节杆菌催化甾体化合物C_（1,2）脱氢规律的研究
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摘要
本文研究了简单节杆菌TCCC 10037(Arthrobacter simplex)和球形节杆菌TCCC 11039(Arthrobacter globiformis)对八种3-酮甾体底物的C1,2位脱氢转化反应,并研究了在四种不同的反应体系中,即完整细胞体系、双液相体系、原生质体体系和破碎细胞抽提液体系中的脱氢转化反应。
研究球形节杆菌的生长特性,发现球形节杆菌与简单节杆菌的生长状况极其相似,对球形节杆菌的转化条件及工艺进行了优化。
研究节杆菌在完整细胞体系下的C1,2位脱氢转化。简单节杆菌和球形节杆菌对不同甾体底物的C1,2位脱氢转化各不相同。同一微生物转化不同官能团的甾体底物的转化率有很大差异；不同微生物催化同一种甾体底物的转化率也不同。
研究节杆菌在双液相体系下的C1,2位脱氢转化,即增大甾体底物溶解度对转化的影响。构建了有机相为四氯化碳,水相为已培养至符合投料条件的发酵液,有机／水相比率为3：7(v：v)的两相系统,其中含有5g／L的甾体底物和0.2 g／L的甲萘醌。双液相体系中的C1,2位脱氢转化率比普通直接转化的转化率有所降低；简单节杆菌在双液相体系中的转化率大多明显高于球形节杆菌。
研究节杆菌在原生质体体系下的C1,2位脱氢转化,即细胞壁对C1,2位脱氢转化的影响。原生质体的最佳制备条件为：青霉素浓度1.5U/mL,青霉素处理时间8-10小时,溶菌酶浓度10mg／mL,溶菌酶酶解时间80-100分钟。原生质体体系下的C1,2位脱氢转化率明显低于完整细胞转化与双液相转化。
研究节杆菌在破碎细胞抽提液体系下的C1,2位脱氢转化,即研究细胞壁、细胞膜对C1,2位脱氢转化的影响。破碎细胞抽提液的最佳制备条件为：超声波输出功率380W,工作／间歇时间6s／5s,总时间7min,缓冲液为l/15mol/L pH 7.0的PBS缓冲液。节杆菌在细胞破碎体系中C1,2位脱氢转化率低于完整细胞转化和双液相转化,但高于原生质体转化。
可以看出在完整细胞中的C1,2位脱氢转化率>双液相体系>破碎细胞体系>原生质体体系,保证细胞的完整性对脱氢转化至关重要,采用Matlab软件建立甾体底物转化率与理化性质之间的数学模型,从数学模型中显示各底物的熔点和吸光系数对C1,2位脱氢转化有着正向促进作用,而比旋度对转化具有较强的负向抑制作用。
Study on the biotransformation of the 3-ketosteroid-C1,2-dehydrogenation by Arthrobacter simplex TCCC 10037 and Arthrobacter globiformis TCCC 11039 on four kinds of reaction system, which include whole-cell biotransformation, biphase biotransformation, protoplast biotransformation and ultrasonication biotransformation.
The growth characteristics of A. globiformis was studied. We found that it's growth conditions very similar to the A. simplex and transformation conditions were optimized.
3-ketosteroid was dehydrogenate with whole-cell by Arthrobacter. Biotransformation rate of 3-ketosteroid with different functional groups by A. simplex and A. globiformis have significant difference.
In order to increase the solubility of steroid substrates, an organic/aqueous two-liquid-phase system was developed to perform the biotransformation. The biphasic system composed of 70%(v/v) broth, and 30%(v/v) carbon tetrachloride containing 5g/L steroid substrate and 0.2g/L menadione as external electron acceptor. The bioconversion rate on biphasic system are lower than that on whole-cell system. And the bioconversion rate of 3-ketosteroid by A. simplex are significantly larger than that by A. globiformis.
To investigate the effect of the cell wall on biotransformation, the protoplast of strains were prepared. As a result, penicillin 1.5U/mL, penicillin treatment time 8-10 h, lysozyme 10mg/mL, lytic time 80-100 min. The biotransformation rate of protoplast was significantly decreased.
The broken cell extract was prepared by ultrasonication, in order to make the substrates direct contact with C1,2-dehydrogenase. The optimal condition on cell wall disruption output power 380 W, radiation/intermission time 6 s/5 s, total time 7 min, and 1/15mol/L pH7.0 PBS buffer.
Experimental results showed the sequence of reaction system, sorted from the highest to the lowest in the order of bioconversion rate, are whole-cell, biphase, ultrasonication, protoplast. It is very important to ensure the cell's integrality in the process of bioconversion. A mathematical model describing the bioconversion rule during the process ofΔ1-dehydrogenation with whole cells was established using MatLab software. The model showed that melting point and absorption coefficient increase the rate of biotransformation, while specific rotation decreases the rate of bioconversion.
