Studies on critical analysis of factors influencing improved production of protoplasts from Trichoderma reesei mycelium
TL;DR: 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 enzyme, 2 h; temperature of protoplasts, 30°C; phosphate buffer, 25 m m , pH 6.5; KCl as osmotic stabilizer, 0.7 m
About: This article is published in Enzyme and Microbial Technology.The article was published on 1992-03-01. It has received 43 citations till now. The article focuses on the topics: Trichoderma longibrachiatum & Mycelium.
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TL;DR: Protease inhibitors, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), antipain hydrochloride, and a mixture of inhibitors, but not pepstatin A, fully or partially nullified the biocontrol effect of T39.
Abstract: The role of protease of Trichoderma harzianum in the biocontrol of Botrytis cinerea was examined. Two isolates of T. harzianum were compared for their ability to produce protease in liquid culture medium and on the surface of bean leaves. The biocontrol agent T. harziaum T39 produced 58 mU/ml of protease and T. harzianum NCIM1185 produced 54 mU/ml on the 5th day of growth in liquid culture medium. On bean leaves, combinations of B. cinerea and T. harzianum isolates were examined for the synthesis of protease. The protease activities were 0.9 and 0.6 mU/ml for T. harzianum T39 and NCIM1185, respectively, and 0.5 mU/ml for B. cinerea alone after 48 h of incubation. In the presence of T. harzianum T39 culture liquid containing protease, a 55% reduction in B. cinerea germination and a 80% reduction in the germ tube length were observed after 17 h of incubation in vitro. When T. harzianum isolates were added to B. cinerea on bean leaves, increased synthesis of protease was observed (1.0 and 1.2 mU/ml for T39 and NCIM1185, respectively). In the presence of T. harzianum NCIM1185 protease, although the rate of germination was reduced, B. cinerea attained 98% germination after 17 h of incubation. The hydrolytic enzymes produced by B. cinerea, endo-polygalacturonase (PG) and exoPG were partially deactivated by protease from the T. harzianum isolates. Carboxymethyl cellulase was deactivated only by protease of NCIM1185. On the surface of bean leaves, the protease (obtained from liquid culture medium of T. harzianum isolates) resulted in 56–100% reduction of disease severity. The culture liquid containing protease synthesized on the surface of bean leaves treated with B. cinerea and with T. harzianum was collected and added to fresh leaves infected by B. cinerea. There was 56–100% and 30–75% reduction of disease severity with liquid droplet collected from the leaves treated with T. harzianum T39 and NCIM1185, respectively. Increased control of disease was obtained by combining the conidia of T. harzianum isolates with protease obtained from culture media. Protease inhibitors, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), antipain hydrochloride, and a mixture of inhibitors, but not pepstatin A, fully or partially nullified the biocontrol effect of T39. T39 was found to be a poor producer of chitinase and β-1,3-glucanase in vitro. These enzymes were not detected on leaves treated with T39. Involvement of protease in biocontrol of B. cinerea is suggested.
248 citations
TL;DR: LMWC enzyme-directed properties suggest chitinase is more predictable and flexible to produce the specified MV of LMWC, and E. coli strains showed much higher susceptibility to LMWCs than Staphylococcus aureus strains did and both species showed intra-species sensitivity diversity toward LMWc.
131 citations
TL;DR: A brief survey of recent progress in the regulation and cloning of microbial chitinase genes is given and emphasis is placed on the post-translational modification and localization of the recombinant protein in the host.
Abstract: A range of chitinase genes from microorganisms have been cloned and the potential uses of these genetically manipulated organisms are being investigated by various researchers Fungi and yeast are better producers of chitinase than bacteria Since fungi grow at a slower rate, there have been efforts to clone the fungal chitinase genes into fast-growing bacteria This review gives a brief survey of recent progress in the regulation and cloning of microbial chitinase genes Emphasis is placed on the post-translational modification and localization of the recombinant protein in the host Various amino acid domains are present in this protein The mode of catalytic activity of the recombinant protein in comparison to the wild-type protein is discussed in the available literature The different mechanisms involved in the regulation of chitinase genes from various microorganisms is discussed by the researchers The scope of future research and conclusions yet to be obtained in this particular area are also outlined in this review
105 citations
Cites background from "Studies on critical analysis of fac..."
