青霉素酰化酶(penicillin acylase, PA)主要用于β-内酰胺类抗生素半合成工业生产。该文从雷氏普罗维登斯菌(Providencia rettgeri)获得pga基因,在毕赤酵母(Komagataella pastoris)GS115中进行异源表达,并对重组酶PA进行产酶条件优化。异源表达构建的结果表明,重组毕赤酵母发酵上清液酶活力为2 155.59 U/L,沉淀酶活力为1 375.75 U/L。重组酶的最佳诱导产酶条件为温度29 ℃,时间72 h,甲醇浓度1.5%,重组酶PA的活性为(5 003.72±50.623) U/L,酶活力较优化前提高了1.57倍,纯化后蛋白大小为72 kDa,蛋白浓度为1.58 mg/mL,纯化后重组酶的最适反应温度为60 ℃,最适pH值为7,Mn2+对其有强烈抑制作用,Al3+对其有强烈激活作用。该研究结果可为青霉素酰化酶的异源表达及进一步工业应用提供指导。
Penicillin acylase is mainly used in the semi-synthetic industrial production of β-lactam antibiotics.This thesis, the pga gene was obtained from Providencia rettgeri, heterologously expressed in Komagataella pastoris GS115, and the enzyme production conditions were optimized for the recombinant enzyme PA.The results of the heterologous expression constructs showed that the enzyme activity of reorganization K. pastoris fermentation supernatant was 2 155.59 U/L, and the precipitated enzyme activity was 1 375.75 U/L. The optimal conditions for induction of enzyme production of recombinant enzyme were 29 ℃, 72 h, and methanol concentration of 1.5%, and the activity of recombinant enzyme PA was (5 003.72±50.623) U/L, which was 1.57-fold higher than the pre-optimization enzyme activity, and the purified The protein size was 72 kDa, the protein concentration was 1.58 mg/mL, the optimum reaction temperature of the purified recombinant enzyme was 60 ℃, the optimum pH was 7, and it was strongly inhibited by Mn2+ and activated by Al3+.The results of this study can provide guidance for the heterologous expression of penicillin acylase and further industrial applications.
[1] PAN X, XU L, LI Y R, et al.Strategies to improve the biosynthesis of β-lactam antibiotics by penicillin G acylase:Progress and prospects[J].Frontiers in Bioengineering and Biotechnology, 2022, 10:936487.
[2] SAMBYAL K, SINGH R V.Exploitation of E.coli for the production of penicillin G amidase:A tool for the synthesis of semisynthetic β-lactam antibiotics[J].Journal, Genetic Engineering & Biotechnology, 2021, 19(1):156.
[3] KAMIYA T, TERAJI T, HASHIMOTO M, et al.Synthesis of new.beta.-lactam antibiotics[J].Journal of the American Chemical Society, 1975, 97(17):5020-5021.
[4] BRUNS W, HOPPE J, TSAI H, et al.Structure of the penicillin acylase gene from Escherichia coli:A periplasmic enzyme that undergoes multiple proteolytic processing[J].Journal of Molecular and Applied Genetics, 1985, 3(1):36-44.
[5] LJUBIJANKIĆ G, KONSTANTINOVIĆ M, GLIŠIN V.The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases[J].DNA Sequence, 199, 3(3):195-200.
[6] CHENG T F, CHEN M L, ZHENG H B, et al.Expression and purification of penicillin G acylase enzymes from four different micro-organisms, and a comparative evaluation of their synthesis/hydrolysis ratios for cephalexin[J].Protein Expression and Purification, 2006, 46(1):107-113.
[7] GASSLER T, SAUER M, GASSER B, et al.The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2[J].Nature Biotechnology, 2020, 38(2):210-216.
[8] AHMAD M, HIRZ M, PICHLER H, et al.Protein expression in Pichia pastoris:Recent achievements and perspectives for heterologous protein production[J].Applied Microbiology and Biotechnology, 2014, 98(12):5301-5317.
[9] BORČINOVÁ M, RASCHMANOVÁ H, ZAMORA I, et al.Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris[J].Applied Microbiology and Biotechnology, 2020, 104(13):5787-5800.
[10] ZHANG X Y, CHEN S T, LIN Y, et al.Metabolic engineering of Pichia pastoris for high-level production of lycopene[J].ACS Synthetic Biology, 2023, 12(10):2961-2972.
[11] ARAYA-GARAY J M, FEIJOO-SIOTA L, ROSA-DOS-SANTOS F, et al.Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene[J].Applied Microbiology and Biotechnology, 2012, 93(6):2483-2492.
[12] GAO Y, ZHAO H L, FENG X, et al.Expression of recombinant human lysozyme-tachyplesin i (hlyz-tp i) in Pichia pastoris and analysis of antibacterial activity[J].Biomedical and Environmental Sciences, 201, 26(4):319.
[13] VICI A C, DA CRUZ A F, FACCHINI F D A, et al.Beauveria bassiana Lipase A expressed in Komagataella (Pichia) pastoris with potential for biodiesel catalysis[J].Frontiers in Microbiology, 2015, 6:1083.
[14] WEN Y, SHI X L, YUAN Z Y, et al.Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli[J].Protein Expression and Purification, 2004, 38(1):24-28.
[15] 吴卓颖. 产青霉素G酰化酶枯草芽孢杆菌工程菌株的构建及其发酵条件的优化[D].兰州:甘肃农业大学, 2015.
WU Z Y.Construction of Bacillus subtilis engineering strain producing penicillin G acylase and optimization of its fermentation conditions[D].Lanzhou:Gansu Agricultural University, 2015.
[16] 李成成, 黄义德.毕赤酵母表达系统及其发酵策略[J].福建轻纺, 2023(10):31-36.
LI C C, HUANG Y D.Pichia pastoris expression system and its fermentation strategy[J].The Light & Textile Industries of Fujian, 2023(10):31-36.
[17] AVELAR M, OLVERA C, ACEVES-ZAMUDIO D, et al.Recombinant expression of a laccase from Coriolopsis gallica in Pichia pastoris using a modified α-factor preproleader[J].Protein Expression and Purification, 2017, 136:14-19.
[18] 李怿韬, 曾伟主, 唐蕾.发酵过程优化提高毕赤酵母合成血红素[J].食品与发酵工业, 2024, 50(22):60-66.
LI Y T, ZENG W Z, TANG L.Enhancing heme biosynthesis in Pichia pastoris by fermentation process optimization[J].Food and Fermentation Industries, 2024, 50(22):60-66.
[19] 邓庆博. 细胞表面展示脲基酰胺水解酶降低黄酒中EC含量的研究[D].天津:天津科技大学, 2021.
DENG Q B.Study on Reducing the content of EC in Huangjiu by displaying urea amide hydrolase on cell surface[D].Tianjin:Tianjin University Of Science&technology,2021.
[20] KARBALAEI M, REZAEE S A, FARSIANI H.Pichia pastoris:A highly successful expression system for optimal synthesis of heterologous proteins[J].Journal of Cellular Physiology, 2020, 235(9):5867-5881.
[21] SEVO M, DEGRASSI G, SKOKO N, et al.Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris[J].FEMS yeast research, 200, 1(4):271-277.
[22] 王婕妤. 阿魏酸酯酶在毕赤酵母中的异源表达及酶学特性分析[D].无锡:江南大学, 2021.
WANG J Y.Heterologous expression of feruloyl esterase in Pichia pastoris and analysis of its enzymatic characteristics[D].Wuxi:Jiangnan University, 2021.
