ABSTRACT
Aspergillus fumigatus wild-type phytase has many favorable properties, such as a good thermorstability and a broad pH optimum. However, the specific activity of the enzyme is relative low. A. fumigatus Q23L phytase resulted in a remarkable increase in specific activity around pH4.5 - 7.0, but the pH stability of Q23L was lower than A. fumigatus wild-type phytase. To increase the pH stability of Q23L, the mutant Q23LG272E was constructed by site-directed mutagenesis with PCR. The gene of A. fumigatus wild-type phytase and the mutant genes encoding the Q23LG272E and the Q23L were correctly expressed in Pichia pastoris GS115. Enzymes were purified and their enzymatic properties were determined. The results revealed that the specific activity of the Q23L improved remarkably, which increased from 51 u/mg of the wild type to 109 u/mg at pH5.5. Meanwhile, the pH stability of Q23L, decreased evidently, especially from pH3.0 to pH4.0.The pH stability of Q23LG272E in pH3.0 - 4.5 and pH6.5 - 7.0 has been improved compared with Q23L. The specific activity of Q23LG272E basically maintained at the level of Q23L. Analysis of 3-D structure and sequence similarity were used to reveal the presumable factors influencing the enzymatic properties of Q23LG272E, and discussion for the relationship between structure and function of phytase was given.
Subject(s)
6-Phytase , Chemistry , Genetics , Metabolism , Amino Acid Substitution , Aspergillus fumigatus , Genetics , Biocatalysis , Electrophoresis, Polyacrylamide Gel , Fungal Proteins , Chemistry , Genetics , Metabolism , Hydrogen-Ion Concentration , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins , Genetics , Metabolism , Mutation , Pichia , Genetics , Polymerase Chain Reaction , Protein Conformation , Protein Engineering , Methods , Recombinant Proteins , Metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A homology modeling of xylanase XYNB from Streptomyces olivaceoviridis A1 was made by Swiss-Model. The hydrophobic Interaction between beta-sheet B1 and B2 in the tertiary structure model of XYNB was compared with other thermophilic xylanase. A T11Y mutation was introduced in XYNB by site-dirrected mutagenesis to improve the thermostability of the enzyme. The XYNB and mutant xylanase (XYNB') expressed in Pichia pastoris were purified and their enzymatic properties were determined. The result revealed that the thermostability of XYNB' was obviously higher than that of XYNB. The optimal temperature of XYNB' for its activity was 60 degrees C, similar to XYNB. But, compare to XYNB, the optimal pH value, the Km value and the specific activity of XYNB' had also been changed. The research results suggested that the aromatic interaction between beta-sheet B1 and B2 in xylanase should increase enzyme thermostability. The mutant xylanase XYNB' is a good material for further research in the relationship between structure and function of xylanase.
Subject(s)
Bacterial Proteins , Chemistry , Genetics , Endo-1,4-beta Xylanases , Chemistry , Genetics , Enzyme Stability , Hot Temperature , Hydrophobic and Hydrophilic Interactions , Mutagenesis, Site-Directed , Mutant Proteins , Chemistry , Pichia , Genetics , Metabolism , Protein Conformation , Protein Folding , Protein Structure, Tertiary , Genetics , Recombinant Proteins , Genetics , Streptomyces , Genetics , beta-Glucosidase , Chemistry , GeneticsABSTRACT
The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.
Subject(s)
Bacillus , Genetics , Bacterial Proteins , Genetics , Metabolism , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Pichia , Genetics , Metabolism , alpha-Amylases , Genetics , MetabolismABSTRACT
High-level expression of phytase with high specific activity is an effective way to improve phytase fermentation potency and reduce its production cost. The gene appA encoding Escherchia coli phytase AppA with high specific activity was modified and artificially synthesized according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence of the AppA. The modified gene, appA-m, was inserted in the Pichia pastoris expression vector pPIC9, then introduced into the host Pichia pastoris by electroporation. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The result of Southern blotting analysis of the recombinant yeast indicated that only one copy of the appA-m gene was integrated into the genome of Pichia pastoris. The result of Northern analysis of the recombinant yeast showed that the modified gene was effectively transcribed. SDS-PAGE analysis of the phytase expressed in Pichia pastoris revealed that the phytase was overexpressed and secreted into the medium supernatant. There are three phytase proteins with apparent molecular weight in approximately 50kD, 52kD and 54kD respectively in the media, which are larger in the size than the native phytase from E. coli. The results of N-terminal sequecing and deglycosylation of the expressed phytase in Pichia pastoris proved that the expressed phytase were glycosylated protein with different glycosylation degree. The expressed phytase Pichia pastoris shared similar pH and temperature optima to those of the natural phytase from E. coli and had highly resistant to pepsin digestion. In 5-L fermentor, after induced by 0.5% methanol for 120 h, the expression level of phytase protein was 2.5 mg/mL, and the phytase activity (fermentation potency) exceeded 7.5 x 10(6) IU/mL, which was the highest among those of all kinds of recombinant strains reported now.
Subject(s)
6-Phytase , Genetics , Metabolism , Escherichia coli , Escherichia coli Proteins , Genetics , Fermentation , Pichia , Genetics , Plasmids , Recombinant ProteinsABSTRACT
The gene xynA encoding xylanase was cloned from Streptomyces olivaceoviridis A1. The xynA with and without origin signal peptide sequence were fused behind pel B signal peptide in the plasmid pET-22b(+) respectively, then transfered into the host E. coli. The xylanase expressed in E. coli had normal bioactivity. Further, the xynA without origin signal peptide sequence was cloned into the plasmid pPIC9 under the control of AOX1 promoter and introduced into the host Pichia pastoris by electroporation. The results of SDS-PAGE and activity assay of the xylanase expressed by recombinant P. pastoris showed that the xynA had been overexpressed and secreted, and the xylanase expressed had normal bioactivity. The expression level of xylanase in recombinant P. pastoris exceeded 0.2mg/mL in shake culture.