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Materials and Methods
Micro-organism and crude lipase
A Gram-negative coccobacillary bacterial isolate BTG1-99 was obtained from Department of Biotechnology, Himachal Pradesh University, Summer-Hill, Shimla-171 005 (India). The culture was maintained by repeated subculturing on a mineral based (MB) medium supplemented with 1% (v/v) cotton-seed-oil as the sole carbon-source. The MB-broth contained (gL-1); NaNO3 3.0, K2HPO4 0.1, MgSO4.7H20 0.5, KCl 0.5 and FeSO4.7H2O 0.01. The oil was initially emulsified with gum Arabic (0.5%, w/v) and then added to the MB-broth. The pH of the medium was adjusted to 7.0. The medium was autoclaved at 1.2 bar for 20 min at 121OC. The bacteria produced a maximum lipase activity at 48 h post-inoculation in MB-broth. The inoculated MB-broth (1L) was harvested free of cells at 48 h post-inoculation by centrifugation (10,000x g for 20 min. at 4OC). The supernatant was filtered through a Millipore membrane filter of 0.22m m porosity. The filtrate was concentrated by lyophlization (FTS Freeze Dryer, U.S.A.) and the protein concentration was measured by a standard method (Lowry et al., 1951).
The concentrated/lyophilized cell-free culture broth was henceforth referred to as crude lipase.
Assay of lipase activity
The lipase activity was assayed in the cell-free culture broth and celite or silica immobilized-lipase by colorimetric method of Winkler and Stuckmann (1979). The stock solutions of p-nitrophenyl palmitate (pNPP; 20 mM), p-nitrophenyl caprylate (pNPC; 10 mM) and p-nitrophenyl acetate (pNPA; 20 mM) were prepared in iso-propanol. For the free-lipase assay, 75 m l of p-NPP stock-solution and 5 to 50 m l of crude enzyme (cell-free broth) were taken in a glass tube. The final volume of this reaction mixture was made to 3 ml with Tris-HCl buffer (0.05 M, pH 8.5). The test tubes were incubated at 45oC for 20 min in a water-bath. Appropriate control (activated matrix without enzyme) was also incubated with each assay. The absorbance of this control was subtracted from the absorbance of the corresponding test sample. The absorbance of p-nitrophenol released was measured at 410 nm (Shimadzu UV/Visible Spectrophotometer, Japan). The unknown concentration of p-nitrophenol released was determined from a reference curve of p-nitrophenol. Each of the assays was performed in triplicate unless otherwise stated and mean values were presented. The lipase activity associated with the immobilized matrices was represented as Ug-1 of matrix.
One unit of lipase activity was defined as microgram(s) of p-nitrophenol released by one ml free-enzyme (or 1 g of matrix immobilized enzyme) at 45oC under assay conditions.
Immobilization of bacterial lipase on matrices
The lipase of the Gram-negative bacterial isolate was immobilized on native and activated (alkylated) matrices i.e. silica and celite. Each of the matrices was activated by exposure to either glutaraldehyde or formaldehyde (1% or 2% in distilled water, v/v). The suspension-containing matrix (1 g of celite or silica) and 4 ml of activating agent were incubated for 1 h at 37OC. Each of the activated matrices was then given five washings with Tris-HCl buffer (0.05 M, pH 8.5) to get rid-off the unbound activating agent.
Approximately, 2 ml (~340 U) of the enzyme was then added to each of the activated and non-activated matrices (1 g) and the suspension was incubated for another one hour at 37oC. The unbound lipase was removed by four washings with Tris-HCl buffer (0.05 M, pH 8.5). Finally, the immobilized matrices were kept suspended in Tris-HCl buffer at 4oC till further use.
Immobilization kinetics
A study was conducted to find out the rate of immobilization of lipase on both activated and un-activated matrices. The concentration of the free lipase in the supernatant was estimated at intervals till 1 h. This was done to workout the kinetics and efficiency of the process of immobilization of lipase on to the activated matrices.
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