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

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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

0.

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

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