Panigrahi Priyabrata, Chand Deepak, Mukherji Ruchira, Ramasamy Sureshkumar, Suresh C G
Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India.
J Ind Microbiol Biotechnol. 2015 Nov;42(11):1493-506. doi: 10.1007/s10295-015-1690-x. Epub 2015 Sep 29.
Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in β-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.
青霉素酰化酶是制药行业用于制造半合成青霉素的酶。在这类应用中，对热稳定和嗜碱酶的需求持续存在。我们利用各种蛋白质稳定因子，对已知的青霉素G酰化酶（PGA）的热稳定性质进行了计算分析。最初考虑二硫键的存在，以便从数据库中筛选假定的热稳定PGA，同时还考虑了其他各种因素进行比较，如高的精氨酸与赖氨酸比例、热不稳定氨基酸含量较低、β-转角中脯氨酸的存在、更多数量的离子对和其他非键相互作用。设计的一种改进的共识方法可以通过中稳定和热稳定PGA之间的位点特异性比较，进一步确定稳定残基位置。从分析中鉴定出的最有可能的热稳定酶是反硝化副球菌的PGA（PdPGA）。对其进行了克隆、表达，并通过生化和生物物理实验测试了其热稳定性质。基于共识位点特异性序列的方法预测PdPGA比大肠杆菌PGA更耐热，但不如木糖氧化无色杆菌的PGA耐热。实验数据表明，尽管热稳定性因素有利于更高的稳定性，但PdPGA的热稳定性相对低于木糖氧化无色杆菌PGA。尽管PdPGA是中稳定的，但它在碱性pH下具有活性和稳定性是一个优势。最后，在PdPGA中可以鉴定出几个残基位置，通过选择性突变可以提高该酶的热稳定性。