...Chitinases are reported to dissolve cell walls of various fungi, a property that has been used for the generation of fungal protoplasts (Anjani kumari and Panda 1992; Ramaguera et al. 1993). Chitinase from Streptomyces was found to be active in the generation of protoplasts from Asperigillus oryzae and Fusarium solani (Skujins et al. 1965). An enzyme complex from Bacillus circulans WL-12 with high chitinase activity was eective in generating protoplasts from Phaa rhodozyme (Johnson et al. 1979). A mixture of commercially available chitinase and cellulase was used to release viable protoplasts from Caprinus macrorhizus (Yanagi and Takebe 1984). Chitinase from Trichoderma harzianum was the most ecient generator of protoplasts from dierent fungi (Kitomoto et al. 1988; Anjani kumari and Panda 1992). Chitinase-producing organisms are used in agriculture as an eective biocontrol agent against a number of phytopathogenic fungi (Kapat et al. 1996a, b; Elad et al. 1982). Chitinase from T. harzianum was found to be active against the broadest range of pathogens and soilborne fungi (Irene et al. 1994). Aeromonas caviae controlled infection by Rhizoctonia solani and Fusarium oxysporum in cotton and Sclerotium rolfsii in beans by producing chitinase (Inbar and Chat 1991). Chitinase produced by Serratia marcescens was eective against pathogenic fungi S. rolfsii (Ordentlich et al. 1988). A culture ®ltrate of Aphanocladium album strongly inhibited growth of Necteria haematcocca in the pea (Kunz et al. 1992). S. marcescens chitinase was eective against larvae of Galleria mellonela (Lysenko 1976). Appl Microbiol Biotechnol (1999) 51: 141±151 Ó Springer-Verlag 1999...
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...Chitinase from Trichoderma harzianum was the most e cient generator of protoplasts from di erent fungi (Kitomoto et al. 1988; Anjani kumari and Panda 1992)....
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...Biotechnological aspects of chitinase and their applications Chitinases are reported to dissolve cell walls of various fungi, a property that has been used for the generation of fungal protoplasts (Anjani kumari and Panda 1992; Ramaguera et al. 1993)....
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TL;DR: This study proved that T. harzianum Th-3013 as a biocontrol agent showed significant reduction in onion purple blotch disease compared with the tested fungicide.
Abstract: Nine isolates of Trichoderma were collected from Assiut Governorate,Egypt, as leaf surface and endophytic fungi associated with onion florastalks. Four isolates were identified as Trichoderma harzianum, while fiveisolates were belonging to Trichoderma longibrachiatum. The antagonisticactivity of these isolates against onion purple blotch pathogen Alternariaporri was studied in vitro using dual culture assay. All tested Trichodermaisolates showed mycoparasitic activity and competitive capability againstthe mycelial growth of A. porri. Mycoparastic activity of Trichoderma wasmanifested morphologically by the overgrowth upon the mycelial growthof the pathogen and microscopically by production of coiling hyphaearound pathogen hyphae. Isolates of T. harzianum exhibited high abilityto compete on potato dextrose agar (PDA) medium causing the maximumrate of pathogen inhibition (73.12%), while isolates of T. longibrachiatumshowed inhibition rate equalling 70.3%. Chitinase activity of Trichodermawas assayed, and T. harzianum Th-3013 showed the maximum value con-tributing 2.69 U/min. Application of T. harzianum Th-3013 to control pur-ple blotch disease in vivo under greenhouse conditions caused diseasereduction up to 52.3 and 79.9% before and after 48 h of pathogen inocu-lation, respectively, while the fungicide Ridomil Gold Plus caused diseasereduction comprising 56.5 and 71.7%, respectively. This study proved thatT. harzianum Th-3013 as a biocontrol agent showed significant reductionin onion purple blotch disease compared with the tested fungicide.IntroductionPurple blotch disease, caused by Alternaria porri (Ellis)Cif., is one of the most serious diseases that wide-spread in many parts of the world, restricted to Alliumspp. and more prevalent in warm and humid environ-ments (Cramer 2000; Suheri and Price 2000). Purpleblotch disease caused significant reduction in bulband seed yield of the onion crop (Gupta and Pathak1988). The disease is more severe on seed crop ascompared to bulb crop causing sometimes 100% loos-ing of the seed production (Singh