Dataset Information

Production and Characteristics of a Novel Xylose- and Alkali-tolerant GH 43 ?-xylosidase from Penicillium oxalicum for Promoting Hemicellulose Degradation.

ABSTRACT: ?-xylosidase is a pivotal enzyme for complete degradation of xylan in hemicelluloses of lignocelluloses, and the xylose- and alkali-tolerant ?-xylosidase with high catalytic activity is very attractive for promoting enzymatic hydrolysis of alkaline-pretreated lignocellulose. In this study, a novel intracellular glycoside hydrolase family 43 ?-xylosidase gene (xyl43) from Penicillium oxalicum 114-2 was successfully high-level overexpressed in Pichia pastoris, and the secreted enzyme was characterized. The ?-xylosidase Xyl43 exhibited great pH stability and high catalytic activity in the range of pH 6.0 to 8.0, and high tolerance to xylose with the Ki value of 28.09?mM. The Xyl43 could effectively promote enzymatic degradation of different source of xylan and hemicellulose contained in alkaline-pretreated corn stover, and high conversion of xylan to xylose could be obtained.

SUBMITTER: Ye Y

PROVIDER: S-EPMC5599605 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Production and Characteristics of a Novel Xylose- and Alkali-tolerant GH 43 β-xylosidase from Penicillium oxalicum for Promoting Hemicellulose Degradation.

Ye Yanxin Y Li Xuezhi X Zhao Jian J

Scientific reports 20170914 1

β-xylosidase is a pivotal enzyme for complete degradation of xylan in hemicelluloses of lignocelluloses, and the xylose- and alkali-tolerant β-xylosidase with high catalytic activity is very attractive for promoting enzymatic hydrolysis of alkaline-pretreated lignocellulose. In this study, a novel intracellular glycoside hydrolase family 43 β-xylosidase gene (xyl43) from Penicillium oxalicum 114-2 was successfully high-level overexpressed in Pichia pastoris, and the secreted enzyme was character ...[more]

PMID: 28912429

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Project description:BACKGROUND:?-D-xylosidase is a vital exoglycosidase with the ability to hydrolyze xylooligosaccharides to xylose and to biotransform some saponins by cleaving outer ?-xylose. ?-D-xylosidase is widely used as one of the xylanolytic enzymes in a diverse range of applications, such as fuel, food and the pharmaceutical industry; therefore, more and more studies have focused on the thermostable and xylose-tolerant ?-D-xylosidases. RESULTS:A thermostable ?-xylosidase gene (xln-DT) of 1509 bp was cloned from Dictyoglomus thermophilum and expressed in E.coli BL21. According to the amino acid and phylogeny analyses, the ?-xylosidase Xln-DT is a novel ?-xylosidase of the GH family 39. The recombinant ?-xylosidase was purified, showing unique bands on SDS-PAGE, and had a protein molecular weight of 58.7 kDa. The ?-xylosidase Xln-DT showed an optimal activity at pH 6.0 and 75 °C, with p-nitrophenyl-?-D-xylopyranoside (pNPX) as a substrate. Xln-DT displayed stability over a pH range of 4.0-7.5 for 24 h and displayed thermotolerance below 85 °C. The values of the kinetic parameters K m and V max for pNPX were 1.66 mM and 78.46 U/mg, respectively. In particular, Xln-DT displayed high tolerance to xylose, with 60% activity in the presence of 3 M xylose. Xln-DT showed significant effects on the hydrolyzation of xylobiose. After 3 h, all the xylobiose tested was degraded into xylose. Moreover, ?-xylosidase Xln-DT had a high selectivity for cleaving the outer xylose moieties of natural saponins, such as notoginsenoside R1 and astragaloside IV, which produced the ginsenoside Rg1 with stronger anti-fatigue activity and produced cycloastragenol with stronger anti-aging activity, respectively. CONCLUSION:This study provides a novel GH 39 ?-xylosidase displaying extraordinary properties of highly catalytic activity at temperatures above 75 °C, remarkable hydrolyzing activity of xylooligosaccharides and rare saponins producing ability in the pharmaceutical and commercial industries.

Project description:Cellulolytic enzyme hydrolysis of lignocellulose biomass to release fermentable sugars is one of the key steps in biofuel refining. Gene expression of fungal cellulolytic enzymes is tightly controlled at the transcriptional level. Key transcription factors such as activator ClrB/CLR2 and XlnR/XYR1, as well as repressor CreA/CRE1 play crucial roles in this process. The putative protein methyltransferase LaeA/LAE1 has also been reported to regulate the gene expression of the cellulolytic enzyme. The formation and gene expression of the cellulolytic enzyme was compared among Penicillium oxalicum wild type (WT) and seven mutants, including ?laeA (deletion of laeA), OEclrB (clrB overexpression), OEclrB?laeA (clrB overexpression with deletion of laeA), OExlnR (xlnR overexpression), OExlnR?laeA (xlnR overexpression with deletion of laeA), ?creA (deletion of creA), and ?creA?laeA (double deletion of creA and laeA). Results revealed that LaeA extensively affected the expression of glycoside hydrolase genes. The expression of genes that encoded the top 10 glycoside hydrolases assayed in secretome was remarkably downregulated especially in later phases of prolonged batch cultures by the deletion of laeA. Cellulase synthesis of four mutants ?laeA, OEclrB?laeA, OExlnR?laeA, and ?creA?laeA was repressed remarkably compared with their parent strains WT, OEclrB, OExlnR, and ?creA, respectively. The overexpression of clrB or xlnR could not rescue the impairment of cellulolytic enzyme gene expression and cellulase synthesis when LaeA was absent, suggesting that LaeA was necessary for the expression of cellulolytic enzyme gene activated by ClrB or XlnR. In contrast to LaeA positive roles in regulating prominent cellulase and hemicellulase, the extracellular ?-xylosidase formation was negatively regulated by LaeA. The extracellular ?-xylosidase activities improved over 5-fold in the OExlnR?laeA mutant compared with that of WT, and the expression of prominent ?-xylosidase gene xyl3A was activated remarkably. The cumulative effect of LaeA and transcription factor XlnR has potential applications in the production of more ?-xylosidase.

Project description:BackgroundThere is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood.ResultsScreening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM-5.3 mM; Vmax-122 U/mg; kcat-107; Topt-50 °C; pHopt-6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications.ConclusionsHere we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.

Project description:?-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant ?-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above.A ?-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of ?-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 ?-xylosidase was a novel ?-xylosidase of GH3. The optimal activity of the Tth xynB3 ?-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 ?-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 ?-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 ?-xylosidase in 200 ?L reaction system for 3 h, almost all the substrate was biodegraded into xylose.The article provides a useful and novel ?-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.

Dataset Information

Production and Characteristics of a Novel Xylose- and Alkali-tolerant GH 43 ?-xylosidase from Penicillium oxalicum for Promoting Hemicellulose Degradation.

Publications

Production and Characteristics of a Novel Xylose- and Alkali-tolerant GH 43 β-xylosidase from Penicillium oxalicum for Promoting Hemicellulose Degradation.

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