Studies on an alkali-thermostable xylanase from Aspergillus fumigatus MA28.
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ABSTRACT:
SUBMITTER: Bajaj BK
PROVIDER: S-EPMC3339620 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Xylanase with a high thermostability will satisfy the needs of raising the temperature of hydrolysis to improve the rheology of the broth in industry of biomass conversion. In this study, a xylanase gene (xyn10A), predicted to encode a hydrolase domain of GH10, a linker region and a CBM1 domain, was cloned from a superior lignocellulose degrading strain Aspergillus fumigatus Z5 and successfully expressed in Pichia pastoris X33. Xyn10A has a specific xylanase activity of 34.4 U mg-1, and is optimally active at 90 °C and pH 6.0. Xyn10A shows quite stable at pHs ranging from 3.0 to 11.0, and keeps over 40% of xylanase activity after incubation at 70 °C for 1 h. Removal of CBM1 domain has a slight negative effect on its thermostability, but the further cleavage of linker region significantly decreased its stability at high temperature. The transfer of CBM1 and linker region to another GH10 xylanase can help to increase the thermostability. In addition, hydrolase domains between the two Xyn10A proteins naturally formed a dimer structure, which became more thermostable after removing the CBM1 or/and linker region. This thermostable Xyn10A is a suitable candidate for the highly efficient fungal enzyme cocktails for biomass conversion.
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Project description:Xylanases (EC 3.2.1.8) catalyze the hydrolysis of beta-1,4-glycosidic linkages within xylan, a major hemicellulose component in the biosphere. The extracellular endoxylanase (XylnA) from the alkalophilic Bacillus sp. strain NG-27 belongs to family 10 of the glycoside hydrolases. It is active at 343 K and pH 8.4. Moreover, it has attractive features from the point of view of utilization in the paper pulp, animal feed and baking industries since it is an alkali-thermostable protein. In this study, XylnA was purified from the native host source and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 174.5, b = 54.7, c = 131.5 A, beta = 131.2 degrees, and diffract to better than 2.2 A resolution.
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Project description:Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-?-xylanase that performs optimally at 65-75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (?/?)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.
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Project description:Isogenic bar-coded strains of Aspergillus fumigatus carrying the G54W or M220K mutation in Cyp51A were constructed. In vitro, the growth and conidiation capacities of the mutants were similar to those of the parental strain. Competition studies in the absence of azoles showed that there was no adverse fitness cost for the azole-resistant A. fumigatus strains in vitro or in vivo compared to the parental strain.
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Project description:BACKGROUND:The search for novel thermostable xylanases for industrial use has intensified in recent years, and thermophilic fungi are a promising source of useful enzymes. The present work reports the heterologous expression and biochemical characterization of a novel thermostable xylanase (GH10) from the thermophilic fungus Malbranchea pulchella, the influence of glycosylation on its stability, and a potential application in sugarcane bagasse hydrolysis. RESULTS:Xylanase MpXyn10A was overexpressed in Aspergillus nidulans and was active against birchwood xylan, presenting an optimum activity at pH 5.8 and 80°C. MpXyn10A was 16% glycosylated and thermostable, preserving 85% activity after 24 hours at 65°C, and deglycosylation did not affect thermostability. Circular dichroism confirmed the high alpha-helical content consistent with the canonical GH10 family (?/?)8 barrel fold observed in molecular modeling. Primary structure analysis revealed the existence of eight cysteine residues which could be involved in four disulfide bonds, and this could explain the high thermostability of this enzyme even in the deglycosylated form. MpXyn10A showed promising results in biomass degradation, increasing the amount of reducing sugars in bagasse in natura and in three pretreated sugarcane bagasses. CONCLUSIONS:MpXyn10A was successfully secreted in Aspergillus nidulans, and a potential use for sugarcane bagasse biomass degradation was demonstrated.
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Project description:Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30-35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.Banana peels (BP), an under-utilized waste material, was studied for the production of xylanase and pectinase by Aspergillus fumigates MS16. The factors affecting the co-production of both the enzymes were separately studied for their influence under submerged (Smf) and solid-state fermentation (SSF) of BP. The strain was cultivated in the presence of mineral salt (MS) solution containing BP powder as a sole source of carbon and physical and nutritional factors varied to observe the change in the enzyme titers. The data revealed that the MS-based medium was appropriate for the production of both the enzymes; therefore, in subsequent experiments, the same medium was used. A temperature of 30–35°C was found better for the production of the two enzymes under Smf; however, the titers of pectinase dropped significantly at 40°C. Contrarily, xylanase production was inhibited at 40°C under SSF but not under Smf. Whereas, supplementation of xylan or pectin to BP induced the production of xylanase and pectinase, respectively. Lowering the pH value favored the production of both the enzymes under Smf; however, the production of pectinase improved significantly when a higher concentration of BP (1%) was used compared to the concentration (0.25%) required for the production of xylanase. Interestingly, the enzyme preparation obtained under SSF exhibited optimal activities of both the enzymes at higher temperatures when compared to those obtained under Smf. The data indicated that the physiology of the fungus differed greatly when the cultivation pattern varied from Smf to SSF and, hence, the enzymes produced were characteristically distinct.
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Project description:Aspergillus fumigatus has been understood to be the most common cause of invasive aspergillosis (IA) in all epidemiological surveys. However, recent studies have uncovered a large degree of genetic heterogeneity between isolates morphologically identified as A. fumigatus, leading to the description of a new species, Aspergillus lentulus. Here, we examined the genetic diversity of clinical isolates identified as A. fumigatus using restriction enzyme polymorphism analysis and sequence-based identification. Analysis of 50 clinical isolates from geographically diverse locations recorded the presence of at least three distinct species: A. lentulus, Aspergillus udagawae, and A. fumigatus. In vitro, A. lentulus isolates demonstrated decreased susceptibility to antifungal drugs currently used for IA, including amphotericin B, voriconazole, and caspofungin; A. udagawae isolates demonstrated decreased in vitro susceptibility to amphotericin B. Results of the present study demonstrate that current phenotypic methods to identify fungi do not differentiate between genetically distinct species in the A. fumigatus group. Differential antifungal susceptibilities of these species may account for some of the reported poor outcomes of therapy in clinical studies.
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Project description:BACKGROUND: Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and ?-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful ?-glucosides. Although the ?-glucosidases of different sources have been investigated, the amount of ?-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of ?-glucosidases, which was thermostable and with high catalytic efficiency. RESULTS: In this study, a thermostable native ?-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus Aspergillus fumigatus Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, bgl3, was cloned based on the peptide sequences obtained from the LC-MS/MS results. bgl3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant ?-glucosidase (rBgl3) was obtained by the functional expression of bgl3 in Pichia pastoris X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-?-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan. CONCLUSIONS: An native ?-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of A. fumigatus Z5. The gene bgl3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene bgl3 was expressed in Pichia pastoris X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.
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