Hydrolysis Of Long-chain Triglycerides Research Articles

Characterization & Partial purification of Microbial Lipase enzyme produce by Serratia nematodiphilia isolated from Paper & pulp effluent

As the demand is growing for paper and pulp at a highest peak, it not only imposes a severe threat on the forest resources but also on the environment. The effluent discharged by these paper and pulp industries is of great concern. Along with the biodegradation of theses effluents, we also aimed to produce microbial lipase. Lipases, a class of enzymes which catalyze the hydrolysis of long chain triglycerides. At present, microbial lipases are attaining much awareness with the rapid progress of enzyme technology. Lipases represent the most significant group of biocatalysts for industrial applications. In the present study various samples were collected from the paper and pulp effluent from Vapi, Gujarat. For preliminary understanding, total 18 bacterial isolates (JKB1 to JKB18) and 5 Fungi isolates (S1 to S5) were isolated out of that. Based on their primary screening technique, JKB1, JKB3, JKB5 and JKB21 bacterial isolates were studied for further for lipase production. Results show that isolate JKB3 was more potential and was studied further for characterization of lipase enzyme. It was identified as Serratia nematodaphilia. The results also indicate that the maximum activity was obtained i.e. 59.66 U/ml in optimized conditions like Glucose 2.75 U/ml, Tryptone 32.39 U/ml, Peanut oil 34.71U/ml, pH-7 64.92 U/ml, room temperature. At the end of partial purification, 5.15 fold purification of lipase was obtained. The current study represents the isolation and characterization of lipase enzyme which has a great applicability in paper & pulp effluent in deinking process in making of paper and increasing brightness, intensity and the pulping rate. It can also decrease chemical consumption, prolong machinery life, reduction in pollution, save energy, time and production cost.

TITLE OF MANUSCRIPT: ENHANCEMENT OF LIPOLYTIC CHARACTERISTICS OF A PALM-OIL MILL EFFLUENT-SOURCED KODAMAEA OHMERI PL2

The hydrolysis of long-chain triglycerides and fats is catalyzed by lipases, important enzymes which have essential biotechnological, medical, and industrial roles. Fungi were isolated from palm-oil mill wastes and screened for their lipid-hydrolyzing potentials. