Production and characterization of cellobiohydrolase from a novel strain of Penicillium purpurogenum KJS506.

A high cellobiohydrolase (CBH)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 according to the morphology and comparison of internal transcribed spacer rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, a maximum CBH activity of 2.6 U mg-protein(-1), one of the highest among CBH-producing microorganisms, was obtained. The optimum temperature and pH for CBH production were 30 °C and 4.0, respectively. The increased production of CBH in P. purpurogenum culture at 30 °C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. The internal amino acid sequences of P. purpurogenum CBH showed a significant homology with hydrolases from glycoside hydrolase family 7. The extracellular CBH was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast-protein liquid chromatography. The purified CBH was a monomeric protein with a molecular weight of 60 kDa and showed broad substrate specificity with maximum activity towards p-nitrophenyl ß-D: -cellobiopyranoside. P. purpurogenum CBH showed t (1/2) value of 4 h at 60 °C and V (max) value of 11.9 µmol min(-1) mg-protein(-1) for p-nitrophenyl-D: -cellobiopyranoside. Although CBHs have been reported, the high specific activity distinguishes P. purpurogenum CBH.

Characterization of beta-glucosidase from a strain of Penicillium purpurogenum KJS506.

A novel beta-glucosidase (BGL)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal BGL activity of 12.3 U ml(-1), one of the highest levels among BGL-producing microorganisms was observed. The optimum temperature and pH for BGL production were 32 degrees C and 4, respectively. An extracellular BGL was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants, and the purified BGL showed higher activity (V (max) = 934 U mg protein(-1)) than most BGLs from other sources. The complete ORF of bgl3 was cloned from P. purpurogenum by a modified thermal asymmetric interlaced polymerase chain reaction. The bgl3 gene consists of a 2,571-bp ORF and encodes a putative protein containing 856 amino acids with a calculated molecular mass of 89,624 Da. The putative gene product was identified as a member of glycoside hydrolase family 3. The present results should contribute to improved industrial production of BGL by P. purpurogenum KJS506.

Characterization of endo-beta-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601.

A novel endo-beta-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein(-1), one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28 degrees C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28 degrees C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene (eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601.

Purification and characterization of a thermostable beta-1,3-1,4-glucanase from Laetiporus sulphureus var. miniatus.

A beta-1,3-1,4-glucanase from the fungus Laetiporus sulphureus var. miniatus was purified as a single 26 kDa band by ammonium sulfate precipitation, HiTrap Q HP, and UNO Q ion-exchange chromatography, with a specific activity of 29 U/mg. The molecular mass of the native enzyme was 52 kDa as a dimer by gel filtration. beta-1,3-1,4-Glucanase showed optimum activity at pH 4.0 and 75 degrees . The half-lives of the enzyme at 70 degrees and 75 degrees were 152 h and 22 h, respectively. The enzyme showed the highest activity for barley beta- glucan as beta-1,3-1,4-glucan among the tested polysaccharides and p-nitrophenyl-beta-D-glycosides with a K(m) of 0.67 mg/ml, a k(cat) of 13.5 s(-1) and a k(cat)/K(m) of 20 mg/ml/s.

Purification and characterization of a beta-1,4-glucosidase from a newly isolated strain of Fomitopsis pinicola.

An efficient beta-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50 degrees C. The enzyme showed high substrate specificity and high catalytic efficiency (kcat=2,990 s-1, Km=1.76mM, kcat/Km=1,700 mM-1 s-1) for p-nitrophenyl-beta-d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity.