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

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 o...

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Main Authors: Kezia Abib Yerah Tjandra, Kartika Sari Dewi, Asrul Muhamad Fuad, Trisanti Anindyawati
Format: Article
Language:English
Published: Universitas Gadjah Mada, Yogyakarta 2020-12-01
Series:Indonesian Journal of Biotechnology
Subjects:
Online Access:https://jurnal.ugm.ac.id/ijbiotech/article/view/55604
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author Kezia Abib Yerah Tjandra
Kartika Sari Dewi
Asrul Muhamad Fuad
Trisanti Anindyawati
author_facet Kezia Abib Yerah Tjandra
Kartika Sari Dewi
Asrul Muhamad Fuad
Trisanti Anindyawati
author_sort Kezia Abib Yerah Tjandra
collection DOAJ
description 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.
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spelling doaj.art-074b7397134c488f9fe5d2c186c769e72023-02-06T03:00:10ZengUniversitas Gadjah Mada, YogyakartaIndonesian Journal of Biotechnology0853-86542089-22412020-12-0125212713410.22146/ijbiotech.5560428763Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoterKezia Abib Yerah Tjandra0Kartika Sari Dewi1Asrul Muhamad Fuad2Trisanti Anindyawati3Department of Industrial Biotechnology, Brawijaya University, Jalan Veteran, Malang 165145, IndonesiaResearch Centre for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km.46, Cibinong 16911, IndonesiaResearch Centre for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km.46, Cibinong 16911, IndonesiaResearch Centre for Biotechnology, Indonesian Institute of Sciences, Jalan Raya Jakarta-Bogor Km.46, Cibinong 16911, IndonesiaTrichoderma 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.https://jurnal.ugm.ac.id/ijbiotech/article/view/55604endoglucanase iigap promotertrichoderma reeseipichia pastorisnelson-somogyi assay
spellingShingle Kezia Abib Yerah Tjandra
Kartika Sari Dewi
Asrul Muhamad Fuad
Trisanti Anindyawati
Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
Indonesian Journal of Biotechnology
endoglucanase ii
gap promoter
trichoderma reesei
pichia pastoris
nelson-somogyi assay
title Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
title_full Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
title_fullStr Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
title_full_unstemmed Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
title_short Expression and characterization of Trichoderma reesei endoglucanase II in Pichia pastoris under the regulation of the GAP promoter
title_sort expression and characterization of trichoderma reesei endoglucanase ii in pichia pastoris under the regulation of the gap promoter
topic endoglucanase ii
gap promoter
trichoderma reesei
pichia pastoris
nelson-somogyi assay
url https://jurnal.ugm.ac.id/ijbiotech/article/view/55604
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