Isolation and Physicochemical Characterization of Laccase fromGanoderma lucidum-CDBT1 Isolated from Its Native Habitat in Nepal.
At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely,Ganoderma lucidum-CDBT1, Ganoderma japonicum,andLentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, andG. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation fromG. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced byG. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants,Km,Vmax, andKcat, determined using 2,2'-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110μM, 36μmol/min/mg, and 246 min-1, respectively. The isolated thermostable laccase will be used in future experiments for delignification process.
Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal.
Prabin
0000-0003-4467-4069
P
Shrestha
Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal.
Bishnu
0000-0003-3841-0796
B
Joshi
Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal.
Jarina
J
Joshi
Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal.
Rajani
R
Malla
Central Department of Biotechnology, Tribhuvan University, Kirtipur, Nepal; Department of Chemistry and Earth Sciences, Qatar University, Doha, Qatar; Department of Chemistry and Biochemistry, St. Cloud State University, St. Cloud, MN 56301, USA.
Lakshmaiah
0000-0002-9718-5114
L
Sreerama
D017753:Ecosystem; D004789:Enzyme Activation; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification; D042845:Laccase / Q000737:chemistry* / Q000302:isolation & purification*; D009390:Nepal; D012275:Oryza / Q000382:microbiology*; D020940:Reishi / Q000145:classification* / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity
D005656:Fungal Proteins; D042845:Laccase