Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

Kezia Abib Yerah Tjandra; Kartika Sari Dewi; Asrul Muhamad Fuad; Trisanti Anindyawati

doi:10.22146/ijbiotech.55604

Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter

https://doi.org/10.22146/ijbiotech.55604

Kezia Abib Yerah Tjandra⁽¹⁾, Kartika Sari Dewi^(2*), Asrul Muhamad Fuad⁽³⁾, Trisanti Anindyawati⁽⁴⁾

(1) Department of Industrial Biotechnology, Brawijaya University, Jalan Veteran, Malang 165145, Indonesia
(2) Research Centre for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km.46, Cibinong 16911, Indonesia
(3) Research Centre for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km.46, Cibinong 16911, Indonesia
(4) Research Centre for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km.46, Cibinong 16911, Indonesia
(*) Corresponding Author

Abstract

Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentrations. Among these cellulases, the highest catalytic efficiency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial application. The characterization of the EGII is necessary since it is widely used in high-temperature reactions in the industries. In this study, the recombinant EGII protein was expressed in Pichia pastoris and it has a molecular mass of approximately 52 kDa. Recombinant EGII was purified using Ni-NTA affinity chromatography and characterized by SDS-PAGE and western blot analyses. The enzyme activity of recombinant EGII was measured using the Nelson Somogyi method to determine its optimum pH and temperature. The result showed that the maximum EGII expression was achieved after 72 h of culture incubation. The crude enzyme has optimum activity at pH 5.0, resulting in 16.3 U/mL and 14.6 U/mL activity at 40 °C and 50 °C, respectively. While the purified enzyme gave the specific activity of 115.7 U/mg under the optimum condition. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase activity for 89% and 80% at 40 °C and 50 °C, respectively.

Keywords

Endoglucanase II; GAP promoter; Trichoderma reesei; Pichia pastoris; Nelson-Somogyi assay

Full Text:

PDF

References

Adney WS, Jeoh T, Beckham GT, Chou YC, Baker JO, Michener W, Brunecky R, Himmel ME. 2009. Probing the role of Nlinked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis. Cellulose. 16(4):699–709. doi:10.1007/s1057000993051.

Akbarzadeh A, Ranaei Siadat SO, Motallebi M, Zamani MR, Barshan Tashnizi M, Moshtaghi S. 2014. Characterization and high level expression of acidic endoglucanase in Pichia pastoris. Appl Biochem Biotechnol. 172(4):2253–2265. doi:10.1007/s1201001306726.

Bai R, Zhang Y, Wang C, Zhang F, Zhang Z, Sun F, Zhang Z. 2016. Gene optimization and efficient expression of Trichoderma reesei Cel5A in Pichia pastoris. Shengwu Gongcheng Xuebao 32(10):1381– 1394. doi:10.13345/j.cjb.160017.

Bajaj BK, Pangotra H, Wani MA, Sharma P, Sharma A. 2009. Partial purification and characterization of a highly thermostable and pH stable endoglucanase from a newly isolated Bacillus strain M9. Indian J Chem Technol. 16(5):382–387.

Boonvitthya N, Bozonnet S, Burapatana V, O’Donohue MJ, Chulalaksananukul W. 2013. Comparison of the heterologous expression of Trichoderma reesei endoglucanase II and cellobiohydrolase II in the yeasts Pichia pastoris and Yarrowia lipolytica. Mol Biotechnol. 54(2):158–169. doi:10.1007/s120330129557

Charoenrat T, Khumruaengsri N, Promdonkoy P, Rattanaphan N, Eurwilaichitr L, Tanapongpipat S, Roongsawang N. 2013. Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and pH control of proteolysis. J Biosci Bioeng. 116(2):193–198. doi:10.1016/j.jbiosc.2013.02.020.

Dotsenko AS, Gusakov AV, Volkov PV, Rozhkova AM, Sinitsyn AP. 2016. Nlinked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity. Biotechnol Bioeng. 113(2):283–291. doi:10.1002/bit.25812.

Garvey M, Klinger J, Klose H, Fischer R, Commandeur U. 2014. Expression of recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants. J Visualized Exp. (88):e51711. doi:10.3791/51711.

Gusakov AV, Kondratyeva EG, Sinitsyn AP. 2011. Comparison of Two Methods for Assaying Reducing Sugars in the Determination of Carbohydrase Activities. Int J Anal Chem. 2011:1–4. doi:10.1155/2011/283658.

Hadiyanto H, Ariyanti D, Aini AP, Pinundi DS. 2013. Batch and fedbatch fermentation system on ethanol production from whey using Kluyveromyces marxi anus. Int J Renewable Energy Dev. 2(3):127–131. doi:10.14710/ijred.2.3.127131.

Han C, Wang Q, Sun Y, Yang R, Liu M, Wang S, Liu Y, Zhou L, Li D. 2020. Improvement of the catalytic activity and thermostability of a hyperthermostable endoglucanase by optimizing Nglycosylation sites. Biotechnol Biofuels. 13(1). doi:10.1186/s13068020 16684.

