Expression and characterizaton of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulaton of the GAP promoter Kezia Abib Yerah Tjandra 1 , Kartka Sari Dewi 2,* , Asrul Muhamad Fuad 2 , Trisant Anindyawat 2 1 Department of Industrial Biotechnology, Brawijaya University, Jalan Veteran, Malang 165145, Indonesia 2 Research Centre for Biotechnology, Indonesian Insttute of Sciences, Jalan Raya Jakarta‐Bogor Km.46, Cibinong 16911, Indonesia *Corresponding author: kartka.sari.dewi@lipi.go.id SUBMITTED 22 April 2020 REVISED 30 August 2020 ACCEPTED 7 October 2020 ABSTRACT Trichoderma reesei is known to be one of the organisms capable for producing various types of cellulase in high concentratons. Among these cellulases, the highest catalytc efciency of endoglucanases II (EGII, EC 3.2.1.4) are considered important for industrial applicaton. The characterizaton of the EGII is necessary since it is widely used in high‐temperature reactons 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 purifed using Ni‐NTA afnity chromatography and characterized by SDS‐PAGE and western blot analyses. The enzyme actvity of recombinant EGII was measured using the Nelson Somogyi method to determine its optmum pH and temperature. The result showed that the maximum EGII expression was achieved afer 72 h of culture incubaton. The crude enzyme has optmum actvity at pH 5.0, resultng in 16.3 U/mL and 14.6 U/mL actvity at 40 °C and 50 °C, respectvely. While the purifed enzyme gave the specifc actvity of 115.7 U/mg under the optmum conditon. Finally, our study demonstrated that recombinant EGII could retain the endoglucanase actvity for 89% and 80% at 40 °C and 50 °C, respectvely. KEYWORDS Endoglucanase II; GAP promoter; Trichoderma reesei; Pichia pastoris; Nelson‐Somogyi assay Indonesian Journal of Biotechnology VOLUME 25(2), 2020, 127‐134 | RESEARCH ARTICLE 1. Introducton Trichoderma reesei is a wellstudied fungus that is capa ble of producing large amounts of various cellulases. This fungus secretes at least six types of cellulases that con sist of two types of cellobiohydrolases and four endoglu canases (Knott et al. 2014). These enzymes are exten sively used in several industries, such as laundry deter gent, textile and pulp, paper industry, and potential for bioenergy production. Thereby, this fungus is industri ally relevant to meet the target production level of cellu lases. Nowadays, the engineering of cellulase into a high performance enzyme for biomass hydrolysis and other in dustrial applications becomes the major research priority. However, various industrial processes and conditions (par ticularly in different temperature and pH conditions) re main a challenge. The cellulase excreted from T. reesei cannot withstand a long period of exposure at high tem perature and pH during its reaction process, which leads to the leveling off of enzymatic activity (Akbarzadeh et al. 2014). Among cellulases produced by T. reesei, endoglu canase II (EGII; EC 3.2.1.4) is predominant and showed the highest catalytic proficiency. The EGs activity of T. reesei is known to decrease about 55% when EGII was absent in the secretory complex of EGs (Qin et al. 2008; Boonvitthya et al. 2013). This evidence revealed that the presence of EGII is crucial for lignocellulosic biomass hy drolysis and other industrial applications. Several stud ies reported that EGII production and characteristic im provement were performed by improving the strain of se creting microorganisms, protein engineering, and recom bination (Ito et al. 2004; Liang et al. 2011; Charoenrat et al. 2013). Thereby, there is still ample scope for im provement, particularly to produce EGII in a heterologous expression system to facilitate protein engineering work. Several heterologous expression has been carried out to produce endoglucanases in various host microorganisms, including Escherichia coli, Yarrowia lipolytica, Saccha romyces cerevisiae, and Pichia pastoris (Nakazawa et al. 2008; Qin et al. 2008; Boonvitthya et al. 2013; Akbarzadeh et al. 2014). Yeast is commonly used for its ability to in crease protein stability since the glycosylation process has occurred. In consequence, the structural and thermal sta bility of protein may increase due to the covalent bond for mation. The covalent bond formation causes less dynamic fluctuation and reduces protein molecules’ flexibility (Qin and Qu 2014). Pichia pastoris has negligible native protein produc Indones J Biotechnol 25(2), 2020, 127‐134 | DOI 10.22146/ijbiotech.55604 www.jurnal.ugm.ac.id/ijbiotech Copyright © 2020 THE AUTHOR(S). This artcle is distributed under a Creatve Commons Atributon‐ShareAlike 4.0 Internatonal license.