研究球形节杆菌的生长特性,发现球形节杆菌与简单节杆菌的生长状况极其相似,对球形节杆菌的转化条件及工艺进行了优化。
研究节杆菌在完整细胞体系下的C1,2位脱氢转化。简单节杆菌和球形节杆菌对不同甾体底物的C1,2位脱氢转化各不相同。同一微生物转化不同官能团的甾体底物的转化率有很大差异；不同微生物催化同一种甾体底物的转化率也不同。
研究节杆菌在双液相体系下的C1,2位脱氢转化,即增大甾体底物溶解度对转化的影响。构建了有机相为四氯化碳,水相为已培养至符合投料条件的发酵液,有机／水相比率为3：7(v：v)的两相系统,其中含有5g／L的甾体底物和0.2 g／L的甲萘醌。双液相体系中的C1,2位脱氢转化率比普通直接转化的转化率有所降低；简单节杆菌在双液相体系中的转化率大多明显高于球形节杆菌。
研究节杆菌在原生质体体系下的C1,2位脱氢转化,即细胞壁对C1,2位脱氢转化的影响。原生质体的最佳制备条件为：青霉素浓度1.5U/mL,青霉素处理时间8-10小时,溶菌酶浓度10mg／mL,溶菌酶酶解时间80-100分钟。原生质体体系下的C1,2位脱氢转化率明显低于完整细胞转化与双液相转化。
研究节杆菌在破碎细胞抽提液体系下的C1,2位脱氢转化,即研究细胞壁、细胞膜对C1,2位脱氢转化的影响。破碎细胞抽提液的最佳制备条件为：超声波输出功率380W,工作／间歇时间6s／5s,总时间7min,缓冲液为l/15mol/L pH 7.0的PBS缓冲液。节杆菌在细胞破碎体系中C1,2位脱氢转化率低于完整细胞转化和双液相转化,但高于原生质体转化。
可以看出在完整细胞中的C1,2位脱氢转化率>双液相体系>破碎细胞体系>原生质体体系,保证细胞的完整性对脱氢转化至关重要,采用Matlab软件建立甾体底物转化率与理化性质之间的数学模型,从数学模型中显示各底物的熔点和吸光系数对C1,2位脱氢转化有着正向促进作用,而比旋度对转化具有较强的负向抑制作用。
Study on the biotransformation of the 3-ketosteroid-C1,2-dehydrogenation by Arthrobacter simplex TCCC 10037 and Arthrobacter globiformis TCCC 11039 on four kinds of reaction system, which include whole-cell biotransformation, biphase biotransformation, protoplast biotransformation and ultrasonication biotransformation.
The growth characteristics of A. globiformis was studied. We found that it's growth conditions very similar to the A. simplex and transformation conditions were optimized.
3-ketosteroid was dehydrogenate with whole-cell by Arthrobacter. Biotransformation rate of 3-ketosteroid with different functional groups by A. simplex and A. globiformis have significant difference.
In order to increase the solubility of steroid substrates, an organic/aqueous two-liquid-phase system was developed to perform the biotransformation. The biphasic system composed of 70%(v/v) broth, and 30%(v/v) carbon tetrachloride containing 5g/L steroid substrate and 0.2g/L menadione as external electron acceptor. The bioconversion rate on biphasic system are lower than that on whole-cell system. And the bioconversion rate of 3-ketosteroid by A. simplex are significantly larger than that by A. globiformis.
To investigate the effect of the cell wall on biotransformation, the protoplast of strains were prepared. As a result, penicillin 1.5U/mL, penicillin treatment time 8-10 h, lysozyme 10mg/mL, lytic time 80-100 min. The biotransformation rate of protoplast was significantly decreased.
The broken cell extract was prepared by ultrasonication, in order to make the substrates direct contact with C1,2-dehydrogenase. The optimal condition on cell wall disruption output power 380 W, radiation/intermission time 6 s/5 s, total time 7 min, and 1/15mol/L pH7.0 PBS buffer.
Experimental results showed the sequence of reaction system, sorted from the highest to the lowest in the order of bioconversion rate, are whole-cell, biphase, ultrasonication, protoplast. It is very important to ensure the cell's integrality in the process of bioconversion. A mathematical model describing the bioconversion rule during the process ofΔ1-dehydrogenation with whole cells was established using MatLab software. The model showed that melting point and absorption coefficient increase the rate of biotransformation, while specific rotation decreases the rate of bioconversion.
引文
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