Arshad Rubina, Farooq Shafqat, Ali Syed Shahid
Nuclear Institute for Agriculture and Biology, P.O. Box 128, Jhang Road, Faisalabad, Pakistan.
Pak J Biol Sci. 2007 Sep 15;10(18):3190-4. doi: 10.3923/pjbs.2007.3190.3194.
The aim of present study was to optimize conditions for conversion of penicillin G into 6-APA using intact crude cells of locally collected PGA producing bacterial strains as biocatalyst. Corn steep liquor medium supplemented with phenylacetic acid was used for PGA production. For enzymatic conversion of penicillin G into 6-APA by PGA impregnated bacterial cells, a maximum reaction time of 4 h was found adequate. The procedure for extraction and crystallization of 6-APA from the enzyme reaction mixture was standardized. Isolation process was carried out under controlled pH conditions and 6-APA crystals were recovered from the reaction mixture via filtration, concentration and drying. The maximum PGA activity was observed in Escherichia coli strain BDCS-N-FMu12 (6.4 mg 6-APA h(-1) mg(-1) wet cells) whereas Bacillus megaterium (ATCC 14945 used as check) exhibited only 2.4 mg 6-APA h(-1) mg(-1) wet cells. The overall yield of 6-APA crystals obtained after enzymatic conversion of penicillin G ranged between 37-55 and 47-68% in foreign and local strains, respectively. BDCS-N-FMu12 was identified as the best PGA producer with 68% 6-APA conversion whereas ATCC 14945 showed the lowest conversion (37%). The recovery of 6-APA (68%) obtained by using crude intact cells as cheap biocatalyst appeared promising. The process of enzyme fermentation and 6-APA crystallization optimized during this study seems cost-effective and environment-friendly. However, further studies are required to scale up the 6-APA biosynthesis reaction for achieving 80-90% conversion of penicillin G into 6-APA by PGA hyper-producing locally collected strains of E. coli.
本研究的目的是利用本地收集的产青霉素酰化酶（PGA）细菌菌株的完整粗细胞作为生物催化剂，优化将青霉素G转化为6-氨基青霉烷酸（6-APA）的条件。以添加苯乙酸的玉米浆培养基用于生产PGA。对于用负载PGA的细菌细胞将青霉素G酶促转化为6-APA，发现4小时的最大反应时间就足够了。对从酶反应混合物中提取和结晶6-APA的程序进行了标准化。分离过程在控制的pH条件下进行，通过过滤、浓缩和干燥从反应混合物中回收6-APA晶体。在大肠杆菌菌株BDCS-N-FMu12中观察到最大的PGA活性（6.4 mg 6-APA h⁻¹ mg⁻¹湿细胞），而巨大芽孢杆菌（用作对照的ATCC 14945）仅表现出2.4 mg 6-APA h⁻¹ mg⁻¹湿细胞。青霉素G酶促转化后获得的6-APA晶体的总产率在国外菌株和本地菌株中分别为37 - 55%和47 - 68%。BDCS-N-FMu12被鉴定为最佳的PGA生产者，6-APA转化率为68%，而ATCC 14945的转化率最低（37%）。使用粗完整细胞作为廉价生物催化剂获得6-APA（68%）的回收率似乎很有前景。本研究中优化的酶发酵和6-APA结晶过程似乎具有成本效益且环境友好。然而，需要进一步研究扩大6-APA生物合成反应规模，以通过本地收集的高产PGA的大肠杆菌菌株实现青霉素G向6-APA 80 - 90%的转化率。
