Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris

Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris

B-xylosidase is one of the essential enzymes for complete breakdown of xylan since it degrades short xylo-oligosaccharides and hydrolyzed xylobiose by endoxylanases to xylose. B-xylosidase {Bxl 1) gene of Trichoderma reesei ATCC 58350 containing an open reading frame of 2, 331 nucleotides were ampli...

Full description

Saved in:
Bibliographic Details
Main Author: Muhammad Zaidi, Nor Fadzilah
Format: Thesis
Published: 2010
Subjects:
TP Chemical technology
Tags: Add Tag
No Tags, Be the first to tag this record!
id my-utm-ep.26737
record_format uketd_dc
spelling my-utm-ep.267372017-08-16T02:58:35Z Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris 2010 Muhammad Zaidi, Nor Fadzilah TP Chemical technology B-xylosidase is one of the essential enzymes for complete breakdown of xylan since it degrades short xylo-oligosaccharides and hydrolyzed xylobiose by endoxylanases to xylose. B-xylosidase {Bxl 1) gene of Trichoderma reesei ATCC 58350 containing an open reading frame of 2, 331 nucleotides were amplified by polymerase chain reaction (PCR) and cloned into Pichia pPLCZaA vector. The 5x/l-pPICZaA construct was linearized and integrated at 5’ alcohol oxidase 1 (A0X1) locus in Pichia pastoris (P. pastoris) X33 genome. Effect of cultural conditions on recombinant Bxll production was studied. Production of recombinant Bxll by P. pastoris X33 was best achieved in buffered complex methanol medium (BMMY) pH 6 and induction with 3% methanol for every 24 hours at 30°C. The theoretical molecular weight of the recombinant Bxll was 88.8 kDa, but exhibited a molecular weight of 107.5 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for recombinant Bxll were pH 4 and 50°C, respectively. The enzyme was stable at pH 4 to pH 9 and retained 80% of its activity after incubation at 50°C for 30 minutes. The activity of recombinant Bxll was enhanced by Na2+, K+, Fe2+, Ca2+, Mg2+, Zn2+, NH2+, ethylenediamine-tetraacetate (EDTA), C02+, Ni2+, dimethyl sulfoxide (DMSO), urea, Triton X and Tween 80 and inhibited by Cu2+ and Fe2+. The Michaelis-Menten constant, Km and maximum velocity, Vmax for recombinant Bxll activities towards p-nitrophenyl p-D-xyloside (pNPX) were 8.98 mM and 8.51 (imol/min/mL, respectively. Recombinant Bxll alone can degrade pretreated oil palm empty fruit bunch and react better when combined with recombinant xylanase. 2010 Thesis http://eprints.utm.my/id/eprint/26737/ http://libraryopac.utm.my/client/en_AU/main/search/results?qu=Expression+and+characterization+of+recombinant+beta-xylosidase+from+Trichoderma+reesei+ATCC+58350+in+Pichia+pastoris&te= masters Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering Faculty of Chemical and Natural Resources Engineering
institution Universiti Teknologi Malaysia
collection UTM Institutional Repository
topic TP Chemical technology
spellingShingle TP Chemical technology
Muhammad Zaidi, Nor Fadzilah
Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris
description B-xylosidase is one of the essential enzymes for complete breakdown of xylan since it degrades short xylo-oligosaccharides and hydrolyzed xylobiose by endoxylanases to xylose. B-xylosidase {Bxl 1) gene of Trichoderma reesei ATCC 58350 containing an open reading frame of 2, 331 nucleotides were amplified by polymerase chain reaction (PCR) and cloned into Pichia pPLCZaA vector. The 5x/l-pPICZaA construct was linearized and integrated at 5’ alcohol oxidase 1 (A0X1) locus in Pichia pastoris (P. pastoris) X33 genome. Effect of cultural conditions on recombinant Bxll production was studied. Production of recombinant Bxll by P. pastoris X33 was best achieved in buffered complex methanol medium (BMMY) pH 6 and induction with 3% methanol for every 24 hours at 30°C. The theoretical molecular weight of the recombinant Bxll was 88.8 kDa, but exhibited a molecular weight of 107.5 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for recombinant Bxll were pH 4 and 50°C, respectively. The enzyme was stable at pH 4 to pH 9 and retained 80% of its activity after incubation at 50°C for 30 minutes. The activity of recombinant Bxll was enhanced by Na2+, K+, Fe2+, Ca2+, Mg2+, Zn2+, NH2+, ethylenediamine-tetraacetate (EDTA), C02+, Ni2+, dimethyl sulfoxide (DMSO), urea, Triton X and Tween 80 and inhibited by Cu2+ and Fe2+. The Michaelis-Menten constant, Km and maximum velocity, Vmax for recombinant Bxll activities towards p-nitrophenyl p-D-xyloside (pNPX) were 8.98 mM and 8.51 (imol/min/mL, respectively. Recombinant Bxll alone can degrade pretreated oil palm empty fruit bunch and react better when combined with recombinant xylanase.
format Thesis
qualification_level Master's degree
author Muhammad Zaidi, Nor Fadzilah
author_facet Muhammad Zaidi, Nor Fadzilah
author_sort Muhammad Zaidi, Nor Fadzilah
title Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris
title_short Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris
title_full Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris
title_fullStr Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris
title_full_unstemmed Expression and characterization of recombinant beta-xylosidase from Trichoderma reesei ATCC 58350 in Pichia pastoris
title_sort expression and characterization of recombinant beta-xylosidase from trichoderma reesei atcc 58350 in pichia pastoris
granting_institution Universiti Teknologi Malaysia, Faculty of Chemical and Natural Resources Engineering
granting_department Faculty of Chemical and Natural Resources Engineering
publishDate 2010
_version_ 1747815498611949568

Similar Items