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CLONING OF Aspergillus Niger BglA AND EXPRESSION OF RECOMBINANT β β β β β-GLUCOSIDASE IN METHYLOTROPHIC YEAST Pichia Pastoris
Abstract and Figures
Full length cDNA of bglA gene encoding Aspergillus niger ATCC10574 β-glucosidase was isolated and sequenced. The cDNA has a length of 2583 bp which encodes a polypeptide of 860 amino acid residues with predicted pI value of 4.6 and molecular weight of 93 kDa. Amino acid analysis of BGLA from four different isolates of A. niger, isolates ATCC10574, ATCC1015, B1 and CBS513.88, detected a total of 29 amino acids differences. The degree of differences varies between different variants, from 0.46% up to 2.9%. Around 34% of these differences were located in β-glucosidase two conserved domains, the glycosyl hydrolase family 3 N-terminal and the C-terminal domains. Both of the domains are important for the catalytic activity of the enzyme and these differences might contribute to different biophysical and biochemical enzyme properties. Heterologous expression of BGLA in methylotrophic yeast, Pichia pastoris has been carried out using methanol as inducer resulting in the production of recombinant protein with molecular weight around 90 kDa. β-glucosidase activity was detected from the culture filtrate using UV- stimulated fluorescence of cleaved fluorescence substrate, 4-methylumbelliferyl-β-D-glucopyranoside (MUGlc). The specific activity of the crude recombinant enzyme for cellobiose hydrolysis was 18 U/mg.
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... The bglA gene from A. niger encoding for βglucosidase has previously been cloned into the pPICZαC vector and transformed into P. pastoris strain X33 (Kamaruddin et al., 2008). The expression of the recombinant protein in P. pastoris was carried out as described by Al-Rashed et al. (2010) with some modifications. ...
This study describes the expression of β-glucosidase (BglA) from Aspergillus niger in Pichia pastoris, a methylotrophic yeast strain, under the regulation of an alcohol oxidase promoter. The heterologous expression of BglA was optimized in a shake flask. Optimal conditions were achieved using an initial cell density (OD 600) of 4-5 and an inducer concentration of 2.5% methanol for 72 hours. A recombinant protein with a molecular weight of ~116 kDa was produced. This recombinant BglA has optimal activity at 60°C in sodium acetate buffer at pH 4. This enzyme is stable between pH 3.0-6.0 and retained more than 50% of its maximum activity at pH 6.0 after incubation at 60°C for 30 min. However, it lost almost 80% of its maximal activity at pH 7.0 under the same conditions. A thermostability assay of this enzyme revealed that BglA is relatively stable up to 60°C. This enzyme retained 50% of its original activity at 60°C but was completely inactive after incubation at 70°C for 30 min. BglA showed highest activity and specificity towards the synthetic substrate p-nitrophenol-β-Dglucopyranoside with a specific activity of 347.62 U mg -1 and a specificity constant of 466.19 mL mg -1s-1 . BglA had a specific activity of 6.2 U mg -1 and a specificity constant of 6.01 mL mg -1s-1 for cellobiose. © 2015, Malaysian Society of Applied Biology. All rights reserved.
... The bglA gene from A. niger encoding for βglucosidase has previously been cloned into the pPICZαC vector and transformed into P. pastoris strain X33 (Kamaruddin et al., 2008). The expression of the recombinant protein in P. pastoris was carried out as described by Al-Rashed et al. (2010) with some modifications. ...
This study describes the expression of β-glucosidase (BglA) from Aspergillus niger in Pichia pastoris, a methylotrophic
yeast strain, under the regulation of an alcohol oxidase promoter. The heterologous expression of BglA was optimized in a
shake flask. Optimal conditions were achieved using an initial cell density (OD600) of 4-5 and an inducer concentration of
2.5% methanol for 72 hours. A recombinant protein with a molecular weight of ~116 kDa was produced. This recombinant
BglA has optimal activity at 60°C in sodium acetate buffer at pH 4. This enzyme is stable between pH 3.0-6.0 and retained
more than 50% of its maximum activity at pH 6.0 after incubation at 60°C for 30 min. However, it lost almost 80% of its
maximal activity at pH 7.0 under the same conditions. A thermostability assay of this enzyme revealed that BglA is relatively
stable up to 60°C. This enzyme retained 50% of its original activity at 60°C but was completely inactive after incubation at
70°C for 30 min. BglA showed highest activity and specificity towards the synthetic substrate p-nitrophenol-β-Dglucopyranoside
with a specific activity of 347.62 U mg-1 and a specificity constant of 466.19 mL mg-1s-1. BglA had a
specific activity of 6.2 U mg-1 and a specificity constant of 6.01 mL mg-1s-1 for cellobiose.
