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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Format: | Article |
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Universitas Gadjah Mada, Yogyakarta
2020-12-01
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Series: | Indonesian Journal of Biotechnology |
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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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language | English |
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publishDate | 2020-12-01 |
publisher | Universitas Gadjah Mada, Yogyakarta |
record_format | Article |
series | Indonesian Journal of Biotechnology |
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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