A Comparative Study of Lytic Enzymes Activity Extracted from Different Trichoderma Species and Their Role in Industry

Published: 2021-07-07 12:55:03
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We have selected three strains of Trichoderma species to compare its enzymatic activity, these strains were, T. viride, TRN4 (lab mutant) and TB1 (isolated from banana soil rhizosphere). Trichoderma strains were cultivated by using novel neem cake wheat bran substrate for SSF. The strains were then harvested and the enzymes were extracted using different solvents. Enzyme activity was analyzed by plate well diffusion assay technique and the observations suggested TB1 enzymes shows higher enzymatic activity than other strains (T. viride and TRN4).Introduction: Trichoderma species produces a wide array of enzymes. Some strains are used industries to produce a large quantity of enzymes.
Trichodermaproduces various types of enzymes like chitinase, cellulases, hemicellulase, protease, glucanases, xylanase, amylase, lipase etc. Our recent study aimed to study on lytic enzymes produced by isolated Trichodermaand its significance role in agriculture and other industries. Mainly focused on chitinases, lipases, amylases and proteases enzyme. The extracellular hydrolytic enzymes played important role in mycoparasitism and antibiosis. Trichoderma species T. harzianum, T. viride, T. ressei are known as producers of extracellular hydrolytic enzymes. Enzymes play an important role in soil management, organic matter decomposition and nutrient cycling. Some enzymes only facilitate the breakdown of organic matter (e.g., hydrolase, glucosidase), while others are involved in nutrient mineralization (e.g., amylase, urease, phosphatase, sulfates).Materials and Methodology
Micro Organism – The strain used for lytic enzymes production was T. viride, TRN4 and TB1. T. viride collected from National Collection of Industrial Microbiology (NCIM) Pune, TRN4provided by R&D, Kilpest India Limited(UVlab mutant) and TB1 isolated from banana soil rhizosphere.
Collection of Soil sample: Soil samples were collected from the field of Kilpest India Limited, Bhopal, India.
Rhizosphere soilwas taken from different plants (Banana plant, Garlic, Chick pea and Radish plant) from the 5-12 cm depth.
Isolation of Trichoderma strain (TB1) from Soil Rhizosphere: Potato Dextrose Agar media was prepared and autoclaved at 121º C, 15 psi for 20 minutes. Hot media was poured into pre- sterilized petri plates using aseptic condition the plates were allowed to cool causing media to solidify in plates. 1gm of soil was dissolved in 10 ml of water for making serial dilution. Serial dilution series was prepared. Aseptically 20µl of soil suspension were transferred into PDA plates and spreaded uniformly. Plates were incubated at 28ºC for 5-6 days. After the 5-6 days number of colonies were observed in PDA plates. Morphological study was done. Colonies that produced green color conidia was picked. Subsequently sub culturing was done to obtain pure cultures.
Revive Cultures of the strains [T. viride and TRN4 (mutant)]: Revive the T. viride and TRN4 on PDA plates from glycerol stock and then routinely maintained on PDA plates. 20 µl of culture was taken from glycerol stock and spreaded uniformly on PDA plate. Plates were incubated at 28º C for 5-6 days. After the 5-6 days colonies were observed in PDA plates. Morphological study was done. Colonies that produced green color conidia was picked. Subsequently sub culturing was done to obtain pure cultures.
Solid state fermentation (SSF) of T. viride, TRN4 and TB1 using wheat bran and neem cake: Wheat bran and neem cake used as a substrate for SSF. Wet substrate mixture was transferred in 152 X 152 mm plate and autoclaved at 121 ºC at 15 psi for 20 minutes. After sterilization, the substrate were aseptically inoculated with 5 days old culture plate of T. viride, TRN4 and TB1 and plates were incubated at 28 ºC in BOD for 7 days.
Enzyme Extraction from Trichoderma strains (T. viride, TRN4 and TB1) in different solvents: T.viride, TRN4 and TB1 was harvested from culture plate and grinded using electrical heavy duty grinder. Approx. 30 gm of Trichoderma strains was mixed with 60 ml of different solvents to prepare a thick paste which transferred to four 250 ml flasks kept on shaker for 2 hour at 100 rpm. Then slurry was filtered by using muslin cloth and filter paper. This filtrate was transferred into eppendroff tube and then centrifuged for 1 min at 13000 rpm. Supernatant was collected and used for the enzyme assay.
