构建重组枯草芽孢杆菌催化制备D-对羟基苯甘氨酸

目的: 在Bacillus subtilis中表达异源D-海因酶基因(hyd)和D-氨甲酰水解酶基因(adc),构建重组细胞作为催化剂,用于生产D-对羟基苯甘氨酸(D-HPG)。方法: 构建hyd表达质粒,考察培养基中二价金属离子对D-海因酶活性的影响。过表达acoR基因,考察AcoR蛋白胞内水平与P_acoA-hyd基因拷贝数的关系。筛选表达adc基因的启动子,构建hyd和adc基因共表达质粒,考察双酶活性菌株的催化特性。结果: 成功构建了海因酶表达质粒pHPS和pUBS,培养基中添加0.8mmol/L的MnCl₂·4H₂O,使168N/pUBS菌株的D-海因酶活性达到956U/gDCW。整合表达P_cdd-acoR基因,使LSL02/pUBS菌株的D-海因酶活性达到1 470U/gDCW。单拷贝P_AE-adc基因的表达水平相对最高。双酶共表达质粒pUBSC被成功构建,菌株LSL02/pUBSC的最适催化温度为40℃45℃,催化活性能够持续12h,当底物起始浓度为20g/L时,反应12h生成的D-HPG达到14.32g/L,转化率达到95%,收率超过80%。结论: 构建具有D-海因酶和D-氨甲酰水解酶双酶活性的重组Bacillus subtilis作为全细胞催化剂,用于海因酶法生产D-HPG,具有技术上的可行性和优势。

Construction of Recombinant Bacillus subtilis as Catalyst for Preparing D- p-Hydroxyphenylglycine

Objective: To construct the recombinant Bacillus subtilis with D-hydantoinase(DHase, hyd ene) and D-carbamoylase(DCase, adc gene) activity as catalyst to produce D-p-hydroxyphenylglycine(D-HPG) by hydantoinase process. Methods: The hyd gene expression plasmids were constructed. The effects of divalent metal ions in medium on the DHase activity was investigated. The acoR gene was over-expressed to investigate the correlation between the activator protein AcoR and P_acoA-hyd gene expression. The optimal promoter used to express adc gene was screened from the P_AE, P_spoVG, P_cdd and P_lytR. The hyd and adc gene co-expression plasmid was constructed and its catalytic properties was characterized. Results: The hyd gene expression plasmid pHPS and pUBS were successfully constructed. Mn ²⁺ has a strong activation effect on DHase and the activity of the 168N/pUBS reached 956U/gDCW when 0.8mmol/L MnCl₂·4H₂O was added to the medium. Adding a copy of the P_cdd-acoR gene, DHase activity of the LSL02/pUBS reached 1 470 U/gDCW. The LN04 strain integrated with the P_AE-adc had the highest DCase activity. The co-expression plasmid pUBSC was constructed, and under the optimum conditions of pH 8.0 and 40℃, with initial substrate concentration of 20g/L, the catalytic activity of LSL02/pUBSC could last for 12h to generate 14.32g/L of D-HPG with the conversion rate of 95%, yield of 82.4%. Conclusion: The recombinant strain with higher dual enzyme activity can be obtained when heterologous hyd and adc were expressed in Bacillus subtilis and it is technically feasible and has the application prospect for preparing D-HPG by hydantoinase process.