利用粘红酵母转化反式肉桂酸生成L-苯丙氨酸的研究
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摘要
L-苯丙氨酸是具有生理活性的芳香族氨基酸，是人体和动物不能自身合成的必需氨基酸之一。苯丙氨酸广泛应用于食品加工、饲料添加剂、医药等行业中。L-苯丙氨酸与天冬氨酸缩合制成的二肽甲酯天冬甜肽目前已被多个国家获准销售。这种甜味剂的广阔市场使苯丙氨酸的需求量不断加大。天冬甜肽最近被证实具有提神醒脑及减肥等功效，更加促进了苯丙氨酸的市场需求。
采用合成培养基，确定了粘红酵母产酶模式为滞后合成型，研究了苯丙氨酸解氨酶的产酶动力学，建立了产酶动力学的数学模型，方程式为：dE/dt=βX。研究了参数β的简便求法及意义。分析了影响产酶动力学参数的因素，发现诱导物添加时间、添加量等因素都影响产酶动力学方程的参数β。
基于对粘红酵母PAL产酶动力学方程的分析，优化了影响方程参数的因素。菌体在第18小时达到最大出芽率。此时转接，菌体在产酶培养基中生长与产酶情况最好。通过单因子实验与正交实验优化得到的最佳天然培养基配方为(g／L)；葡萄糖0.4，酵母膏8.0，玉米浆30.0，NaCl 5；KH_2PO_4 1。七水合硫酸镁、七水合硫酸亚铁、六水合硫酸锰等无机盐对于菌体生长与产酶皆无明显影响。0.10g／L钼酸钠能够促进粘红酵母的生长。1.0g／L K_2HPO_4对于促进菌体生长的效果优于1.0 g／L KH_2PO_4的效果。D-苯丙氨酸对于PAL的产生具有安慰诱导作用。菌龄为8h时添加诱导物，诱导物由终浓度为0.40g／L的L-Tyr与0.10g／L的DL-Phe组成。通过产酶条件优化，最终使得酶活力增加了75％，非生长偶联的比产酶速率提高了122％，生物量提高了5.4％，培养时间缩短了45％。
通过单因素实验和正交实验优化了转化条件。转化液成分优化为5.6mol／L[NH_4~+]、14.5％[HCO_3~-]并调pH10.5(t-Ca浓度为0.1％)。选择0.05 g／L氯化十六烷基吡啶加入转化液中可以促进转化反应。
研究了谷氨酸钠、海藻酸钠、聚乙二醇、甘油、锌粉、氮气对PAL稳定性的影响。通过单因子实验和正交实验，优化确定在底物反应液中添加1.0g／L锌粉和20.0g／L谷氨酸钠。最终，苯丙氨酸积累浓度提高到27.52～31.90g／L。
L-Phenylalanine (L-Phe), an essential aromatic amino acid, had found wide use in food processing, feed additive and pharmaceutical industry. Aspartame, an important sweetener produced from L-phenylalanine was sanctified to sell by many countries whose wide application greatly increased the demand of L-phenylalanine.
Properties of yeast were tested in synthetic medium by a serial of experiments, during which the enzyme-producing mode was determined, finding that phenylalanine ammonia lyase (PAL) was a kind of enzyme produced in the lag-phase. Based upon this kind of lag-phase-producing-mode model, PAL-producing dynamics was studied and the non-structure mathematic model was set up. The equation of this PAL-producing mathematic dynamics model listed herewith:
The easier approach to work out the parameter and the significance of P were discussed. Factors, such as the inducer adding time, the amount of inducer, were analyzed. In Yamada medium, a kind of synthetic medium used in this paper in which Rhodotorula glutinis was cultured and PAL was induced, 0.40g/L L-Tyr, the inducer, was added in lag-phase.
Experiments heretofore had set up the theoretical basis for optimizing PAL-producing conditions. Based upon the PAL-producing dynamic equation, experiments about optimizing PAL-producing conditions were performed. Yeast was transferred when the budding rate of Rhodotorula glutinis came up to the largest amount in the 18th hour in seed medium considering the culture's highest enzymatic activity in complex inducing medium. By single-factor experiments and orthogonal experiments, complex inducing medium was optimized (g/L) as follow: glucose 0.4, yeast extract 8.0, corn steep liquor 30.0, NaCl 5, KH2PO4 1.
Salts, including MgSO4 '7H2O, FeSO4 '7H2O, MnSO4 -6H2O had little effects on the growth of Rhodotorula glutinis in complex inducing medium. 1.0g/L.K2HPO4 had better effect on the growth of Rhodotorula glutinis than 1.0g/L.KH2PO4 did.
D-phenylalanine had the gratuitous effect on the inducing of PAL. After yeast was cultured in complex inducing medium for 8h, 0.40g/L L-Tyr and 0. lOg/L DL-Phe were added to complex inducing medium. By optimizing the PAL-producing condition, the non-growth-coupled specific PAL-producing rate was increased by 122 %, and the biomass was increased by 4.5%.
In this paper, trans-cinnamic acid was conversed by resting yeast cell in
conversion-solution. The conversion solution composition was optimized which was: 5.6mol/L [NH4+], 14.5% [HCO3~] . (The concentration of trans-cinnamic acid was 1.0% and pH was 10.5). Mycostatin had little or negative effect on PAL-producing. Cetylpyridinium chloride can increase the bioconversion rate.