Determination of enzyme activity of crude enzyme fractions of T. viride, TRN4 and TB1 by using Plate well diffusion assay
Protease Enzyme assay: Plates of Gelatin Agar media were prepared under the aseptic condition. Formed wells in each plates using sterilized cork borer. Inoculated different volumes (40 µl and 50 µl) in wells and incubated at room temperature for 24 and 48 hours to observe enzyme activity. Indicator- HgCl2 was used as indicator.
Indicator was poured in media plate, uniformly distributed. After the 20 min zones were observed. The resulting diameters of the clearing zone were measured by using ZOI (zones of inhibition) scale.
Amylase Enzyme assay: Plates of Starch Agar media were prepared under the aseptic condition. Formed wells in each plates using sterilized cork borer. Inoculated different volumes (40 µl and 50 µl) in wells and incubated at room temperature for 24 and 48 hours to observe zonal activity. Indicator- Iodine solution was used as an indicator. Indicator was poured in media plate, uniformly distributed. After the 15 min zones was observed.
The resulting diameters of the clearing zone were measured byusing ZOI (zones of inhibition) scale.
Cellulase Enzyme assay: Plates of CMC Agar media were prepared under the aseptic condition. Formed wells in each plates using sterilized cork borer. Inoculated different volumes (40 µl and 50 µl) in wells and incubated at room temperature for 24 and 48 hours to observe zonal activity. Indicator- Congo red solution used as indicator and 1M NaCl used as a counter stain for Cellulase enzyme activity. Congo red solution was poured in media plate, uniformly distributed for 10 min. Then counter stain was poured in plate for 10 min. Zones was observed.
The resulting diameters of the clearing zone were measured by using ZOI (zones of inhibition) scale.
Chitinase Enzyme assay: Plates of Chitinase detection media were prepared under the aseptic condition. Formed wells in each plates using sterilized cork borer. Inoculated different volumes (40 µl and 50 µl) in wells and incubated at room temperature for 24 and 48 hours to observe zonal activity. Indicator- Bromocresol purple was used as an indicator. Indicator was poured in media plate, uniformly distributed. After the 15 min zones was observed.
The resulting diameters of the clearing zone were measured by using ZOI (zones of inhibition) scale. (T. virideTRN4TB1) (TRN4 (40 & 50 µl)TB1 (40 & 50 µl))Lipase Enzyme assay: Plates of Lipase detection media were prepared under the aseptic condition. Formed wells in each plates using sterilized cork borer. Inoculated different volumes (40 µl and 50 µl) in wells and incubated at room temperature for 24 and 48 hours to observe zonal activity. Indicator- Methyl red was used as an indicator. Indicator was poured in media plate, uniformly distributed. After the 15 min zones was observed.
Results and Discussion
The study aimed at choosing a better strain of Trichoderma species, by checking the activity of lytic enzymes. We collected soil samples from different plants soil rhizosphere (Banana, garlic, radish, chickpea) from field of Kilpest India Limited, Bhopal. We successfully isolated Trichoderma strain TB1 from banana soil rhizosphere and other strains Trichoderma viride and TRN4 revived on PDA plates, were provided by R&D, Kilpest India Limited, Bhopal. SSF was done for extracting the enzymes using wheat bran and neem cake (1:1). Enzyme was successfully extracted in different solvents (phosphate buffer, methanol, 1N Saline and 10% ethanol) using filtration and centrifugation process. The activity of lytic enzymes was successfully observed in plate well diffusion assay. We prepared different agar media for different enzymes for example gelatin agar media used for protease enzyme assay and starch agar media used for amylase assay. We compared the activity of lytic enzymes of Trichoderma strains TB1, T. viride and TRN4 by determining zones diameter. The newly isolated Trichoderma strain TB1 showed better enzymatic activity in phosphate buffer than T. viride and TRN4.
CONCLUSION
The newly isolated Trichoderma strains TB1 showed higher enzymatic activity than T. viride and TRN4 in laboratory. The application of enzymes isolated from Trichoderma strains may become a reality in future as they can be produced cheaply in large quantities on an industrial scale. The enzymes are used for plant growth, protection from pathogens, antagonistic activity and to increase soil fertility.

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