β-glukanaz Enziminin Pichia pastoris Ekspresyon Sisteminde Hücre Dışı Üretimi ve Saflaştırılması

Yıl 2021, , 620 - 630, 31.08.2021

Mert Karaoglan , Fidan Erden Karaoğlan

https://doi.org/10.18185/erzifbed.972627

Cited By: 2

Öz

Bu çalışmada, Rhizomucor miehei -1,3-1,4-glukanaz geni, Pichia pastoris ekspresyon sisteminde AOX1 promotorunun regülasyonu altında ifade edilmiş ve en yüksek üretim seviyesini sağlayan klon belirlenmiştir. Kodon optimize edilmiş gen, pPICZA ekspresyon vektörüne bağlanmış ve kompetent P. pastoris KM71H hücrelerine aktarılmıştır. Farklı konsantrasyonlarda zeosin içeren plakalardan otuz transformant seçilerek klonlar protein üretimi için test tüplerinde kültüre alınmıştır. Süpernatant örneklerinde glukanaz enzim aktivitesi ölçülmüş ve en yüksek glukanaz aktivitesi gösteren on klon belirlenmiştir. Ayrıca süpernatant örneklerindeki proteinler SDS-PAGE ile analiz edilmiş ve glukanaz enzimi, yaklaşık 34 ve 38 kDa olmak üzere 2 bant halinde gözlenmiştir. En yüksek glukanaz enzim üretimine sahip klon ile 200 mL indüksiyon ortamında (BMMY) 72 saat boyunca protein üretimi gerçekleştirilmiş ve rekombinant glukanaz enzimi Ni-NTA afinite kromatografisi ile saflaştırılmıştır. Saflaştırma işleminden sonra analiz edilen klonun 79,6 mg/L glukanaz üretim seviyesine ulaştığı belirlenmiştir. Saflaştırma işleminin her aşamasından alınan örneklerin SDS-PAGE analizi, süpernatant örneklerinde de gözlenmiş olan iki protein bandının glukanaz enzimini temsil ettiğini göstermiştir.

Anahtar Kelimeler

B-glukanaz, pichia pastoris, rhizomucor miehei, protein saflaştırma

Destekleyen Kurum

ERZİNCAN BİNALİ YILDIRIM ÜNİVERSİTESİ

Proje Numarası

FBA-2019-596

Extracellular Production and Purification of the β-glucanase in Pichia pastoris Expression System

Öz

In this study, Rhizomucor miehei -1,3-1,4-glucanase gene was expressed under the regulation of AOX1 promoter in the Pichia pastoris expression system, and the clone providing the highest production level was determined. The codon-optimized gene was ligated into expression vector pPICZA and transferred into competent P. pastoris KM71H cells. Thirty transformants were selected from plates containing different concentrations of zeocin and cultured in test tubes for protein production. Glucanase enzyme activities in supernatant samples were measured and ten clones showing the highest glucanase activity were determined. The proteins in supernatant samples were also analyzed by SDS-PAGE and the glucanase enzyme was observed in 2 bands, approximately 34 and 38 kDa. Protein production was performed for 72 hours in 200 mL induction medium (BMMY) with the clone providing the highest glucanase enzyme production and the recombinant glucanase enzyme was purified by Ni-NTA affinity chromatography. After purification, it was determined that the analyzed clone reached 796 mg/L glucanase production level. SDS-PAGE analysis of samples from each step of the purification procedure showed that the two protein bands also observed in the supernatant samples represent glucanase enzyme.

