Characterization of Smallest Active Monomeric Lipase from Novel Rhizopus Strain: Application in Transesterification

Abstract

An extracellular lipase-producing fungus was isolated from oil-rich soil. This fungus belongs to the genus Rhizopus and clades with Rhizopus oryzae. Lipase was purified to homogeneity from this novel fungal source using ammonium sulphate precipitation followed by Q-Sepharose chromatography. The extracellular lipase was purified 8.6-fold, and enzymatic properties were studied. The molecular mass of the purified enzyme was estimated to be 17 kD by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and 16.25 kD by matrix-assisted laser desorption ionization/time-of-flight analysis. The native molecular mass was estimated to be 17.5 kD by gel filtration, indicating the protein to be monomer. The optimum pH and temperature for the enzyme catalysis were 7.0 °C and 40 °C, respectively. Enzyme was stable in pH range 6.0-7.0 and retains 95-100% activity when incubated at 50 °C for 1 h. The pI of the purified lipase was 4.2. Enzyme was stable in the organic solvents such as ethanol, hexane and methanol for 2 h. Purified enzyme was used for transesterification of oleic acid in the presence of ethanol for production of oleic acid ethyl ester with a conversion efficiency of 66% after 24 h at 30 °C.

Biochemical Characterisation of a Newly Isolated Low Molecular Weight Lipase from Rhizopus oryzae NRRL 3562

“…In hexane and diethyl ether the enzyme was found to be more than 65% active. Methanol and ethanol stability was also reported by Kantak and Prabhune [24] and Yoo et al [15] in Rhizopus sp. and Ralstonia sp., respectively.…”

“…Though the optimum pH of the enzyme was found to be 9 (100%), the stability was found to be maximum at pH 7 (100%). Compared to most of the Rhizopus lipases, whose optimum pH lie in the range 7-8.5 [20, 21,24], the present lipase was found to be most active in alkaline pH. Its optimum pH was equal to that of Pseudomonas fluorescens JCM5963, Pseudomonas sp.…”

“…The optimum temperature of the enzyme was found to be more than that of most of Aspergillus lipases [7,28]. A newly isolated Rhizopus strain also shows same optimum temperature, but beyond 50 °C its activity falls sharply [24]. Thus, the present enzyme could be efficiently used for processes requiring stability at moderately high temperatures.…”

“…Recently, a small lipase of 17 kDa produced from Rhizopus sp. has been reported by Kantak and Prabhune [24]. Though small lipase of 11 kDa has been reported in bacterial system by Castro-Ochoa et al [11], presence of such small lipase has not been so far reported in fungal system.…”

Pilot-scale submersed cultivation of R. microsporus var. oligosporus in thin stillage, a dry-grind corn-to-ethanol co-product

“…In one study after the bacteria were induced into the fungi, it was grown for 2--3 days at which point they found the toxins. In our case of a batch process where the fungi is grown for 24h this would likely not be a (Kantak & Prabhune 2012). This makes the enzyme a prime candidate for transesterfication, an imperative step in the conversion of oil into biodiesel.…”