Dataset Information

Expression, Docking, and Molecular Dynamics of Endo-?-1,4-xylanase I Gene of Trichoderma virens in Pichia stipitis.

ABSTRACT: It is essential that major carbohydrate polymers in the lignocellulosic biomass are converted into fermentable sugars for the economical production of energy. Xylan, the major component of hemicelluloses, is the second most naturally abundant carbohydrate polymer comprising 20-40% of the total biomass. Endoxylanase (EXN) hydrolyzes xylan into mixtures of xylooligosaccharides. The objective of this study was to genetically modify Pichia stipitis, a pentose sugar fermenting yeast species, to hydrolyze xylan into xylooligosaccharides via cloning and heterologous extracellular expression of EXNI gene from locally isolated Trichoderma virens species. Pichia stipitis was engineered to carry the EXNI gene of T. virens using pGAPZ? expression vector. The open reading frame encodes 191 amino acids and SDS-PAGE analysis revealed a 24?kDA recombinant protein. The EXNI activity expressed by recombinant P. stipitis clone under standard conditions using 1% beechwood xylan was 31.7?U/ml. Molecular docking and molecular dynamics simulations were performed to investigate EXNI-xylan interactions. Free EXNI and xylan bound EXNI exhibited similar stabilities and structural behavior in aqueous medium. Furthermore, this in silico work opens avenues for the development of newer generation EXN proteins that can perform better and have enhanced catalytic activity.

SUBMITTER: Wickramasinghe GHIM

PROVIDER: S-EPMC5569632 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Expression, Docking, and Molecular Dynamics of Endo-β-1,4-xylanase I Gene of Trichoderma virens in Pichia stipitis.

Wickramasinghe Gammadde Hewa Ishan Maduka GHIM Rathnayake Pilimathalawe Panditharathna Attanayake Mudiyanselage Samith Indika PPAMSI Chandrasekharan Naduviladath Vishvanath NV Weerasinghe Mahindagoda Siril Samantha MSS Wijesundera Ravindra Lakshman Chundananda RLC Wijesundera Wijepurage Sandhya Sulochana WSS

BioMed research international 20170810

It is essential that major carbohydrate polymers in the lignocellulosic biomass are converted into fermentable sugars for the economical production of energy. Xylan, the major component of hemicelluloses, is the second most naturally abundant carbohydrate polymer comprising 20-40% of the total biomass. Endoxylanase (EXN) hydrolyzes xylan into mixtures of xylooligosaccharides. The objective of this study was to genetically modify Pichia stipitis, a pentose sugar fermenting yeast species, t ...[more]

PMID: 28856159

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Project description:Cellulose, a linear polymer of ? 1-4, linked glucose, is the most abundant renewable fraction of plant biomass (lignocellulose). It is synergistically converted to glucose by endoglucanase (EG) cellobiohydrolase (CBH) and ?-glucosidase (BGL) of the cellulase complex. BGL plays a major role in the conversion of randomly cleaved cellooligosaccharides into glucose. As it is well known, Saccharomyces cerevisiae can efficiently convert glucose into ethanol under anaerobic conditions. Therefore, S.cerevisiae was genetically modified with the objective of heterologous extracellular expression of the BGLI gene of Trichoderma virens making it capable of utilizing cellobiose to produce ethanol.The cDNA and a genomic sequence of the BGLI gene of Trichoderma virens was cloned in the yeast expression vector pGAPZ? and separately transformed to Saccharomyces cerevisiae. The size of the BGLI cDNA clone was 1363 bp and the genomic DNA clone contained an additional 76 bp single intron following the first exon. The gene was 90% similar to the DNA sequence and 99% similar to the deduced amino acid sequence of 1,4-?-D-glucosidase of T. atroviride (AC237343.1). The BGLI activity expressed by the recombinant genomic clone was 3.4 times greater (1.7 x 10-3 IU ml-1) than that observed for the cDNA clone (5 x 10-4 IU ml-1). Furthermore, the activity was similar to the activity of locally isolated Trichoderma virens (1.5 x 10-3 IU ml-1). The estimated size of the protein was 52 kDA. In fermentation studies, the maximum ethanol production by the genomic and the cDNA clones were 0.36 g and 0.06 g /g of cellobiose respectively. Molecular docking results indicated that the bare protein and cellobiose-protein complex behave in a similar manner with considerable stability in aqueous medium. The deduced binding site and the binding affinity of the constructed homology model appeared to be reasonable. Moreover, it was identified that the five hydrogen bonds formed between the amino acid residues of BGLI and cellobiose are mainly involved in the integrity of enzyme-substrate association.The BGLI activity was remarkably higher in the genomic DNA clone compared to the cDNA clone. Cellobiose was successfully fermented into ethanol by the recombinant S.cerevisiae genomic DNA clone. It has the potential to be used in the industrial production of ethanol as it is capable of simultaneous saccharification and fermentation of cellobiose. Homology modeling, docking studies and molecular dynamics simulation studies will provide a realistic model for further studies in the modification of active site residues which could be followed by mutation studies to improve the catalytic action of BGLI.

Dataset Information

Expression, Docking, and Molecular Dynamics of Endo-?-1,4-xylanase I Gene of Trichoderma virens in Pichia stipitis.

Publications

Expression, Docking, and Molecular Dynamics of Endo-<i>β</i>-1,4-xylanase I Gene of <i>Trichoderma virens</i> in <i>Pichia stipitis</i>.

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