By single-factor experiments, the effects on the stability of PAL of sodium glutamate, sodium alginate, polyethylene glycol, glycerin, zinc powder, nitrogen gas were studied. By orthogonal experiment and divergence analysis, 1 .Og/L zinc powder and 20g/L sodium glutamate added to conversion-solution, the concentration of L-Phenylalanine was increased by from 18.7g/L to 27.52~31.90g/L, finding that those two effectors, zinc powder and sodium glutamate, had significant effects on the stability of PAL.
采用合成培养基，确定了粘红酵母产酶模式为滞后合成型，研究了苯丙氨酸解氨酶的产酶动力学，建立了产酶动力学的数学模型，方程式为：dE/dt=βX。研究了参数β的简便求法及意义。分析了影响产酶动力学参数的因素，发现诱导物添加时间、添加量等因素都影响产酶动力学方程的参数β。
基于对粘红酵母PAL产酶动力学方程的分析，优化了影响方程参数的因素。菌体在第18小时达到最大出芽率。此时转接，菌体在产酶培养基中生长与产酶情况最好。通过单因子实验与正交实验优化得到的最佳天然培养基配方为(g／L)；葡萄糖0.4，酵母膏8.0，玉米浆30.0，NaCl 5；KH_2PO_4 1。七水合硫酸镁、七水合硫酸亚铁、六水合硫酸锰等无机盐对于菌体生长与产酶皆无明显影响。0.10g／L钼酸钠能够促进粘红酵母的生长。1.0g／L K_2HPO_4对于促进菌体生长的效果优于1.0 g／L KH_2PO_4的效果。D-苯丙氨酸对于PAL的产生具有安慰诱导作用。菌龄为8h时添加诱导物，诱导物由终浓度为0.40g／L的L-Tyr与0.10g／L的DL-Phe组成。通过产酶条件优化，最终使得酶活力增加了75％，非生长偶联的比产酶速率提高了122％，生物量提高了5.4％，培养时间缩短了45％。
通过单因素实验和正交实验优化了转化条件。转化液成分优化为5.6mol／L[NH_4~+]、14.5％[HCO_3~-]并调pH10.5(t-Ca浓度为0.1％)。选择0.05 g／L氯化十六烷基吡啶加入转化液中可以促进转化反应。
研究了谷氨酸钠、海藻酸钠、聚乙二醇、甘油、锌粉、氮气对PAL稳定性的影响。通过单因子实验和正交实验，优化确定在底物反应液中添加1.0g／L锌粉和20.0g／L谷氨酸钠。最终，苯丙氨酸积累浓度提高到27.52～31.90g／L。
L-Phenylalanine (L-Phe), an essential aromatic amino acid, had found wide use in food processing, feed additive and pharmaceutical industry. Aspartame, an important sweetener produced from L-phenylalanine was sanctified to sell by many countries whose wide application greatly increased the demand of L-phenylalanine.
Properties of yeast were tested in synthetic medium by a serial of experiments, during which the enzyme-producing mode was determined, finding that phenylalanine ammonia lyase (PAL) was a kind of enzyme produced in the lag-phase. Based upon this kind of lag-phase-producing-mode model, PAL-producing dynamics was studied and the non-structure mathematic model was set up. The equation of this PAL-producing mathematic dynamics model listed herewith:
The easier approach to work out the parameter and the significance of P were discussed. Factors, such as the inducer adding time, the amount of inducer, were analyzed. In Yamada medium, a kind of synthetic medium used in this paper in which Rhodotorula glutinis was cultured and PAL was induced, 0.40g/L L-Tyr, the inducer, was added in lag-phase.
Experiments heretofore had set up the theoretical basis for optimizing PAL-producing conditions. Based upon the PAL-producing dynamic equation, experiments about optimizing PAL-producing conditions were performed. Yeast was transferred when the budding rate of Rhodotorula glutinis came up to the largest amount in the 18th hour in seed medium considering the culture's highest enzymatic activity in complex inducing medium. By single-factor experiments and orthogonal experiments, complex inducing medium was optimized (g/L) as follow: glucose 0.4, yeast extract 8.0, corn steep liquor 30.0, NaCl 5, KH2PO4 1.
Salts, including MgSO4 '7H2O, FeSO4 '7H2O, MnSO4 -6H2O had little effects on the growth of Rhodotorula glutinis in complex inducing medium. 1.0g/L.K2HPO4 had better effect on the growth of Rhodotorula glutinis than 1.0g/L.KH2PO4 did.
D-phenylalanine had the gratuitous effect on the inducing of PAL. After yeast was cultured in complex inducing medium for 8h, 0.40g/L L-Tyr and 0. lOg/L DL-Phe were added to complex inducing medium. By optimizing the PAL-producing condition, the non-growth-coupled specific PAL-producing rate was increased by 122 %, and the biomass was increased by 4.5%.
In this paper, trans-cinnamic acid was conversed by resting yeast cell in
conversion-solution. The conversion solution composition was optimized which was: 5.6mol/L [NH4+], 14.5% [HCO3~] . (The concentration of trans-cinnamic acid was 1.0% and pH was 10.5). Mycostatin had little or negative effect on PAL-producing. Cetylpyridinium chloride can increase the bioconversion rate.
By single-factor experiments, the effects on the stability of PAL of sodium glutamate, sodium alginate, polyethylene glycol, glycerin, zinc powder, nitrogen gas were studied. By orthogonal experiment and divergence analysis, 1 .Og/L zinc powder and 20g/L sodium glutamate added to conversion-solution, the concentration of L-Phenylalanine was increased by from 18.7g/L to 27.52~31.90g/L, finding that those two effectors, zinc powder and sodium glutamate, had significant effects on the stability of PAL.
引文
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