Background:
Cell walls of the starchy endosperm and young vegetative tissues of barley (Hordeum vulgare) contain high levels of (1-->3,1-->4)-beta-D-glucans. The (1-->3,1-->4)-beta-D-glucans are hydrolysed during wall degradation in germinated grain and during wall loosening in elongating coleoptiles. These key processes of plant development are mediated by several polysaccharide endohydrolases and exohydrolases.
Results:
. The three-dimensional structure of barley beta-D-glucan exohydrolase isoenzyme ExoI has been determined by X-ray crystallography. This is the first reported structure of a family 3 glycosyl hydrolase. The enzyme is a two-domain, globular protein of 605 amino acid residues and is N-glycosylated at three sites. The first 357 residues constitute an (alpha/beta)8 TIM-barrel domain. The second domain consists of residues 374-559 arranged in a six-stranded beta sandwich, which contains a beta sheet of five parallel beta strands and one antiparallel beta strand, with three alpha helices on either side of the sheet. A glucose moiety is observed in a pocket at the interface of the two domains, where Asp285 and Glu491 are believed to be involved in catalysis.
Conclusions:
The pocket at the interface of the two domains is probably the active site of the enzyme. Because amino acid residues that line this active-site pocket arise from both domains, activity could be regulated through the spatial disposition of the domains. Furthermore, there are sites on the second domain that may bind carbohydrate, as suggested by previously published kinetic data indicating that, in addition to the catalytic site, the enzyme has a second binding site specific for (1-->3, 1-->4)-beta-D-glucans.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Rochelle salt, normally present in the dinitrosalicylic acid reagent for reducing sugar, interferes with the protective action of the sulfite, but is essential to color stability. The difficulty may be resolved either by eliminating Rochelle salt from the reagent and adding it to the mixture of reducing sugar and reagent after the color is developed, or by adding known amounts of glucose to the samples of reducing sugar to compensate for the losses sustained in the presence of the Rochelle salt. The optimal composition of a modified dinitrosalicylic acid reagent is given.
An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322. The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The Km was 0·1 mmol l−1 for cellobiose. Enzyme activity was inhibited by several divalent metal ions.
A gene encoding an endo-β-1,4-glucanase, which is highly resistant to high temperature, protease and surfactant treatment, was isolated from Aspergillus niger IFO31125 and designated as eng1. The deduced amino acid sequence encoded by eng1 showed high homology with the sequence of a not-well-characterized cellulase encoded by eglB which has not yet been shown to be a stable enzyme. To confirm the sequence of the gene encoding the highly stable endo-β-1,4-glucanase, the cloned gene was expressed in the yeast Saccharomyces cerevisiae, in which no cellulase activity was found, and the gene product was purified and subjected to enzymatic characterization. The enzyme retained 56% of the initial activity after 1 h of incubation at 80°C and was stable in the range of pH 3.0–10.0. The optimal temperature for enzyme activity was 70°C and the optimal pH was 6.0. The enzyme was highly protease-resistant and retained more than 80% of the initial activity after protease treatment for 3 d at 40°C. The enzyme was also resistant to various surfactants. From these results, eng1 was confirmed to encode a very stable endo-β-1,4-glucanase.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
We have used a targeted gene deletion event to remove the coding region for the bgl1 gene encoding an extracellular beta-glucosidase from the genome of the cellulolytic fungus Trichoderma reesei. The bgl1 null mutants were used to investigate the role of beta-glucosidase in the hydrolysis of cellulose and induction of the other cellulolytic enzyme components. In the absence of extracellular beta-glucosidase, growth of bgl1 null strains on several carbon sources was the same as that of the parent (as measured by mycelial dry weight). However, levels of extracellular protein and total endoglucanase production were seen to lag relative to those levels observed in the control strain. The mRNA levels of the CBHI, CBHII, EGI, and EGII cellulase genes (cbh1, cbh2, egl1 and egl3) showed a corresponding lag in induction, suggesting that the absence of extracellular beta-glucosidase has an effect on the co-ordinate regulation of the other cellulase genes at the level of transcription. The addition of a potent inducer of the cellulase complex (sophorose) resulted in normal rates of cellulase gene mRNA production and extracellular protein release. This indicates that the absence of beta-glucosidase is not affecting some intrinsic cellular ability to produce mRNA or secrete protein. These data suggest that a functional beta-glucosidase is at least partially responsible for the efficient induction of the depolymerase enzymes of the cellulase complex. The observation that the cellulase complex is induced, albeit after a lag, suggests that other enzymes are present that can substitute for the function of beta-glucosidase during induction.