Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium

doi:10.1016/0141-0229(92)90073-W

Enzyme and Microbial Technology

Volume 14, Issue 3, March 1992, Pages 241-248

https://doi.org/10.1016/0141-0229(92)90073-W Get rights and content

Abstract

Efficient synthesis of a large number of viable protoplasts from Trichoderma longibrachiatum (Trichoderma reesei) mycelium has been found to be influenced by a number of factors. In this case, Trichoderma harzianum NCIM 1185 culture filtrate has been used to prepare protoplasts from Trichoderma longibrachiatum QM 9414. A method has been devised to isolate a large number of viable protoplasts from the mycelium. Detailed analysis of different factors affecting the synthesis of protoplasts from T. longibrachiatum has not yet been reported. The most suitable conditions for protoplasting were as follows: age of the organism in slant, 3 days; mycelium age, 20 h; volume of lytic enzymes, 190 ml; mycelial weight (dry equivalent), 1.66 g; time of contact with lytic enzymes, 2 h; temperature of protoplasting, 30°C; phosphate buffer, 25 mm, pH 6.5; KCl as osmotic stabilizer, 0.7 m. Proper spreading of the mycelium in contact with the lytic enzymes was also found to be necessary.

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Studies on a N-acetyl-β-D-glucosaminidase produced by Fusarium oxysporum F3 grown in solid-state fermentation
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Fusarium oxysporum F3 produced N-acetyl-β-d-glucosaminidase when grown on wheat bran and chitin as carbon sources in solid-state fermentation. The initial moisture content and pH of growth medium were 65% and 6.0, respectively, and the enzyme yield 23.6 U g⁻¹ carbon source. Two isozymes of N-acetyl-β-d-glucosaminidase, called N-acetyl-β-d-glucosaminidases I and II, were isolated from the culture filtrate of F. oxysporum F3. The filtrate was subjected to ammonium sulphate fractionation followed by anion exchange, gel filtration, hydrophobic interaction and cation exchange chromatography. The optimum pH of isozymes I and II was 5.0 and 6.0, respectively, whereas maximum activity of both isozymes was obtained at 40 °C. The K_m of isozymes I and II was 49.6 and 48.6 μM and the V_max 1.24 and 0.26 μmol mg⁻¹ min⁻¹, respectively, on p-nitrophenyl N-acetyl-β-d-glucosaminide as substrate. The molecular mass of isozymes I and II was calculated to be 67 kDa by SDS–PAGE.
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PaperStudies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium

Abstract

Enzyme Microb. Technol.

Enzyme Microb. Technol.

Anal. Biochem.

Exp. Mycol.

J. Biotechnol.

Process Biochem.

Annu. Rev. Microbiol.

J. Gen. Microbiol.

Mol. Gen. Genet.

Mol. Gen. Genet.

J. Gen. Microbiol.

Can. J. Microbiol.

Eur. J. Appl. Microbiol. Biotechnol.

Nature

Experientia

Paper
Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium