INCREASING RECOMBINANT PENICILLIN G ACYLASE PRODUCTION: GENETIC, PROTEIN ENGINEERING, AND PRODUCTIVITY IMPROVEMENT

Main Article Content

Is Helianti

Abstract

B-lactam derived antibiotics are the most used globally for treatment against different infections caused by pathogenic bacteria and comprises 65% of the world antibiotics. Recently, penicillin G acylase (PGA) is used as biocatalyst for those B-lactam antibiotics production by which 6-aminopenicillanic acid (6-APA) or 7-aminodeacetoxycephalosporanic acid (7-ADCA) as the building blocks is produced. Commercialized PGA from native microbial resources are still limited to E. coli. Therefore, genetic engineering approach such as cloning and expression in other microbial hosts were assessed to enhance bacterial strains that produce PGA. However, such improvement could increase immature precursors accumulation and lowering the enzyme yield, activity, or stability. This review focus on the review of PGA recombinant produced by several microbial host, their expression levels, and improvement achieved by some modification such as replacement of signal peptide and promoter continued to protein engineering to utilize the enzymes in synthetizing amoxicillin rather than to hydrolyses Penicillin G.

Article Details

How to Cite
Helianti, I. (2023). INCREASING RECOMBINANT PENICILLIN G ACYLASE PRODUCTION: GENETIC, PROTEIN ENGINEERING, AND PRODUCTIVITY IMPROVEMENT. Jurnal Bioteknologi Dan Biosains Indonesia, 9(2), 295–309. Retrieved from https://ejournal.brin.go.id/JBBI/article/view/1775
Section
Articles

References

Adriano, W.S., Silva, J.A., Giordano, R.L., Gonçalves, L. R. 2005. Stabilization of penicillin G acylase by immobilization on glutaraldehyde-activated chitosan. Brazilian Journal of Chemical Engineering 22(4):529-38.

Akkaya, Ö., Öztürk, S., Bolhuis. A., Gümü?el, F. 2012. Mutations in the translation initiation region of the pac gene resulting in increased levels of activity of penicillin G acylase. World Journal of Microbiol Biotechnol 28(5):2159–2164. doi: 10.1007/s11274-012-1021-6

Alvaro, G., Fernandez-Lafuente, R., Blanco, R.M, Guisán, J.M. 1990. Immobilization-stabilization of penicillin G acylase from Escherichia coli. Applied Biochemistry and Biotechnology 26(2):181-95. doi: 10.1007/BF02921533.

Anuj, K., Chandel, L., Venkateswar, R., Lakshmi, N.M., Om, V. S. 2008. The realm of penicillin G acylase in ?-lactam antibiotics. Enzyme and Microbial Technology Volume 42(3)199-207.

Baghban, R., Farajnia, S., Rajabibazl, M., Ghasemi, Y., Mafi, A., Hoseinpoor, R., Rahbarnia, L., Aria, M. 2019. Yeast expression systems: overview and recent advances. Molecular Biotechnology 61(5):365-84.

Bagherinejad, M. R., Korbekandi, H., Tavakoli, N., Abedi, D. 2012. Immobilization of penicillin G acylase using permeabilized Escherichia coli whole cells within chitosan beads. Research in Pharmaceutical Sciences 7(2):79–85.

Balasingham, K., Warburton, D., Dunnill, P.. Lilly, M.D. 1972. The isolation and kinetics of penicillin amidase from Escherichia coli. Biochimica et Biophysica Acta (BBA)-Enzymology 276(1):250-256.

Baneyx, F., Mujacic, M. 2004. Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology 22(11):1399-408. doi: 10.1038/nbt1029.

Be?ka, S., Št?pánek, V., Vyasarayani, R.W., Grulich, M., Maršálek, J., Plhá?ková, K., Dobišová M, Marešová H, Pla?ková M, Valešová R, Palyzová A. 2014. Penicillin G acylase from Achromobacter sp. CCM 4824. Applied Microbiology and Biotechnology 98(3):1195-203.

Bor?inová, M., Raschmanová, H., Zamora, I., Looser, V., Marešová, H., Hirsch, S., Kyslík, P., Kovar, K. 2020. Production and secretion dynamics of prokaryotic Penicillin G acylase in Pichia pastoris. Applied Microbiology and Biotechnology 104(13):5787-800. doi: 10.1007/s00253-020-10669-x.

Buchholz, K. 2016. A breakthrough in enzyme technology to fight penicillin resistance—industrial application of penicillin amidase. Applied Microbiology and Biotechnology 100(9):3825-39. doi: 10.1007/s00253-016-7399-6

Cai, G., Zhu, S., Yang, S., Zhao, G., Jiang, W.. 2004. Cloning, overexpression, and characterization of a novel thermostable penicillin G acylase from Achromobacter xylosoxidans: probing the molecular basis for its high thermostability. Applied and Environmental Microbiology 70(5):2764-70.

