Screening and Production of Manganese Peroxidase from Fusarium sp. on Residue Materials

In this study, we report the manganese peroxidase production ability from a Fusarium sp. strain using an inexpensive medium of agriculture residues of either rice straw or wood chips as carbon source. The highest manganese peroxidase activity on rice straw medium and on wood chips was 1.76 U/mL by day 9 and 1.91 U/mL by day 12, respectively.

Heterologous Expression of Phanerochaete chrysoporium Glyoxal Oxidase and its Application for the Coupled Reaction with Manganese Peroxidase to Decolorize Malachite Green

cDNA of the glx1 gene encoding glyoxal oxidase (GLX) from Phanerochaete chrysosporium was isolated and expressed in Pichia pastoris. The recombinant GLX (rGLX) produces H2O2 over 7.0 nmol/min/mL using methyl glyoxal as a substrate. Use of rGLX as a generator of H2O2 improved the coupled reaction with recombinant manganese peroxidase resulting in decolorization of malachite green up to 150 microM within 90 min.

Heterologous Expression of Endo-1,4-beta-xylanaseA from Phanerochaete chrysosporium in Pichia pastoris

The cDNA of endo-1,4-beta-xylanaseA, isolated from Phaenerocheate chrysosporium was expressed in Pichia pastoris. Using either the intrinsic leader peptide of XynA or the alpha-factor signal peptide of Saccharomyces cerevisiae, xylanaseA is efficiently secreted into the medium at maximum concentrations of 1,946 U/L and 2,496 U/L, respectively.

Characterization of Aspergillus niger Mutants Deficient of a Protease

Aspergillus niger has been used as a host to express many heterologous proteins. It has been known that the presence of an abundant protease is a limiting factor to express a heterologous protein. The protease deficient mutant of A. niger was obtained using UV-irradiation. A total of 1x105 spores were irradiated with 10~20% survival dose of UV, 600 J/m2 at 280 nm, and the resulting spores were screened on the casein-gelatin plates. Ten putative protease deficient mutants showing the reduced halo area around colonies were further analyzed to differentiate the protease deficient mutant from other mutant types. Among ten putative mutants, seven mutants showed significant growth defect on nutrient rich medium and two mutants appeared to be the secretory mutants, which resulted in the impaired secretion of extracellular proteins including proteases. A mutant pro--20 showed reduced halo zone without any notable changes in growth rate. In addition, the starch-degrading and glucose oxidase activities in the culture filtrate of pro--20 mutant showed the similar range as that of the parental strain, which suggested that the pro--20 mutant ought to be the protease deficient mutant rather than a secretory mutant. The reduced proteolytic activity of the pro--20 was demonstrated using SDS-fibrin zymography gel. The reduced extracellular proteolysis was quantified by casein degradation assay and, comparing with the parental strain, less than 30% residual extracellular protease activity was detected in the culture filtrate of the pro--20 mutant. The bio-activity of an exogenously supplemented hGM-CSF (human Granulocyte-Macrophage Colony Stimulating Factor) in the culture filtrate of pro--20 mutant was detected until eight times more diluted preparations than that of the parental strain.

Improved Transformation of the Filamentous Fungus Aspergillus niger Using Agrobacterium tumefaciens

Since it is known that Agrobacterium tumefaciens, which has long been used to transform plants, can transfer the T-DNA to yeast Saccharomyces cerevisiae during tumourigenesis, a variety of fungi were subjected to transformation to improve their transformation frequency. In this study, I report the A. tumefaciens-mediated transformation of filamentous fungus Aspergillus niger. Transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of the Aspergillus nidulans trpC promoter and terminator as a selectable marker, led to the selection of 50~100 hygromycin B-resistant transformants per 1x10(7) conidia of A. niger. This efficiency is improved 10~20 fold more than reported elsewhere. In order to avoid the difficulties in selection transformant from the over-growing non-transformant, I used top agar containing 900 microg/ml of hygromycin. Genomic PCR and Southern analysis showed that all transformants contained single T-DNA insert per fungal genome. This technique offers an easier and more efficient method than that of using protoplast.