Abstract
Laccase enzyme is a ligninolytic enzyme which has an ability to oxidize phenolic compounds of lignocellulosic biomass. The aim of the study was to identify, optimize a thermo-tolerant isolate and laccase enzyme production using Response Surface Methodology- Box Behnken design respectively. The strain S3 was isolated from the compost soil pit and the crude laccase enzyme was extracted from the strain. The parameters such as pH, incubation period and CuSO4 concentration has been optimized using Box-Behnken design and the outcome shows that the enzyme with 4.71 U mL− 1 was stable at pH 6 and 8, the optimum CuSO4 concentration was 100 mM and the incubation period was 7 days at the temperature of 46֯ C. The genomic DNA of the strain was amplified using ITS-1 and 4 primers which showed a product size of 700 bp. The amplicon was sequenced and the blast analysis showed a sequence similarity of 96.6% with the Flammulina velutipes. The protein was isolated and the SDS analysis showed a molecular weight of 120 kDa.The crude laccase enzyme produced from the strain S3 Flammulina velutipes was formulated and optimized at varying parameters to increase the yield of laccase enzyme. We conclude that our results will assist to a wide application in bio-fuel production.
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All data available on request.
Abbreviations
- ITS:
-
Internal transcribed spacer
- LiP:
-
Lignin peroxidase
- MnP:
-
Manganese peroxidase
- RSM:
-
Response surface methodology
- CuSO4 :
-
Copper sulphate
- NaCl:
-
Sodium chloride
- K2HPO4 :
-
Potassium hydrogen phosphate
- NH4H2PO4 :
-
Ammonium dihydrogen phosphate
- (NH4)2SO4 :
-
Ammonium sulfate
- MgSO4⋅7H2O:
-
Magnesium sulfate heptahydrate
- KNO3 :
-
Potassium nitrate
- Cu+ :
-
Copper ions
- ABTS- 2:
-
2’-azinobis-3-ethyl-benzothiozoline-6 sulfonic acid
- DF:
-
Dilution factor
- BSA:
-
Bovine serum albumin
- CTAB:
-
Cetyl trimethyl ammonium bromide
- PCR:
-
Polymerase chain reaction
- SDS:
-
Sodium dodecyl-sulfate
- SDS PAGE:
-
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis
- TCA:
-
Trichloro-acetic acid
- ANOVA:
-
Analysis of variance
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Acknowledgements
This work has been supported by EEQ/2019/000225. The authors wish to thank Dr. G. Hemaprabha, Director, ICAR Sugarcane Breeding Institute and Dr. A. Selvi, Principal Scientist, Biotechnology Section Head, ICAR Sugarcane Breeding Institute, Coimbatore for their support for carrying out the work.
Funding
“This work was supported by [SERB Grant number EEQ/2019/000225] and the author Dr. Lakshmi Kasirajan received the fund from DST SERB”.
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Lakshmi Kasirajan contributed to the study conception, design, and analysis. Material preparation, data collection and the experiment were performed by Keerthana kamaraj, the first draft of the manuscript was written by LK and all authors read and approved the final manuscript.
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Kasirajan, L., Kamaraj, K. Optimization of laccase enzyme from Flammulina velutipes using response surface methodology box-behnken design. Biologia 78, 2273–2281 (2023). https://doi.org/10.1007/s11756-023-01378-7
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DOI: https://doi.org/10.1007/s11756-023-01378-7