 29:75-83.
[128]Hama S, Yamaji H, Kaieda M, et al. Effect of fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel production [J]. Biochem Eng J, 2004, 21:155-160.
[129]Liu Y, Hama H, Fujita Y, et al. Production of S-lactoylglutathione by high activity wholecell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols [J]. Biotechnol Bioeng, 1999, 64:54-60.
[130]van der Werf MJ, Hartmans S, van den Tweel WJJ. Permeabilization and lysis of Pseudomonas pseudoalcaligenes cells by triton X-100 for efficient production of D-matate [J]. Appl Microbiol Biotechnol, 1995, 43:590-594.
[131]Silveira MM, Jonas R. The biotechnological production of sorbitol [J]. Appl Microbiol Biotechnol, 2002, 59:400-409.
[132]Upadhya R, Nagajyothi, Bhat SG. Stabilization of D-amino caid oxidase and catalase in permeabilized Rhodotorula gracilis cells and its application for the preparation ofα-ketoacids [J]. Biotechnol Bioeng, 2000, 68:430-436.
[133]Cavonas M, Torroglosa T, Kleber H, et al. Effect of salt stress on crotonobetaine and D-carnitine biotransformation of L-carnitine by resting cells of Escherichia coli [J]. J Basic Microbiol, 2003, 43:259-268.
[134]Matsumoto T, Takahash S, Kaieda M, et al. Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production [J]. Appl Microbiol Biotechnol, 2001, 57:515-520.
[135]Breedveld MW, Zevenhuizen LP, Zehnder AJB. Synthesis of cyclicβ-1,2-glucans by Rhizobium leguminosarum Biovartrifolii TA-1: factors influencing excretion [J]. J Bacteriol, 1992, 174:6336-6342.
[136]Isoai A, Kimura H, Reichert A, et al. Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysis prepared with recombinant cells [J]. Biotechnol Bioeng, 2002, 80:22-32.
[137]Ni Y, Chen R. Lipoprotein mutation accelerates substrate-permeability limited toluene dioxygenase- catalyzed reaction [J]. Biotechnol Prog, 2005, 21:799-805.
[138]Liang GB, Liao X, Du GC, et al. Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis [J]. J Appl Microbiol, 2008, 105:1432-1440.
[139]Sonntag K, Eggeling K, De Graaf AA, et al. Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum quantification by 13C- and 1H-NMR spectroscopy [J]. Eur J Biochem, 1993, 213:1325-1331.
[140]Kunioka M, Goto A. Biosynthesis of poly(γ-glutamic acid) from L-glutamic acid, citric acid and ammonium sulfate in Bacillus subtilis IFO3335 [J]. Appl Microbiol Biotechnol, 1995, 44:501-506.
[141]Maeda S, Kunimotob KK, Sasaki C, et al. Characterization of microbial poly (ε-L- lysine) by FT-IR, Raman and solid state 13C NMR spectroscopies [J]. J Mol Struct, 2003,655:149-155.
[142]杨革,王宁,张超.全细胞催化合成医用纺织新材料聚γ-谷氨酸[J].过程工程学报, 2010, 10:777-780.
[143]Chen RR. Permeability issues in whole-cell bioprocesses and cellular membrane engineering [J]. Appl Microbiol Biotechnol, 2007, 74:730-738.
[144]刘志钰,张东旭,堵国成,等. Paecilomyces sp. S152胞外漆酶的合成调控[J].生物技术学报, 2010, 29:952-958.
[145]Liang GB, Du GC, Chen J. A novel strategy of enhanced glutathione production in high cell density cultivation of Candida utilis—Cysteine addition combined with dissolved oxygen controlling [J]. Enzyme Microb Technol, 2008, 42:284-289.
[146]Wei GY, Wang DH, Chen J. Overproduction of glutathione by L-cysteine addition and a temperature-shift strategy [J]. Biotechnol Bioproc E, 2008, 13:347-353.
[147]Yoon SH, Do JH, Lee SY, et al. Production of poly-γ-glutamic acid by fed-batch culture of Bacillus Licheniformis [J]. Biotechnol Lett, 2000, 22:585-588.