产DCase的基因工程菌发酵及酶固定化工艺研究
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摘要
D-对羟基苯甘氨酸(D-pHPG)是半合成青霉素和头孢霉素的重要前体，是目前制药行业竞争激烈的产品之一，化学法制备D-pHPG带来严重的污染和毒害，现在酶法制备得到人们的青睐。酶法是利用D-海因酶(DHase)将苯海因(DL-pHPH)转化为N-氨甲酰基-D-对羟基苯甘氨酸(NC-D-pHPG)，N-氨甲酰基-D-氨基酸酰胺水解酶(DCase)则继续将NC-D-pHPG转化为D-pHPG。但现有的菌种产酶活力不高，故提高酶活力和采取新技术提高酶利用率有着重要研究价值。
本实验菌种spMF0507是产DHase和DCase的野生菌，而DHase活力远远大于DCase，因此可以通过控制适当pH值，使DCase失活，而保留DHase活力生产NC-D-pHPG。此外还有一菌种Espf7是克隆了DCase基因的大肠杆菌，它能表达DCase活性继续将NC-D-pHPG转化为D-pHPG，可DCase是胞内酶，故需要对其进行细胞破碎才能释放出酶。另外利用正交法对spMF0507的培养基组成和发酵培养条件也进行了研究，之后把发酵优化结果投入5升发酵罐进行验证。最后对工程菌Espf7发酵后提取的酶进行了固定化研究，通过测定蛋白质含量和酶活力来确定固定化结果。以下是整个实验结果：
(1) 菌种spMF0507的适宜接种龄是20～24h，pH值控制在9.0～9.5时，可以使DCase失活，DHase活力仍有0.2u·mL~(-1)，在该条件下来制备NC-D-pHPG。但转化过程中，NC-D-pHPG容易被氧化成红色物质，而通氮气能防止其被氧化。提取NC-D-pHPG时加入2％的丙酮能加快结晶速度，结晶率达到97.9％，NC-D-pHPG的纯度达98.5％。
(2) 基因工程菌Espf7在0.02％的氯仿用量下可以通透细胞膜，适宜接种龄为16～20h，接种量2％，初始pH值7.0，培养温度30℃，阿拉伯糖0.01％；甘油0.30％；玉米浆3％；NaCL 0.2％；KH_2PO_4 0.05％；MgSO_4 0.05％；MnCL_2 0.02％；CoCL_2 0.01％，使DCase活力由1.12u·mL~(-1)提高到2.1u·mL~(-1)，提高了87.5％。在发酵罐上控制条件温度30℃、转速500r／min，pH设定在7.0来补料，结果活力提高到3u·mL~(-1)。
(3) 均质机破碎细胞的最佳条件为：压力控制在70MPa，破碎时间15min。以TJS环氧基树脂作为固定载体，最适固定化条件为：TJS用量1g对应133u酶活，固定时间15h，温度28℃，pH值7.5最后固定化酶活为58u·g~(-1)，蛋白固定率可达98％，酶活回收率达到49.3％。国产载体与进口载体固定化相比较：国外固定化酶活力比国内高3u·g~(-1)，但国产的半衰期为35批，而国外为25批，明显国内的固定化酶较稳定。
D-hydroxyphenylglycine(D-pHPG) was the most important precursor used for the synthesis of Semisynthetic penicillin and cephalospor and was a produce competed furiously in the field of pharmacy.Making D-pHPG,enzyme method was better than chemical method which was poisonous.In enzyme method, P-hydroxyphenyldantoin (p-HPH) was transformed into N-Carbamoyl-D-P-hydroxyphenyldantoin(NC-D-pHPG) by Dhydantoinase(DHase), then NC-D-pHPG was transformed into D-pHPG by Ncarbamyl Danimo-acidamidohydrolase (DCase). But now the activity of strain was low,it was very significative that the activity of strain was improved.
The strain of spMF0507 could produce DHase and DCase, but the activity of DHase was higher than the one of DCase.Controlling pH DHase had no activity but DCase for producing NC-D-pHPG.However the Espf7 was gene engineering strain of DCase's genes cloned in E.coli.DCase expressed by Espf7 could transform NC-D-pHPG into D-pHPG.But DCase was in cell membrance,Dialysing cell membrance was investigated.Moreover by orthogonal optimization the culture condition and the culture medium were investigated,then the result of optimization was used to fermentor.In end under of detemining protein and enzyme activity the result of immobilizing DCase was conformed.The results of all experiments were followings:
(1) The pre-inoculation time of spMF0507 was 20~ 24h,under pH9.0-9.5 DCase had no activity but DHase 0.2 u ? ml~(-1) .Otherwise NC-D-pHPG which was oxidated into red matter was prevented by N_2 in the course of transformation. Appending 2% acetone increased crystallization , the crystallized rate was 97.9% and the pure degree of NC-D-pHPG was 99% by HP LC.
(2) The cell membrance of Espf7 was dialysed by 0.02% CHCl_3 the pre-inoculation time of 16~20h,the inoculum ratio at 2%,initial pH of 7.0,temperature of 30°C,arabinose 0.01%; glycerin 0.30%;Corn steep liquor 3%;NaCL 0.2%;KH_2PO_4 0.05%;MgSO_4 0.05%;MnCL_2 0.02%;CoCL_2 0.01%,the activity of DCase was increased from 1.12 u ? ml~(-1) to 2.1 u ? ml~(-1), increased by 87.5%.The activity of DCase reached 3 u ? ml~(-1) under 5L fermentor conditions including: temperature 30°C, the shaking speed of 500r ? min~(-1) fed in pH of 7.0.