Ito J, Fujita Y, Ueda M, Fukuda H, Kondo A. 2004. Improvement of cellulosedegrading ability of a yeast strain displaying Trichoderma ree sei endoglucanase II by recombination of cellulose binding domains. Biotechnol Prog. 20(3):688–691. doi:10.1021/bp034332u.

Jin X, Meng N, Xia LM. 2011. Expression of an endoβ1,4glucanase gene from orpinomyces PC2 in Pichia pastoris. Int J Mol Sci. 12(5):3366–3380. doi:10.3390/ijms12053366.

Kamal S, Khan S, Khan S, Shoaib M, Khan H, Man S, Khan W, Samson M. 2017. Recent view on heterologous expression of thermostable fungal cellulases, focused on expression factory of Pichia pastoris. Int J Basic Med Sci Pharm. 7(2):43—57.

Knott BC, Haddad Momeni M, Crowley MF, MacKenzie LF, Götz AW, Sandgren M, Withers SG, Staìšhlberg J, Beckham GT. 2014. The mechanism of cellulose hydrolysis by a twostep, retaining cellobiohydrolase elucidated by structural and transition path sampling studies. J Am Chem Soc. 136(1):321–329. doi:10.1021/ja410291u.

Koontz L. 2014. TCA precipitation. Methods Enzymol. 541:3–10. doi:10.1016/B9780124201194.00001 X.

Li C, Yang Z, He Can Zhang R, Zhang D, Chen S, Ma L. 2013. Effect of pH on cellulase production and morphology of Trichoderma reesei and the application in cellulosic material hydrolysis. J Biotechnol. 168(4):470–477. doi:10.1016/j.jbiotec.2013.10.003.

Liang C, Fioroni M, RodríguezRopero F, Xue Y, Schwaneberg U, Ma Y. 2011. Directed evolution of a thermophilic endoglucanase (Cel5A) into highly active Cel5A variants with an expanded temperature profile. J Biotechnol. 154(1):46–53. doi:10.1016/j.jbiotec.2011.03.025.

MacauleyPatrick S, Fazenda ML, McNeil B, Harvey LM. 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4):249– 270. doi:10.1002/yea.1208.

Nakazawa H, Okada K, Kobayashi R, Kubota T, Onodera T, Ochiai N, Omata N, Ogasawara W, Okada H, Morikawa Y. 2008. Characterization of the catalytic domains of Trichoderma reesei endoglucanase I, II, and III, expressed in Escherichia coli. Appl Microbiol Biotechnol. 81(4):681–689. doi:10.1007/s00253 0081667z.

Qin Y, Qu Y. 2014. Asn124 of cel5A from Hypocrea jecorina not only provides the Nglycosylation site but is also essential in maintaining enzymatic activity. BMB Rep. 47(5):256–261. doi:10.5483/BMBRep.2014.47.5.166.

Qin Y, Wei X, Liu X, Wang T, Qu Y. 2008. Purifi cation and characterization of recombinant endoglucanase of Trichoderma reesei expressed in Saccharomyces cerevisiae with higher glycosylation and stability. Protein Expression Purif. 58(1):162–167. doi:10.1016/j.pep.2007.09.004.

Ratnakomala S, Fahrurrozi, Yopi. 2019. Enhancement of Cellulase (CMCase) production from marine actinomycetes Streptomyces sp. Bse 79: Optimization of fermentation medium by Response Surface Method ology. IOP Conf Ser Earth Environ Sci. 251(1). doi:10.1088/17551315/251/1/012005.

Safder I, Khan S, Islam Iu, Kazim M. 2018. Pichia pastoris expression system : a potential candidate to ex press protein in industrial and biopharmaceutical do mains. Biomed Lett. 4(1):1–13.

Samanta S, Basu A, Halder UC, Sen SK. 2012. Characterization of Trichoderma reesei endoglucanase ii expressed heterologously in Pichia pastoris for better biofinishing and biostoning. J Microbiol. 50(3):518– 525. doi:10.1007/s1227501212075.

ShentalBechor D, Levy Y. 2008. Effect of glycosylation on protein folding: A close look at thermo dynamic stabilization. Proc Natl Acad Sci USA. 105(24):8256–8261. doi:10.1073/pnas.0801340105.

Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q. 2009. Practical protocols for production of very high yields of recombinant proteins us ing Escherichia coli. Protein Sci. 18(5):936–948. doi:10.1002/pro.102.

Sun FF, Yang H, Bai R, Fang X, Wang F, He J, Tu M. 2018. Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular coexpression of Vitreoscilla hemoglobin. J Chem Technol Biotechnol. 93(1):35– 42. doi:10.1002/jctb.5433.

DOI: https://doi.org/10.22146/ijbiotech.55604