INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH, VOL 3, ISSUE 5, MAY 2014
Production And Partial Characterization Of a
Thermostable, Alkaline And Organic Solvent
Tolerant Lipase From Trichoderma atroviride 676
Thiago Andrade Marques, Cristiani Baldo, Dionísio Borsato, João Batista Buzato, Maria Antonia Pedrine Colabone Celligoi
Abstract—The several industrial applications of lipases have stimulated interest in isolation of new enzymes from novel sources. In this
study, the lipase production from Trichoderma atroviride 676 was optimized by statistical design methods. The response surface
methodology based on 3(3-1) fractional factorial, showed that the yeast extract and MgSO4 played a significant role on lipase production,
yielded an enzymatic activity of 101.75 U/ml. Using Box-Benhken design, the optimization of the temperature levels, pH and agitation rate
resulted in the maximum enzyme production of 175.20 U/ml, obtained at 28 ºC, pH 6.0 and 105 rpm. The enzyme was optimally active at
pH 8.0 and 35ºC, and was stable at pH 3.0-8.0 and temperature of 25ºC-75ºC. In addition, the lipase was highly stable on non-polar
hydrophobic solvents as kerosene, n-dodecane and hexane. The new lipase from T. atroviride 676 could be considered a potential
candidate for industrial and biotechnological applications.
Index Terms— fermentation, lipase, optimization, organic solvents, thermo-alkaline, Trichoderma atroviride 676.
1 INTRODUCTION
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2 MATERIALS AND METHODS
2.1 Microorganism and inoculum preparation
T. atroviride 676 (IOC 4503) was originally isolated from
Amazon forest soil and are available at Coleção de Culturas de
Fungos Filamentosos, Fundação Oswaldo Cruz, Rio de
Janeiro, Brazil. The strain was grown in a potato dextrose agar
medium (PDA), in Erlenmeyer flasks (125 ml), at 28 ºC for 4
days, using an inoculum of 10
6
spores/ml. The strain was
subculture every 15 days, and preserved at 4 ºC.
2.2 Fermentation medium
The lipase production by T. atroviride 676 was investigated
employing the Vogel minimal salts medium supplemented
with Tween 80 (0,5% v/v), containing olive oil at a
concentration of 1% (v/v) and different concentrations of
glycerol, olive oil, tween 80 and Vogel's medium, according to
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