The purine nucleoside phosphorylase (PNPase) is a key enzyme in the purine remediation synthesis pathway. The purine degradation pathway involves oxidative purine ring cleavage, which forms uric acid as an end product in body and increases the risk of gout. To improve the fermentation level of purine nucleoside phosphorylase, in this study, the purine nucleoside phosphorylase gene derived from Kluyveromyces lactis was codon optimized, and the target PNP gene before and after optimization was ligated to the secretory expression vector pBSA43, respectively, and PNP-YP-pBSA43 and PNP-YO-pBSA43 were recombinantly expressed in Bacillus subtilis WB600. The results showed that the enzyme activity was 333.69 U/mL before codon optimization and 351.61 U/mL after optimization. To further improve the recombinant protein expression, the optimal fermentation conditions for PNPase production by the optimized recombinant strain were derived from response surface analysis: initial pH 6.67, 55 h, and 28.7 ℃. Under these conditions, the recombinant purine nucleoside phosphorylase activity was 372.79 U/mL, which was 12% higher than that before optimization. The recombinant expression of purine nucleoside phosphorylase gene from K. lactis with codon optimization significantly improved the enzyme activity and laid the foundation for the efficient expression of purine nucleoside phosphorylase in B. subtilis.
[1] ERION M D, TAKABAYASHI K, SMITH H B, et al.Purine nucleoside phosphorylase.1.structure-function studies[J].Biochemistry, 1997, 36(39):11725-11734.
[2] TIMOFEEV V I, ZHUKHLISTOVA N E, ABRAMCHIK Y A, et al.Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine[J].Acta Crystallographica Section F Structural Biology Communications, 2018, 74(6):355-362.
[3] DESSANTI P, ZHANG Y, ALLEGRINI S, et al.Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase[J].Acta Crystallographica Section D Biological Crystallography, 2012, 68(3):239-248.
[4] STOECKLER J D, POIROT A F, SMITH R M, et al.Purine nucleoside phosphorylase.3.reversal of purine base specificity by site-directed mutagenesis[J].Biochemistry, 1997, 36(39):11749-11756.
[5] KOELLNER G, LUIĆ M, SHUGAR D, et al.Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 Å resolution 1[J].Journal of Molecular Biology, 1997, 265(2):202-216.
[6] LECOQ K, BELLOC I, DESGRANGES C, et al.YLR209c encodes Saccharomyces cerevisiae purine nucleoside phosphorylase[J].Journal of Bacteriology, 2001, 183(16):4910-4913.
[7] TAHIROV T H, INAGAKI E, OHSHIMA N, et al.Crystal structure of purine nucleoside phosphorylase from Thermus thermophilus[J].Journal of Molecular Biology, 2004, 337(5):1149-1160.
[8] VISSER D F, HENNESSY F, RASHAMUSE K, et al.Cloning, purification and characterisation of a recombinant purine nucleoside phosphorylase from Bacillus halodurans Alk36[J].Extremophiles, 2010, 14(2):185-192.
[9] 戴芳萍. 嘌呤核苷磷酸化酶合成利巴韦林的应用研究[D].上海:华东理工大学, 2019.
DAI F P.Study on the synthesis of ribavirin by purine nucleoside phosphorylase[D].Shanghai:East China University of Science and Technology, 2019.
[10] MAHOR D, PRIYANKA A, PRASAD G S, et al.Functional and structural characterization of purine nucleoside phosphorylase from Kluyveromyces lactis and its potential applications in reducing purine content in food[J].PLoS One, 2016, 11(10):e0164279.
[11] 李玉淼, 朱德伟, 朱洪康, 等.嘌呤核苷磷酸化酶的表达及其在啤酒中的应用[J].食品与生物技术学报, 2017, 36(12):1264-1268.
LI Y M, ZHU D W, ZHU H K, et al.Expression of purine nucleoside phosphorylase and its application in beer[J].Journal of Food Science and Biotechnology, 2017, 36(12):1264-1268.
[12] OUYANG X Y, LIU Y J, QU R D, et al.Optimizing protein-glutaminase expression in Bacillus subtilis[J].Current Microbiology, 2021, 78(5):1752-1762.
[13] HE Q, FU A Y, LI T J.Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus subtilis[J].Journal of Industrial Microbiology & Biotechnology, 2015, 42(4):647-653.
[14] 陈灵艳, 陈先进, 蒋烨, 等.猪圆环病毒衣壳蛋白在枯草芽孢杆菌中的表达研究[J].生物技术通报, 2016, 32(5):140-145.
CHEN L Y, CHEN X J, JIANG Y, et al.Research on the expression of porcine circovirus capsid protein in the Bacillus subtilis[J].Biotechnology Bulletin, 2016, 32(5):140-145.
[15] KUMAR N, PANDEY R, PRABHU A A, et al.Genetic and substrate-level modulation of Bacillus subtilis physiology for enhanced extracellular human interferon gamma production[J].Preparative Biochemistry & Biotechnology, 2018, 48(5):391-401.
[16] 王光路, 夏俊刚, 谢希贤, 等.假交替单胞菌XM2107嘌呤核苷磷酸化酶基因克隆表达、重组蛋白纯化及酶学性质[J].微生物学报, 2010, 50(2):222-227.
WANG G L, XIA J G, XIE X X, et al.Molecular cloning, gene expression and characterization of purine nucleoside phosphorylase from Pseudoalteromonas sp.XM2107[J].Acta Microbiologica Sinica, 2010, 50(2):222-227.
[17] ZHANG X Z, ZHANG Y.Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis[J].Microbial Biotechnology, 2011, 4(1):98-105.
[18] 张卫兰, 陶国庆, 刘刚, 等.氨基酸对大肠杆菌BL21产嘌呤核苷磷酸化酶的影响[J].化学与生物工程, 2010, 27(2):53-56.
ZHANG W L, TAO G Q, LIU G, et al.Influence of amino acids on the production of purine nucleoside phosphorylase by E.Coli BL21[J].Chemistry & Bioengineering, 2010, 27(2):53-56.
[19] 杨云彭, 马晓焉, 霍毅欣.密码子优化策略在异源蛋白表达中的应用[J].生物工程学报, 2019, 35(12):2227-2237.
YANG Y P, MA X Y, HUO Y X.Application of codon optimization strategy in heterologous protein expression[J].Chinese Journal of Biotechnology, 2019, 35(12):2227-2237.
[20] TIMOFEEV V I, FATEEV I V, KOSTROMINA M A, et al.The comparative analysis of the properties and structures of purine nucleoside phosphorylases from thermophilic bacterium Thermus thermophilus HB27[J].Journal of Biomolecular Structure and Dynamics, 2022, 40(8):3626-3641.
[21] ANTIPOV A N, MORDKOVICH N N, KHIJNIAK T V, et al.Cloning of nucleoside phosphorylase genes from the extremophilic bacterium Halomonas chromatireducens AGD 8-3 with the construction of recombinant producer strains of these proteins and the study of their enzymatic properties[J].Applied Biochemistry and Microbiology, 2020, 56(1):37-43.
[22] SILVA R G, CARVALHO L P S, OLIVEIRA J S, et al.Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase[J].Protein Expression and Purification, 2003, 27(1):158-164.