Cano-Cabrera, J.C, Palomo-Ligas, L., Flores-Gallegos, A.C, Martínez-Hernández, J.L, Rodríguez-Herrera, R. 2021. Penicillin G acylase production by Mucor griseocyanus and the partial genetic analysis of its pga gene. International Microbiology 24(1):37-45. doi: 10.1007/s10123-020-00137-x.

Cecchini, D.A., Serra, I., Ubiali, D. 2007. New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase. BMC Biotechnoogyl 7:54.

Chen, X.A, Cen, P.L. 2005. A novel three-stage process for continuous production of Penicillin G Acylase by a temperature-sensitive expression system of Bacillus subtilis Phagephi 105. Chemical and Biochemical Engineering Quarterly 19(4):367-72.

Cheng, T., Chen, M., Zheng, H., Wang, J., Yang, S., Jiang, W. 2006. Expression and purification of penicillin G acylase enzymes from four different micro-organisms, and a comparative evaluation of their synthesis/hydrolysis ratios for cephalexin. Protein Expression and Purification 46(1):107-13. doi: 10.1016/j.pep.2005.07.016.

Datla, A., Rajasekar, V.W., Kyslik, P., Becka, S., Krishnakant, A.T., Yogesh, Z.S., Nikunj, K. 2010. Process for the preparation of immobilized recombinant penicillin acylase catalyst from Achromobacter sp. CCM 4824 expressed in E. coli BL21 CCM 7394 and its use for the synthesis of beta-lactam antibiotics. United States patent application US 12/670,792.

De León-Rodríguez, A., Rivera-Pastrana, D., Medina-Rivero, E., Flores-Flores, J.L, Estrada-Baltazar, A., Ordóñez-Acevedo, L.G., de la Rosa, A.P. 2006. Production of penicillin acylase by a recombinant Escherichia coli using cheese whey as substrate and inducer. Biomolecular Engineering 23(6):299-305.

De Vroom, E. 1997. An improved immobilized Penicillin G Acylase. WO/1997/004086

Deak, P.M., Lutz-Wahl, S., Bothe, H., Fischer, L. 2003. Bioreactor cultivation of Escherichia coli for production of recombinant penicillin G amidase from Alcaligenes faecalis. Biotechnology Letters 25(5):397-400. doi: 10.1023/a:1022498314354.

Deng, S., Ma, X., Su, E., Wei, D. 2016. Efficient cascade synthesis of ampicillin from penicillin G potassium salt using wild and mutant penicillin G acylase from Alcaligenes faecalis. Journal of Biotechnology 219:142-148.

Elander, R.P. 2003. Industrial production of beta-lactam antibiotics. Applied Microbiology and Biotechnology 61(5-6):385-392. doi: 10.1007/s00253-003-1274-y

Giordano, R.C., Ribeiro, M.P., Giordano, R.L. 2006. Kinetics of ?-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization. Biotechnology advances 24(1):.27-41.

Hartinger, D., Heinl, S., Schwartz, H.E. et al. 2010. Enhancement of solubility in Escherichia coli and purification of an aminotransferase from Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1. Microbial Cell Factories 9, 62. https://doi.org/10.1186/1475-2859-9-62.

Hassan, M.E. 2016. Production, immobilization and industrial uses of penicillin G acylase. International Journal of Current Research and Review 8(15):11-22.

Hewitt, L., Kasche, V., Lummer, K., Lewis, R.J., Murshudov, G.N., Verma, C.S., Dodson, G.G., and Wilson, K.S. 2000. Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft. Journal of Molecular Biology 302:887-898.

Jia, B., Jeon, C.O. 2016. High-throughput recombinant protein expression in Escherichia coli: current status and future perspectives. Open biology 6(8):160196.

Jiang, Y., Wei, D. 2008. Leakage of recombinant penicillin G acylase from Escherichia coli by adding chloroform under fermentation conditions. World Journal of Microbiology and Biotechnology 24(10):2365-7.

Kang, X.M., Cai, X., Huang, Z.H., Liu, Z.Q., Zheng, Y.G. 2020. Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering. International Journal of Biological Macromolecules 15(143):833-41.

Kasche, V., Galunsky, B., Ignatova, Z. 2003. Fragments of pro?peptide activate mature penicillin amidase of Alcaligenes faecalis. European Journal of Biochemistry 270(23):4721-8.

