Rapid release of protoplasts from Eremothecium ashbyii in comparison with Trichoderma reesei and Penicillium chrysogenum using novozyme and funcelase.
TL;DR: Protoplast release in Eremothecium ashbyii, Trichoderma reesei, and Penicillium chrysogenum was achieved using commercially available enzymes, Novozyme 234 and Funcelase and a rapid release of protoplasts was observed.
About: This article is published in Enzyme and Microbial Technology.The article was published on 1993-08-01. It has received 10 citations till now. The article focuses on the topics: Trichoderma reesei & Penicillium chrysogenum.
Citations
More filters
TL;DR: The regenerated protoplasts from Trichothecium roseum were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.
Abstract: A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 x 10(4) protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose--yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.
50 citations
TL;DR: The fungal strains Graphium putredinis and Trichoderma harzianum and their intergeneric fusant in the production of hydrolytic enzymes - amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) - have probable applications in various industrial processes.
29 citations
TL;DR: Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1−1 KCl and sorbitol were used as osmotic stabilizers, and Endo‐P, Exo-P and pectin lyase production were not modified during the process of regeneration.
Abstract: S Solis, ME FLORES AND C HUITRON 1996 Protoplast release in pectinolytic strain mutants of Aspergillus sp CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml−1) were added in 005 mol 1−1 citrate-phosphate buffer pH 50 containing 07 mol 1−1 KCl and 10 mg ml−1 BSA Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml−1Aureobasidium sp lytic enzymes Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 07 mol 1−1 KCl and sorbitol were used as osmotic stabilizers Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration
16 citations
TL;DR: Neither Cellulase Onozuka RS nor snail gut enzymes from Helix pomatia were able to generate protoplasts either alone or in combination, however, when commercial chitinase was added to either of these two enzymes.
11 citations
TL;DR: This study defines the conditions (wavelength selection and sensitivity) for the spectrofluorimetric quantification of lipids in situ in the macerated mycelia of this fungus in the presence of intracellular autofluorescent riboflavin without the need to extract the lipids from theMycelia.
11 citations
References
More filters
TL;DR: The protoplast Fusion and the Hybridization of Fungal Species and Transformation in Fungi by Using Protoplasts by using Enzymatic and Nonenzymatic methods are studied.
Abstract: INTRODUCTION 21 ISOLATION OF PROTOPLASTS AND THEIR P ROPERTIES 22 Enzymatic Methods for Protoplast Isolation 22 Nonenzymatic Procedures for Protoplast Release 26 Properties of Protopiasts 27 WALL REGENERATION AND REVERSION OF PROTOPLASTS 28 Frequency of Protoplast Reversion 28 Morphology of Protoplast Reversion 29 Ultrastructure and Composition of the Regenerated Wall ..... 30 Biochemical Aspects oj Wall Biogenesis 31 PROTOPLAST FUSION AND GENETIC MANIPULATION 33 Protoplast Fusion and the Hybridization of Fungal Species 33 Transformation in Fungi by Using Protoplasts . .. ........ 36 CONCLUDING REMARKS 36
187 citations
TL;DR: It is shown that high yields of protoplasts can be prepared from a variety of fungi using relatively cheap commercial enzymes.
163 citations
TL;DR: Osmotically fragile protoplasts have been prepared by the action of lytic enzymes on the mycelium of Penicillium chrysogenum and Cephalosporium acremonium by measuring their respiration, ability to maintain intracellular amino-acid pools, and antibiotic production.
Abstract: SUMMARY: Osmotically fragile protoplasts have been prepared by the action of lytic enzymes on the mycelium of Penicillium chrysogenum and Cephalosporium acremonium. The yield of protoplasts, based on DNA content, was up to 18 %. Pretreatment of the mycelium with a thiol compound was necessary with Cephalosporium but not with Penicillium. Electron micrographs indicated that the protoplasts contained all the intracellular organelles of the mycelium and were bounded only by a cytoplasmic membrane. The metabolic activity of the protoplasts, as measured by their respiration, ability to maintain intracellular amino-acid pools, and antibiotic production, was similar to that of control mycelium. l-Valine and l-α-aminoadipic acid, which are precursors of penicillin N and cephalosporin C, were taken up and concentrated by the protoplasts of C. acremonium, although the latter transported l-valine less rapidly than did the corresponding mycelium. Protoplasts of P. chrysogenum took up l-valine but not l-α-aminoadipic acid or its δ-ester. There was little or no transport of the corresponding d-amino acids by protoplasts of either organism.
57 citations
TL;DR: It is concluded that knobs are the segregants from the fusants, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.
Abstract: Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation of the resulting fusants were studied. Combinations of protoplasts prepared from young conidia with double amino acid requirements, one of which was a common requirement and the other uncommon, were fused in the presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only the commonly deficient amino acid. The frequency of fusion was 0.9 x 10 to 4.0 x 10 for the starting conidia and 3.0 x 10 to 4.9 x 10 for the regenerated protoplasts, which was significantly higher than the expected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diverse phenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed from single conidia of the nonparental phenotype contained special spots called "knobs" that have a higher density of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, and recombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob part was essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of a piece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same as that of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these results, we conclude that knobs are the segregants from the fusants. One knob clone showed twice the carboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reesei cells by the protoplast fusion technique.
53 citations
Journal Article•
TL;DR: Separation of the secreted proteins by fast protein liquid chromatography revealed at least seven peaks of carboxymethylcellulase activity and β-glucosidase activity, indicating that some multiplicity of the forms of these enzymes is already present at the secretion stage.
Abstract: Novozym 234, a commercially available enzyme from Trichoderma harzianum, has been used to prepare protoplasts from Trichoderma reesei QM 9414. Optimal conditions were: 20 h old mycelium, 0.9 M-KCl as the osmotic stabilizer. 0.1% (w/v) Novozym 234 and 18 h incubation at 28°C. To prevent damage to the protoplasts during isolation the incubation mixtures were agitated by vibration, so avoiding shaking or stirring. More than 95% of the protoplasts were viable and were also metabolically intact as shown by their ability to take up [14C]leucine and incorporate it into cellular protein. Freshly prepared protoplasts could be induced by sophorose to produce and secrete carboxymethylcellulase and β-glucosidase. Optimal conditions were: 0.9 M-KCl as osmotic stabilizer, phosphate buffer pH 6.0, 7 mM-sophorose and 107 protoplasts ml-1. Separation of the secreted proteins by fast protein liquid chromatography revealed at least seven peaks of carboxymethylcellulase activity and β-glucosidase activity, indicating that some multiplicity of the forms of these enzymes is already present at the secretion stage.
32 citations