• Microbiology and Biotechnology Letters
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• v.21 no.6
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• pp.588-593
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• 1993

Xylanase(1, 4-beta-D-xylan xylanohydrolase` EC 3.2.1.8) II was purified from Penicillium verruculosum by using the techniques of two anion exchange chromatographies, and gel filtration. The molecular weight of this enzyme was about 22, 000 as determined by SDS-electrophoresis. The enzyme showed hydropytic activity toward xylan but did not catalyze hydrolysis of Rho-nitrophenyl-beta-D-xylopyranoside, Rho-nitrophenyl-beta-D-glucopyranoside, Rho-nitrophenyl-beta-D-cellobiopyranoside, and celluloses such as Avicel, cotton, filter paper, carboxymethylcellulose.

• The Korean Journal of Food And Nutrition
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• v.7 no.1
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• pp.1-7
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• 1994

To investigate the characteristics of active sites of the D-xylanase and $\beta$-xylosidase purified from Penicillium verruculosum, effects of various chemicals on the enzyme activity were analyzed. The D-xylanase was activated by Cua), however it was inhibited by metal ions, Hg2+ and Mna+, by chemicals, N-bromosuccinimide, iodine, diethylpyrocarbonate, and 2,3-butanedione. These results suggested that the D-xylanase from Penicillium verruculosum contained tyrosine, histidine, arginine and tryptophan at the active center. The $\beta$-xylosidase was inhibited by Hg2+, N-bromosuccinimide and sodium dodecyl sulfate, however it was not effected by Mn2+ and Cu2). It was suggested that the enzyme contained tryptophan at the active center.

• Microbiology and Biotechnology Letters
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• v.16 no.3
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• pp.182-186
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• 1988

The possibility of strain improvement of cellulolytic fungus, Penicillium verruculosum via protoplast fusion was investigated. The cellulolytic activities of the six fusants, finally selected for their hyper-cellulolytics were 2 times of those of wild type and 1.2 to 4.4 times of those parental auxotrophs. It was confirmed that the nuclear fusion occurred in fusants by their DNA contents and nuclear staining with Giemsa. It was also found that the fusants were aneuploids, and their genetic stability was demonstrated from the subculture for four months.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.163-167
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• 1988

The conditions for the protoplast fusion of auxotrophic mutants of Penicillium verruculosum were determined. A preparation of commercial enzyme Novozym 234 was used to successfully isolate protoplast from the 20hr old mycelium of P. verruculosum. Under optimal condition, the protoplast yield ranged from 2.4$\times$10$^7$ to 3.0$\times$10$^7$ protoplasts from 400mg of damp mycelia of various auxotrophic mutant strains. The regeneration frequency ranged from 26.6 to 42.4% and the spontaneous reversion frequency of the protoplasts on the regeneration minimal medium was less than 10$^7$. The optimal concentration of PEG 6000 was 20%, and exposure of protoplasts to PEG for 10 min was found to be sufficient for protoplast fusion. Optimal pH of fusion mixture was deter-mined as 5.5 and l0mM of calcium chloride in fusion mixture effectively enhanced the protoplast fusion frequency. Under optimal condition, the fusion frequency between various auxotrophs ranged from 1.8$\times$10$^{-3}$ to 3.5$\times$0$^{-3}$.

• The Korean Journal of Mycology
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• v.21 no.1
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• pp.28-37
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• 1993

An endo-type cellulase, carboxymethyl cellulase(CMCase) IV, was purified from culture filtrate of cellulolytic fungus Penicillium verruculosum. The CMCase IV was acidic glycoprotein having carbohydrate of 13% as glucose and pI value of 4.0. The CMCase IV was 52 KDa of molecular weight in SDS-polyacrylamide gel electrophoresis and have optimum temperature and pH of $50^{\circ}C$ and 5.0 for enzyme activity. The CMCase IV liberated glucose and cellobiose as major products of the enzyme against carboxymethyl cellulose(CMC) and seemed to has transglycosylation activity simultaneously. Cellulase activity staining(zymogram) showed that the cellulase components of P. verruculosum were not aggregated in the medium. P. vrttuculosum mRNA was translated in vitro and translation product by the mRNA coding for CMCase IV was identified by immunoprecipitation.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.23-30
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• 1994

Intergeneric hybrids formed between Aspergillus oryzae var oryzae and Penicillium verruculosum F-3 were obtained by nuclear transfer technique. Several auxotrophic mutants isolated fromconidio-spores of the two strains mutagenized with ultraviolet and N-methyl-N-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of hybrid formation by nuclear transfer were 3$\times $10$^{-5}$ $~$1$\times $10$^{-5}$. From observation of genetic stability, conidial size, DNA content, nuclear stain, it was suggested that their karyptypes are aneuploid. The hybrid posses the 1.3$~$2.2 fold higher amylase activities than those of parental strains. It was also revealed that some hybids had different isozyme patterns compared to those of parental strains by amylase activity assays.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.4
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• pp.423-427
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• 1992

During the cultivation of Penicillium verruculosum in the medium containing xylan as a sole carbon source for 26 days, xylanolytic activity and some changes were investigated. Protein content and xylanolytic activity, p-Nitrophenyl-$\beta$-D-xylopyranoside (PNPX), p-Nitrophenyl-$\beta$ -D-glucopyranoside (PNPG) hydrolytic activities were increased until 8 days but reducing sugar content was not correlated to protein content. When crude proteins from the culture broth were separated on SDS-PAGE, distribution of proteins was different from the culture broth of cellobiose octaacetate (COA) medium. The culture broth of xylan medium had high hydrolytic activity on xylan but not on cellulose. Furthermore, xylanolytic products were showed xylose, xylobiose and oligosaccharides on thin layer chromatography, and xylobiose was major product. Those result suggested that xylanolytic activity of culture broth was endo-type hydrolysis. Optimum temperatures of xylanolytic activity and PNPX hydrolytic activity of culture broth were 50~6$0^{\circ}C$ and 60~7$0^{\circ}C$, respectively and optimum pHs were 3.0~4.0 and 4.0~5.0, respectively.

• Microbiology and Biotechnology Letters
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• v.16 no.2
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• pp.156-162
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• 1988

Optimal conditions for the formation and regeneration of protoplasts of the cellulolytic fungus Penicillium verruculosum were investigated. Among the various commercial cell wall lytic enzymes tested, 0.5%(w/ v) Novozym 234 was the most effective for protoplast formation. The highest yield of protoplast exceeding 4.5$\times$10$^6$/m$\ell$ obtained when 400mg of 20 hr-old mycelia was incubated with 0.5%(w/v) Novozym 234 at 3$0^{\circ}C$ for 1 hr. The best osmotic stabilizer for the isolation and re-generation of protoplasts was 0.7M sorbital (pH 5.6) and 0.6M MgSO$_4$(pH 5.6), respectively. When 0.6M MgSO$_4$was added as osmotic stabilizer to the complete medium, the maximum regeneration frequency obtained was 4.6-27.8%. Micromorphological change of giant protoplasts into hyphae was observed during incubation in the regeneration liquid medium.

• Korean Journal of Microbiology
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• v.31 no.1
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• pp.1-8
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• 1993

lntergeneric hybrids formed between Aspergillus niger and Penicillium verruculosum were obtained by nuclear transfer technique. Nuclei isolated from wild type and auxotrophic mutants of donor strains were transferred into the protoplasts of different auxotrophic mutants as recipient strains. Several auxotrophic mutants were isolated from conidiospores of the two strains mutagenized with ultraviolet and N-methyl-N'-nitrosoguanidine. Optimal conditions for formation of intergeneric hybrids were investigated. Frequencies of intergeneric hybrid formation by nuclear transfer were $7{\times}10^{5}~1{\times}10^{5}$. From observations of genetic stability. DNA content. nuclear stain and conidial size. it was suggested that their karyotypes are aneuploid. In addition. the hybrids possess the 1.1~2.3-fold higher cellulase activities than those of parental strains. It was also revealed that some hybrids had different isozyme patterns compared to those of parental strains by CMCase and $\beta$-glucosidase activity assays.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.232-239
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• 1991

Caboxymethyl cellulase (CMCase) I was purified from the induced culture filtrate of Penicllium verruculosum F-3 by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and Bio-gel P-150 filtration. The purified enzyme was assumed to be a glycoprotein consisting of 8.5% carbohydrate and having a molecular weight of 70.000 in SDS-polycrylamide gel electrophoresis (SDS-PAGE). The purified enzyme-specific anti-CMCase I IgG was obtained by rabbit immunization and protein A-sepharose CL-4B chromatography. The fungal poly($A^+$) RNA was isolated from the total RNA of the mycelium grown under cellulase induction conditions by oligo(dT)-cellulosse chromatography. The translation products in vitro were prepared by translating the isolated poly ($A^+$) RNA in rabbit reticulocyte lysate and analyzed by SDS-PAGE and fluorography. Of the translation products, CMCase I was identified by the immunoprecipitation against anti-CMCase I IgG.