Krzeslak, J., Braun, P., Voulhoux, R., Cool, R.H., Quax, W.J. 2009. Heterologous production of Escherichia coli penicillin G acylase in Pseudomonas aeruginosa. Journal of Biotechnology 142 (3-4):250-8.

Lakowitz, A., Godard, T., Biedendieck, R., Krull, R.. 2018. Mini review: recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives. European Journal of Pharmaceutics and Biopharmaceutics 126:27-39. doi: 10.1016/j.ejpb.2017.06.008

Lee, S.M. 2017. An efficient method for the release of recombinant penicillin g amidase from the Escherichia coli periplasm. Journal of Life Science 27(10):1145-1151.

Lin, Y.H., Hsiao, H.C., Chou, C.P. 2002. Strain improvement to enhance the production of recombinant penicillin acylase in high-cell-density Escherichia coli cultures. Biotechnology Progress 18:1458–1461. doi: 10.1021/bp020093j

Liu, R., Fan, J., Zhang, Y., Wang, P., Shen, X. 2016. Immobilization and characterization of penicillin G acylase (PGA immobilized on magnetic Ni.Zn.FeO? nanoparticles. Journal of Nanoscience and Nanotechnology 16(1):182-8.

Marešová, H., Marková, Z., Valešová, R., Sklená?, J., Kyslík, P. 2010. Heterologous expression of leader-less pga gene in Pichia pastoris: Intracellular production of prokaryotic enzyme. BMC Biotechnology 10(1):1-10.

Marešová, H., Palyzová, A., Pla?ková, M., Grulich, M., Rajasekar, V.W., Št?pánek, V., Kyslíková, E., Kyslík, P. 2017. Potential of Pichia pastoris for the production of industrial penicillin G acylase. Folia microbiologica 62(5):417-24. doi: 10.1007/s00253-013-5492-7

Marešová, H., Št?pánek, V., Kyslik, P. 2001. A chemostat culture as a tool for the improvement of a recombinant E. coli strain over?producing penicillin G acylase. Biotechnology and Bioengineering 5;75(1):46-52.

Martín, L., Prieto, M.A., Cortes, E., García, J. 1995. Cloning and sequencing of the pac gene encoding the penicillin G acylase of Bacillus megaterium ATCC 14945. FEMS Microbiology Letters 125(2-3):287-92.

Martínez-Hernández, J.L., Mata-Gómez, M.A., Aguilar-González, C.N., Ilyina, A. 2010. A process to produce penicillin G acylase by surface-adhesion fermentation using Mucor griseocyanus to obtain 6-aminopenicillanic acid by penicillin G hydrolysis. Applied Biochemistry and Biotechnology 160(7):2045-53.

Mayer, J., Pippel, J., Günther, G., Müller, C., Lauermann, A., Knuuti, T., Blankenfeldt, W., Jahn, D., Biedendieck, R. 2019. Crystal structures and protein engineering of three different penicillin G acylases from Gram-positive bacteria with different thermostability. Applied Microbiology and Biotechnology 103(18):7537-52. doi: 10.1007/s00253-019-09977-8

Mcdonough, M.A., Klei, H.E., Kelly, J.A. 1999. Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri. Protein Science 8(10):1971-81.

Nam, D.H. and Ryu, D.D. 1979. Biochemical properties of penicillin amidohydrolase from Micrococcus luteus. Applied and Environmental Microbiology 1979. 38(1):35-8.

Nawaz, A.F., Samra Zafar, S.L., Shahzadi, K., Fatima, Z., Siddique, I. 2021. Use of nanomaterials for the immobilization of industrially important enzymes. Journal of Nanotechnology Research 4:45-57.

Ohashi, H., Katsuta, Y., Nagashima, M., Kamei, T., Yano, M. 1989. Expression of the Arthrobacter viscosus penicillin G acylase gene in Escherichia coli and Bacillus subtilis. Applied and Environmental Microbiology 55(6):1351-6.

Orrego, A.H., Romero-Fernández, M., Millán-Linares, M.D., Pedroche, J., Guisán, J.M., Rocha-Martin, J. 2020. High stabilization of enzymes immobilized on rigid hydrophobic glyoxyl-supports: Generation of hydrophilic environments on support surfaces. Catalysts 10(6):676.

Pan, X., Li, A., Peng, Z., Ji, X., Chu, J., He, B. 2020. Efficient synthesis of ?-lactam antibiotics with in situ product removal by a newly isolated penicillin G acylase. Bioorganic Chemistry 99:103765.

Pan, X., Yu, Q., Chu, J., Jiang, T., He, B. 2018. Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. Coli. .Applied Microbiology and Biotechnology 102:7455–7464.

Panbangred, W., Weeradechapon, K., Udomvaraphant, S., Fujiyama, K., Meevootisom, V. 2000. High expression of the penicillin G acylase gene (pac) from Bacillus megaterium UN1 in its own pac minus mutant. Journal of Applied Microbiology. 89(1):152-7.

Panigrahi, P., Chand, D., Mukherji, R., Ramasamy, S., Suresh, C.G. 2015. Sequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases. Journal of Industrial Microbiology and Biotechnology 42(11):1493-506. doi: 10.1007/s10295-015-1690-x

Pereira, S.C., Bussamara, R., Marin, G., Giordano, R.L., Dupont, J., de Campos Giordano, R. 2012. Enzymatic synthesis of amoxicillin by penicillin G acylase in the presence of ionic liquids. Green Chemistry 14(11):3146-56.

Putri, F.P., Nurhasanah, A., Nurhayati, N., Helianti, I., Syamsu, K. 2015. Medium optimization for penicillin acylase (PAC) production by recombinant B.megaterium MS941 containing pac gene from B.thuringiensis BGSC BD1 using response surface methodology. Microbiology Indonesia 9(2):3.

Quax, W.J., Gist, B.N.V. 1992. Penicillin G acylase, a gene encoding the same and a method for the production of this enzyme. United States patent US 5,168,048.

Rajendran, K., Mahadevan, S., Sekar, S., Paramasamy, G., Mandal, A.B. 2011. Biocalorimetric and respirometric studies on production of Penicillin G acylase from Bacillus badius pac in E. coli DH5 alpha. Biochem Eng J 55:223–9.

Ramos, J.L., Duque, E., Gallegos, M.T., Godoy, P., Ramos-Gonzalez, M.I., Rojas, A., Terán, W., Segura, A. 2002. Mechanisms of solvent tolerance in gram-negative bacteria. Annual Reviews in Microbiology 56(1):743-68.

Ratnaningsih, E., Achmad, S., Liang, O.B. 2008. Characterization of extracellular penicilin G acylase produced by a new local strain of Bacillus subtilis BAC4. HAYATI Journal of Biosciences 15(2):71-6.

Rathore, A. 2011. Penicillin G acylase from Arthrobacter viscosus ATCC 15294 : Production, biochemical aspects and structural studies. http://hdl.handle.net/10603/195973

Rodriguez-Herrera, R., Puc, L.E., Sobrevilla, J.M., Luque, D., Cardona-Felix, C.S., Aguilar-González, C.N., Flores-Gallegos, A.C. 2019. Enzymes in the pharmaceutical industry for ?-lactam antibiotic production. Enzymes in Food Biotechnology: 627-643. doi: 10.1016/B978-0-12-813280-7.00036-0

Sakaguchi, K., and Murao, S. 1950. A preliminary report on a new enzyme, "penicillin-amidase." Journal of Agriculture and Chemistry Society 23:411.

Sambyal, K., Singh, R.V. 2021. Exploitation of E. coli for the production of penicillin G amidase: a tool for the synthesis of semisynthetic ?-lactam antibiotics. Journal of Genetic Engineering and Biotechnology 19(1):1-1.

Scaramozzino, F., Estruch, I., Rossolillo, P., Terreni, M., Albertini, A.M. 2005. Improvement of catalytic properties of Escherichia coli penicillin G acylase immobilized on glyoxyl agarose by addition of a six-amino-acid tag. Applied Environtment and Microbiology. 12:8937-40. doi: 10.1128/AEM.71.12.8937-8940.2005.

Schmidt, F.R. 2010. The ?-Lactam antibiotics: current situation and future prospects in manufacture and therapy industrial applications. Berlin, Heidelberg: Springer. p:101–21.

Šenerovi?, L., Stankovi?, N., Ljubijanki?, G., Vasiljevi?, B. 2009. Glycosylation and pH stability of penicillin G acylase from Providencia rettgeri produced in Pichia pastoris. Archives of Biological Sciences 61(4):581-586.

Ševo, M., Degrassi, G., Skoko, N., Venturi, V., Ljubijanki?, G. 2002. Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris. FEMS yeast research 1(4):271-7.

Srirangan, K., Orr, V., Akawi, L., Westbrook, A., Moo-Young, M., Chou, C.P. 2013. Biotechnological advances on penicillin G acylase: pharmaceutical implications, unique expression mechanism and production strategies. Biotechnology Advance. 31(8):1319-32. doi: 10.1016/j.biotechadv.2013.05.006. Epub 2013 May 27. PMID: 23721991.