(3) DCase was dialysed via pressure of 70MPa,time of 15min. TJS was used as carriers for immobilization of DCase,the optimized conditions were the amount of TJS of
本实验菌种spMF0507是产DHase和DCase的野生菌，而DHase活力远远大于DCase，因此可以通过控制适当pH值，使DCase失活，而保留DHase活力生产NC-D-pHPG。此外还有一菌种Espf7是克隆了DCase基因的大肠杆菌，它能表达DCase活性继续将NC-D-pHPG转化为D-pHPG，可DCase是胞内酶，故需要对其进行细胞破碎才能释放出酶。另外利用正交法对spMF0507的培养基组成和发酵培养条件也进行了研究，之后把发酵优化结果投入5升发酵罐进行验证。最后对工程菌Espf7发酵后提取的酶进行了固定化研究，通过测定蛋白质含量和酶活力来确定固定化结果。以下是整个实验结果：
(1) 菌种spMF0507的适宜接种龄是20～24h，pH值控制在9.0～9.5时，可以使DCase失活，DHase活力仍有0.2u·mL~(-1)，在该条件下来制备NC-D-pHPG。但转化过程中，NC-D-pHPG容易被氧化成红色物质，而通氮气能防止其被氧化。提取NC-D-pHPG时加入2％的丙酮能加快结晶速度，结晶率达到97.9％，NC-D-pHPG的纯度达98.5％。
(2) 基因工程菌Espf7在0.02％的氯仿用量下可以通透细胞膜，适宜接种龄为16～20h，接种量2％，初始pH值7.0，培养温度30℃，阿拉伯糖0.01％；甘油0.30％；玉米浆3％；NaCL 0.2％；KH_2PO_4 0.05％；MgSO_4 0.05％；MnCL_2 0.02％；CoCL_2 0.01％，使DCase活力由1.12u·mL~(-1)提高到2.1u·mL~(-1)，提高了87.5％。在发酵罐上控制条件温度30℃、转速500r／min，pH设定在7.0来补料，结果活力提高到3u·mL~(-1)。
(3) 均质机破碎细胞的最佳条件为：压力控制在70MPa，破碎时间15min。以TJS环氧基树脂作为固定载体，最适固定化条件为：TJS用量1g对应133u酶活，固定时间15h，温度28℃，pH值7.5最后固定化酶活为58u·g~(-1)，蛋白固定率可达98％，酶活回收率达到49.3％。国产载体与进口载体固定化相比较：国外固定化酶活力比国内高3u·g~(-1)，但国产的半衰期为35批，而国外为25批，明显国内的固定化酶较稳定。
D-hydroxyphenylglycine(D-pHPG) was the most important precursor used for the synthesis of Semisynthetic penicillin and cephalospor and was a produce competed furiously in the field of pharmacy.Making D-pHPG,enzyme method was better than chemical method which was poisonous.In enzyme method, P-hydroxyphenyldantoin (p-HPH) was transformed into N-Carbamoyl-D-P-hydroxyphenyldantoin(NC-D-pHPG) by Dhydantoinase(DHase), then NC-D-pHPG was transformed into D-pHPG by Ncarbamyl Danimo-acidamidohydrolase (DCase). But now the activity of strain was low,it was very significative that the activity of strain was improved.
The strain of spMF0507 could produce DHase and DCase, but the activity of DHase was higher than the one of DCase.Controlling pH DHase had no activity but DCase for producing NC-D-pHPG.However the Espf7 was gene engineering strain of DCase's genes cloned in E.coli.DCase expressed by Espf7 could transform NC-D-pHPG into D-pHPG.But DCase was in cell membrance,Dialysing cell membrance was investigated.Moreover by orthogonal optimization the culture condition and the culture medium were investigated,then the result of optimization was used to fermentor.In end under of detemining protein and enzyme activity the result of immobilizing DCase was conformed.The results of all experiments were followings:
(1) The pre-inoculation time of spMF0507 was 20~ 24h,under pH9.0-9.5 DCase had no activity but DHase 0.2 u ? ml~(-1) .Otherwise NC-D-pHPG which was oxidated into red matter was prevented by N_2 in the course of transformation. Appending 2% acetone increased crystallization , the crystallized rate was 97.9% and the pure degree of NC-D-pHPG was 99% by HP LC.
(2) The cell membrance of Espf7 was dialysed by 0.02% CHCl_3 the pre-inoculation time of 16~20h,the inoculum ratio at 2%,initial pH of 7.0,temperature of 30°C,arabinose 0.01%; glycerin 0.30%;Corn steep liquor 3%;NaCL 0.2%;KH_2PO_4 0.05%;MgSO_4 0.05%;MnCL_2 0.02%;CoCL_2 0.01%,the activity of DCase was increased from 1.12 u ? ml~(-1) to 2.1 u ? ml~(-1), increased by 87.5%.The activity of DCase reached 3 u ? ml~(-1) under 5L fermentor conditions including: temperature 30°C, the shaking speed of 500r ? min~(-1) fed in pH of 7.0.
(3) DCase was dialysed via pressure of 70MPa,time of 15min. TJS was used as carriers for immobilization of DCase,the optimized conditions were the amount of TJS of
引文
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