et al. 1992;Schwartz 2004). Although chemical control of onionblotch had been practised and its success depends lar-gely on high frequency of spraying, but today, thereare strict regulations on chemical fungicide use due tocarcinogenic effects, residual toxicity problems, envi-ronmental pollution and development of fungicide-resistant strains (Benitez et al. 2004; Rial-Otero et al.2005). Therefore, there are a large number of studiesthat have been devoted to apply a biological control asnature-friendly alternative method (Siameto et al.2010; Soria et al. 2012). The potential of Trichodermaspecies as biocontrol agents of plant diseases was firstrecognized in the early 1930s (Weindling 1932), andsince then, there have been extensive efforts in thecommercial production of them for disease control ina number of crops (Harman 1996; Gardener and Frav-el 2002). Recently, several investigation proved thatTrichoderma have been used to control many foliar
86 citations
TL;DR: To produce various sizes of COS for versatile biological functions, as seen in this study to inhibit various types of bacteria, is made possible in this established process.
Abstract: BACKGROUND: Chito-oligosaccharide (COS) is generally known to possess many specific biological functions, especially antibacterial activity, depending on its size. To prepare a specific size range of COS, however, has proved difficult. The aim of this study was to establish a method for preparing a specific size range of antibacterially active COS by adjusting the degree of deacetylation (DD) of β-chitosan in a Trichoderma harzianum chitinase-hydrolysing process.
RESULTS: The molecular weight spectrum, elucidated by viscosity-average molecular weight, high-performance liquid chromatography and thin layer chromatography, of COS in chitosan hydrolysate was significantly related to the DD of its original chitosan. Compared with the original form, COS produced at 90% DD showed superior activity against most Gram-negative bacteria tested, with a minimum inhibition concentration (MIC) ranging from 55 ± 27 to 200 ± 122 µ g mL−1. Conversely, most Gram-positive strains tested were less sensitive to COS (MIC > 880 ± 438 µ g mL−1) than to its original form. Among the Gram-positive strains, Staphylococcus xylosus was the only exception in that it showed a high susceptibility to COS and had an MIC as low as 45 ± 11 µ g mL−1.
CONCLUSION: The results indicate that the production of a specific size range of COS product is possible by altering the DD of chitosan in the chitinase-catalysed process. To produce various sizes of COS for versatile biological functions, as seen in this study to inhibit various types of bacteria, is made possible in this established process. Copyright © 2008 Society of Chemical Industry
58 citations
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TL;DR: Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicilium cyaneo-fulvum and on transfer gave genetically stable colonies.
Abstract: Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.
34 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
TL;DR: Novozym 234, a commercially available enzyme from Trichoderma harzianum, has been used to prepare protoplasts from trichodermas reeseiQM 9414 as mentioned in this paper.
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
TL;DR: For enhanced production of cellulase and xylanase by the mixed culture of T. reesei D1-6 and A. wentii Pt 2804, the composition of medium has been optimized.
Abstract: For enhanced production of cellulase and xylanase by the mixed culture ofT. reesei D1-6 andA. wentii Pt 2804, the composition of medium has been optimized.
31 citations
TL;DR: High yields of protoplasts from the 18-hr old mycelium of Trichoderma viride were obtained by using the lytic system, produced by Streptomyces venezuelae RA and Micromonospora chalcea grown on a synthetic medium containing laminarin and chitin.
Abstract: High yields of protoplasts from the 18-hr old mycelium of Trichoderma viride were obtained by using the lytic system, produced by Streptomyces venezuelae RA and Micromonospora chalcea grown on a synthetic medium containing laminarin and chitin, when 0.7 M MgSO4 or (NH4)2SO4 were used as osmotic stabilizers. Regeneration of these protoplasts occurred through the production of an abortive tube and direct germination of the protoplasts. Regeneration could also take place in the medium used to produce protoplasts, but the process was different in many details.
29 citations