Overexpression of Lipase Gene from  Alcaligenes  sp. JG3 and its Activity toward Hydrolysis Reaction

Norman Yoshi Haryono; Winarto Haryadi; Tri Joko Raharjo

doi:10.22146/ijc.45663

Overexpression of Lipase Gene from Alcaligenes sp. JG3 and its Activity toward Hydrolysis Reaction

https://doi.org/10.22146/ijc.45663

Norman Yoshi Haryono⁽¹⁾, Winarto Haryadi⁽²⁾, Tri Joko Raharjo^(3*)

(1) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara BLS 21, Bulaksumur, Yogyakarta 55281, Indonesia
(2) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara BLS 21, Bulaksumur, Yogyakarta 55281, Indonesia
(3) Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara BLS 21, Bulaksumur, Yogyakarta 55281, Indonesia
(*) Corresponding Author

Abstract

Bacterial lipase holds an important role as a new source for many industrial catalysts. The investigation and understanding of the lipase-encoding gene become apparent as the key step for generating high-quality lipase as biocatalyst for many chemical reactions. In this study, bacterial lipase from Alcaligenes sp. JG3 was produced via overexpression gene method. This specific lipase was successfully overexpressed using pQE-30 vector and E. coli M15[pREP4] as host, producing His-tagged protein sized 46 kDa and was able to hydrolyze triacylglycerol from olive oil with the calculated unit activity and specific activity of 0.012 U and 1.175 U/mg respectively. The in silico investigation towards lipase JG3 revealed that it was categorized as ABC transporter protein as opposed to the conventional hydrolase family. Lastly, amino acid sequences SGSGKTT from lipase JG3 was highly conserved sequences and was predicted as the ATP-binding site but the catalytic triad of serine, histidine, and aspartate has not been solved yet.

Keywords

Alcaligenes; lipase gene; enzyme activity; a transporter protein

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References

[1] Lajis, A.F.B., 2018, Realm of thermoalkaline lipases in bioprocess commodity, J. Lipids, 2018, 5659683.

[2] Singh, R., Kumar, M., Mittal, A., and Mehta, P.K., 2016, Microbial enzymes: Industrial progress in the 21^st century, 3 Biotech, 6 (2), 174.

[3] Boshale, H., Shaheen, U., and Kadam, T., 2016, Characterization of a hyperthermostable alkaline lipase from Bacillus sonorensis 4R, Enzyme Res., 2016, 4170684.

[4] Patil, K.J., Chopda, M.Z., and Mahajan, R.T., 2011, Lipase biodiversity, Indian J. Sci. Technol., 4 (8), 971–982.

[5] Lan, D., Yang, N., Wan, W., Shen, Y., Yang, B., and Wang, Y., 2011, A novel cold-active lipase from Candida albicans: Cloning, expression, and characterization of the recombinant enzyme, Int. J. Mol. Sci., 12 (6), 3950–3965.

[6] Anbu, P., and Hur, B.K., 2014, Isolation of an organic solvent-tolerant bacterium Bacillus licheniformis PAL05 that is able to secrete solvent-stable lipase, Biotechnol. Appl. Biochem., 61 (5), 528–534.

[7] Sangeetha, R., Geetha, A., and Arulpandi, I., 2010, Concomitant production of protease and lipase by Bacillus licheniformis VSG1: Production, purification, and characterization, Braz. J. Microbiol., 41 (1), 179–185.

[8] Anobom, C.D., Pinheiro, A.S., De-Andrade, R.A., Aguieiras, E.C.G., Andrade, G.C., Moura, M.V., Almeida, R.V., and Freire, D.M., 2014, From structure to catalysis: Recent development in the biotechnological applications of lipases, Biomed. Res. Int., 2014, 684506.

[9] Bora, L., and Bora, M., 2012, Optimization of extracellular thermophilic highly alkaline lipase from thermophilic Bacillus sp. isolated from hot spring of Arunachal Pradesh, India, Braz. J. Microbiol., 43 (1), 30–42.

[10] Shu, C.H., Xu, C.J., and Lin, G.C., 2006, Purification and partial characterization of a lipase from Antrodia cinnamomea, Process. Biochem., 41 (3), 734–738.

[11] Lestari, P., Handayani, S.N., and Oedjijono, O., 2009, Sifat-sifat biokimiawi ekstrak kasar lipase ekstraseluler dari bakteri Azospirillum sp. JG3, Molekul, 4 (2), 73–82.

[12] Ethica, S.N., 2014, Determination of genes involved in glycerol metabolism of Alcaligenes sp. JG3, Dissertation, Universitas Gadjah Mada, Yogyakarta, Indonesia.

[13] Ethica, S.N., Oedjijono, Semiarti, E., Widada, J., and Raharjo, T.J., 2018, Genotypic and Phenotypic Characterization of Alcaligenes javaensis JG3 Potential as an Effective Biodegrader, BIOTROPIA, 25 (1), 1–10.

[14] Ethica, S.N., Nataningtyas, D.R., Lesteri, P., Istini, Semiarti, E., Widada, J., and Raharjo, T.J., 2013, Comparative evaluation of conventional versus rapid methods for amplifiable genomic DNA isolation of cultured Azospirillum sp. JG3, Indones. J. Chem., 13 (3), 248–253.