Anahtar Kelimeler

β-glucanase, Pichia pastoris, Rhizomucor miehei, Protein purification

Proje Numarası

FBA-2019-596

Kaynakça

• Bailey, M.J. 1988. “A note on the use to dinitrosalicyclic acid for determining the products of enzymatic reactions”. Applied Microbiology and Biotechnology, 29:494-496.
• Bhat, M.K. and Bhat, S. 1997. “Cellulose degrading enzymes and their potential industrial applications”, Biotechnology Advances, 15: 583-620.
• Celestino, K.R.S., Cunha, R.B. and Felix, C.R. 2006. “Characterization of a -glucanase produced by Rhizopus microsporus var. microsporus, and its potential for application in the brewing industry”. BMC Biochemistry, 7:23.
• Chaari, F., Belghith-Fendri, L. and Ellouz-Chaabouni, S. 2015. “Production and in vitro evaluation of oligosaccharides generated from lichenan using immobilized Penicillium occitanis lichenase”. Journal of Molecular Catalysis B; Enzymatic, 116: 153–158.
• Cregg, J.M., Tschopp, J.F., Stillman, C., Siegel, R., Akong, M., Craig, W.S., Buckholz, R.G., Madden, K.R., Kellaris, A., Davis, G.R., Smiley, B.L., Cruze, J., Torregrossa, R., Velicelebi, G. and Thill, G.P. 1987. “High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris”, Biotechnology, 5, 479-485.
• Cregg J.M. (2007). “Pichia Protocols, 2nd ed.”, Methods in Molecular Biology Series, 389, Humana Press Inc., Totowa, NJ. 1-10.
• Daly, R. and Hearn, M.T.W. 2005. “Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production”, Journal of Molecular Recognition, 18, 119–138.
• Hua, C., Yan, Q., Jiang, Z., Li, Y. and Katrolia, P. 2010. “High-level expression of a spesific -1,3,-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris”, Applied Microbiology and Biotechnology, 88:509-518.
• Huanf, Y., Long, Y., Li, S., Lin, T., Wu, J., Zhang, Y. and Lin, Y. 2018. “Investigation on the processing and improving the cleavage efficiency of furin cleavage sites in Pichia pastoris”, Microbial Cell Factories, 17:172.
• Huang, H., Yang, P., Luo, H., Tang., Shao, N., Yuan, T., Wang, Y., Bai, Y. and Yao, B. 2008. “High-level expression of a truncated 1,3-1,4--D-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codon and fermentation”, Applied Microbiology and Biotechnology, 78:95-103.
• Junga, C., Marison I. and Stockar U.V. 2007. “Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures”, Journal of Biotechnology, 130, 236-246.
• Karaoglan, M. and Erden-Karaoglan, F. 2020. “Effect of codon optimization and promoter choice on recombinant endo-polygalacturonase production in Pichia pastoris”. Enzyme and Microbial Technology, 139, 109589.
• Khan, S., Ullah, M.W., Siddique, R., Nabi, G., Manan, S., Yousaf, M. and Hou, H. 2016. “Role of Recombinant DNA Technology to Improve Life”. International Journal of Genomics, 2016/ 2405954.
• Li, H., Wang, S., Zhang, Y. and Chen, L. 2018. “High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide”, Marine drugs, 16:158.
• Lin-Cereghino, J. and Cregg, J.M. 2000. “Heterologous protein expression in the methylotrophic yeast Pichia pastoris”, FEMS Microbiology Reviews, 24(1), 45–66.
• Luang, S., Hrmova, M. and Ketudat Cairns, J.R. 2010. “High-level expression of barley -D-glucan exohydrolase HvExoI from a codon-optimized cDNA in Pichia pastoris”. Protein Expression and Purification, 73(1):90-8.
• Mathlouthi, N., Serge, M., Luc, S., Bernard, Q. and Michel, L. 2002. “Effects of xylanase and -glucanase addition on performance, nutrient digestibility, and physicochemical conditions in the small intestine contents and faecal microflora of broiler chickens fed a wheat and barley-based diet”. Animal Research, 51:395–406.
• Miller, G.H. 1959. “Use of dinitrosalicylic acid reagent for determination of reducing sugar”. Analytical Chemistry, 31:426-429.
• Pham, T.H., Quyen, D.T., Nghiem, N.M. and Vu, T.D. 2011. “Cloning, expression, purification, and properties of an endoglucanase gene (glycosyl hydrolase family 12) from Aspergillus niger VTCC-F021 in Pichia pastoris” Journal of Microbiology and Biotechnology, 21(10):1012-20.
• Planas, A. 2000. “Bacterial 1,3-1,4- -glucanases: structure, function and protein engineering”. Biochimica at Biophysica Acta, 1543:361–82.
• Qiao, J., Zhang, B., Chen, Y. and Cao, Y. 2010. “Codon optimization, expression and characterization of Bacillus subtilis MA139 β-1,3-1,4-glucanase in Pichia pastoris”. Biologia, 65(2), 191-196.
• Ramadan, M.F. 2019 “Enzymes in Fruit Juice Processing”, Enzymes in Food Biotechnology, Elsevier Inc. Academic Press (45-59).
• Tang, Y., Yang, S., Yan, Q., Zhou, P., Cui, J. and Jiang Z.Q. 2012. “Purification and Chaacterization of a Novel -1,3-1,4- glucanase (Lichenase) from Thermophilic Rhizomucor miehei with High Specific Activity and Its Gene sequence”, Journal of Agricultural and Food Chemistry, 60, 2354-2361.
• Teng, D., Fan, Y., Yang, Y.L., Tian, Z.G., Luo, J. and Wang, J.H. 2007. “Codon optimization of Bacillus licheniformis -1,3-1,4-glucanase gene and its expression in Pichia pastoris”. Applied Microbiology and Biotechnology, 74:1074-1083.
• Wang, J.R., Li, Y.Y., Liu, D.N., Liu, J.S., Li, P., Chen, L.Z. and Xu, S.D. 2015. “Codon optimization Significantly Improves the Expression Level of -amylase Gene from Bacillus licheniformis in P. pastoris”, Biomed Research International, 2015/248680.
• Wu, S. and Letchworth, G.J. 2004. “High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol”. Biotechniques, 36: 152–154.
• Yang, S.Q., Xiong, H., Yang, H.Y., Yan, Q.J. and Jiang, Z.Q. 2014. “High-level production of -1,3-1,4-glucanase by Rhizomucor miehei under solid-state fermentation and its potential application in the brewing industry”, Journal of Applied Microbiology 118, 84-91.