Sudhakaran, V.K., Deshpande, B.S., Ambedkar, S.S., Shewale, J.G. 1992. Molecular aspects of penicillin and cephalosporin acylases. Process Biochemistry 27(3):131-43.

Tishkov VI, Savin SS, Yasnaya AS (2010) Protein engineering of penicillin acylase. Acta Naturae 2:47–61. doi: 10.32607/20758251-2010-2-3-47-61

Torres, L.L., Ferreras, E.R., Cantero, Á., Hidalgo, A., Berenguer, J. 2012. Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli. Microbial Cell Factories 11(1):1-2.

Travascio, P., Zito, E., Portaccio, M., Diano, N., Grano, V., Di Martino, S. 2002. Enzyme reaction engineering: effect of methanol on the synthesis of antibiotics catalyzed by immobilized penicillin G acylase under isothermal and non-isothermal conditions. Biotechnology Progress 18:975 – 85

Van Der Laan, J.M., Moody, H.M., Kerkman, R., van der Does, T. 2013. Mutant penicillin G acylases. United States patent US 8,541,199.

Van Dooren, T.J., Smeets, J.C., Moody, H.M., Koninklijke, D.S.M. 2001. Process for the preparation of a?-lactam antibiotic. United States patent US 6,287,799.

Vélez, A.M., da Silva, A.J., Luperni-Horta, A.C., Sargo, C.R., Campani, G.G., Silva, G., de Lima Camargo Giordano, R., Zangirolami, T.C. 2014. High-throughput strategies for penicillin G acylase production in rE. coli fed-batch cultivations. BMC Biotechnology 14:6. doi: 10.1186/1472-6750-14-6.

Verhaert, R.M., Riemens, A.M., Van der Laan, J.M., Van Duin, J., Quax, W.J. 1997. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis. Applied and Environmental Microbiology 63(9):3412-8.

Vohra, P.K., Sharma, R., Kashyap, D.R., Tewari, R. 2001. Enhanced production of penicillin G acylase from a recombinant Escherichia coli. Biotechnology Letter. 23(7):531-5.

Wen, Y., Feng, M., Yuan, Z., Zhou, P. 2005. Expression and overproduction of recombinant penicillin G acylase from Kluyvera citrophila in Escherichia coli. Enzyme and Microbial Technology 37(2):233-7 doi: 10.1016/j.enzmictec.2005.03.005.

Westers, L., Westers, H., Quax, W.J. 2004. Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1694(1-3):299-310.

Wilson, L., Illanes, A., Pessela, B.C., Abian, O., Fernández?Lafuente, R., Guisán, J.M. 2004. Encapsulation of crosslinked penicillin G acylase aggregates in lentikats: evaluation of a novel biocatalyst in organic media. Biotechnology and Bioengineering 86(5):558-562.

Yang, S., Huang, H., Zhang, R., Huang, X., Li, S., Yuan, Z. 2001. Expression and purification of extracellular penicillin G acylase in Bacillus subtilis. Protein Expression and Purification 21(1):60-4. doi: 10.1006/prep.2000.1339.

Yang, Y., Rebekka, Wei, W., Martin, G., Marco, M., Dieter, J., Wolf-Dieter, D. 2006. High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium. Microbial Cell Factories 23(6):299-305. doi: 10.1016/j.bioeng.2006.09.003.

Youshko, M.I. and ?vedas, V.K. 2000. Kinetics of ampicillin synthesis catalyzed by penicillin acylase from E. coli in homogeneous and heterogeneous systems. Quantitative characterization of nucleophile reactivity and mathematical modeling of the process. Biochemistry 65(12):1367-75.

Zhang, M., Shi, M., Zhou, Z., Yang, S., Yuan, Z. Ye, Q. 2006. Production of Alcaligenes faecalis penicillin G acylase in Bacillus subtilis WB600 (pMA5) fed with partially hydrolyzed starch. Enzyme and Microbial Technology 39(4):555-560.

Zhao, J., Wang, Y., Luo, G., Zhu, S. 2011. Immobilization of penicillin G acylase on macro-mesoporous silica spheres. Bioresource Technology 102(2):529-535.

Zhou, Z., Zhou, L.P., Chen, M.J., Zhang, Y.L., Li, R.B., Yang, S., Yuan, Z.Y. 2003. Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis. Acta Biochimica et Biophysica Sinica-Chinese Edition 35(5):416-22.

Zhou, H., Yang, L., Li, W., Wang, F., Li, W., Zhao, J., Liang, X., Liu, H. 2012. Immobilizing penicillin g acylase using silica-supported ionic liquids: The effects of ionic liquid loadings. Industrial and Engineering Chemistry Research 51(40):13173-13181