SCREENING OF POTENTIAL LIGNOCELLULOLYTIC FUNGI ISOLATED FROM HIMALAYAN FORESTS AND TO ASSESS THEIR ROLE IN PINE NEEDLE DEGRADATION
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Date
2019-11
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UHF,NAUNI
Abstract
ABSTRACT
The present study was carried out to isolate lignocellulolytic fungi from the rotten wood of Himachal
Pradesh (i.e. Solan, Shimla, Sirmour, Kangra and Chamba), their screening and optimization of laccase,
cellulase and xylanase production. Among all the fungal isolates, R4, S5, SH2 and SH5 fungal strain were
selected for enzyme production under submerged fermentation. The phenotypic characterization was done for
their tentative identification i.e. Trichoderma sp. R4, Trichoderma sp. S5, Trichoderma sp. SH2 and Rhizopus
sp. SH5. The molecular identification was done by using ITS 5.8S rRNA technique and S5 was identified as
Trichoderma guizhouense |MN17050|. The extracellular hydrolytic enzyme production from these potential
identified fungal strain was then subjected to solid state fermentation by optimizing the different environmental
parameters i.e. temperature, substrate: moisture ratio, incubation time and pH using pine needles biomass as
substrate under classical one factor approach. An enhanced production with laccase (6.45U/g), cellulase
(37.20U/g) and xylanase (380 U/g) activity was obtained by Trichoderma sp. R4. Optimization process was then
switched over to response surface methodology (RSM) and maximum enzyme production of Trichoderma sp.
R4 in RSM with four responses i.e. laccase (6.90U/g), cellulase (37.86U/g), xylanase (398 U/g) and reducing
sugar (47.98mg/g) was obtained. Highest production of enzymes activity was observed in pine needles biomass
and Trichoderma sp. R4. had been used for further purification process. The culture filterate was subsequently
partially purified by ammonium sulphate precipitation at 40% saturation level of laccase, 40% CMCase, 60%
FPase, 50% β-glucosidase and 70% xylanase with purification fold of 3.06 (laccase), 2.20 (cellulase) and 1.59
(xylanase) with 82.94, 62.43 and 63.24 % recovery yield respectively. Gel exclusion chromatography was done
for purification of hydrolytic enzymes with 5.36, 5.51 and 6.33 purification fold and 45.77, 61.33 and 60.23 %
recovery yield for laccase, cellulase and xylanase respectively. The molecular mass of purified laccase
(40.0kDa), cellulase – CMCase (45.0kDa), FPase (31.0kDa), β-glucosidase (29.0kDa) and xylanase (65.0kDa)
was obtained by using SDS-PAGE. The maximum enzymatic degradation of pine needles was obtained in
purified fractions of enzymes of Trichoderma sp. R4 with a release of 76.75 mg/g reducing sugars. The present
study strongly proves the success of optimization of different parameters for enhancement in level of enzyme
i.e. laccase, cellulase, xylanase and degradation of pine needles with a cost-effective approach of enzyme
production using a cheap and abundant pine needle waste as a carbon source.
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