[15] Nataningtyas, D.R., Raharjo, T.J., and Astuti, E., 2019, Three-dimensional structural modeling of the lipase-encoding gene from soil bacterium Alcaligenes sp. JG3 using automated protein homology analysis, Indones. J. Chem., 19 (3), 565–574.

[16] Raharjo, T.J., Haryono, N.Y., Nataningtyas, D.R., Alfiraza, E.N., and Pranowo, D., 2016, Characterization of lipase gene fragment from Alcaligenes sp. JG3 bacterium, Am. J. Biochem. Mol. Biol., 6 (2), 45–52.

[17] Haryono, N.Y., Haryadi, W., and Raharjo, T.J., 2018, Characterization of a putative lipase gene from Alcaligenes sp. JG3 bacterium via cloning, J. Biol. Sci., 18 (5), 216–222.

[18] Ethica, S.N., Semiarti, E., Widada, J., Oedjijono, O., and Raharjo, T.J., 2017, Characterization of moaC and nontarget gene fragments of food-borne pathogen Alcaligenes sp. JG3 using degenerate colony and arbitrary PCRs, J. Food Saf., 37 (4), e12345.

[19] Golaki, B.P., Aminzadeh, S., Kharkane, A.A., Yakhchali, B., Farroch, P., Khaleghinejad, S.H., Tehrani, A.K., and Mehrpooyan, S., 2015, Cloning, expression, purification and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01, Protein Expression Purif., 109, 120–126.

[20] Bacha, A.B., Moubayed, N.M.S., and Abid, I., 2015, Thermostable alkaline and detergent-tolerant lipase from a newly isolated thermophilic Bacillus stearothermophilus, Indian J. Biochem. Biophys., 52, 179–188.

[21] Fotouh, D.M.A., Bayoumi, R.A., and Hassan, M.A., 2016, Production of thermoalkaliphilic lipase from Geobacillus thermoleovorans DA2 and application in the leather industry, Enzyme Res., 2016, 9034364.

[22] Rogalska, E., Dochet, I., and Verger, R., 1997, Microbial lipases: Structures, function and industrial applications, Biochem. Soc. Trans., 25 (1), 161–164.

[23] Kanmani, P., Kumaresan, K., and Aravind, J., 2015, Gene cloning, expression and characterization of the Bacillus amyloliquefaciens PS35 lipase, Braz. J. Microbiol., 46 (4), 1235–1243.

[24] De Simone, A., 2016, Engineering the genetic code of Escherichia coli with methionine analogues and bioorthogonal amino acids for protein immobilization, Thesis, Freie Universität Berlin, Germany.

[25] Mala, J.G.S., and Takeuchi, S., 2008, Understanding structural features of microbial lipase-An overview, Anal. Chem. Insights, 3, 9–19.

[26] Simossis, V.A., and Herina, J., 2004, Intgrating protein secondary structure prediction and multiple sequence alignment, Curr. Protein Pept. Sci., 5 (4), 249–266.

[27] Yang, J., and Zhang, Y., 2015, Protein structure and function prediction using I-TASSER, Curr. Protoc. Bioinf., 52 (1), 5.8.1–5.8.15.

[28] Newstead, S., Fowler, P.W., Bilton, P., Carpenter, E.P., Sadler, P.J., Campopiano, D.J., Sansom, M.S., and Iwata, S., 2009, Insights into how nucleotide-binding domains power ABC transport, Structure, 17 (9), 1213–1222.

[29] Orelle, C., Durmort, C., Mathieu, K., Duchene, B., Aros, S., Fenaille, F., Andre, F., Junot, C., Vernet, T., and Jault, J.M., 2018, A multidrug ABC transporter with a taste for GTP, Sci. Rep., 8 (1), 2309.

[30] Verdon, G., Albers, S.V., Dijkstra, B.W., Driessen, A.J., and Thunnissen, A.M., 2003, Crystal structures of the ATPase subunit of the glucose ABC transporter from Sulfolobus solfataricus: Nucleotide-free and nucleotide-bound conformations, J. Mol. Biol., 330 (2), 343–358.

[31] Bell, P.J., Sunna, A., Gibbs, M.D., Curach, N.C., Nevalainen, H., and Bergquist, P.L., 2002, Prospecting for novel lipase gene using PCR, Microbiology, 148 (1), 2283–2291.

[32] Arpigny, J.L., and Jaeger, K.E., 1999, Bacterial lipolytic enzymes: Classification and properties, Biochem. J., 343 (1), 177–183.

[33] Meier, R., Drepper, T., Svensson, V., Jaeger, K.E., and Baumann, U., 2007, A calcium-gated lid and a large beta-roll sandwich are revealed by the crystal structure of extracellular lipase from Serratia marcescens, J. Biol. Chem., 282 (43), 31477–31483.

[34] Barbe, S., Lafaquière, V., Guieysse, D., Monsan, P., Reamud-Siméon, M., and André, I., 2009, Insights into lid movements of Burkholderia cepacia lipase inferred from molecular dynamics simulations, Proteins, 77 (3), 509–523.

[35] Wilken, S., 2015, Structure and mechanism ABC transporter, F1000Prime Rep., 7, 14.

[36] Akatsuka, H., Kawai, E., Omori, K., and Shibatami, T., 1995, The three genes lipB, lip C and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide, J. Bacteriol., 177 (22), 6381–6389.

DOI: https://doi.org/10.22146/ijc.45663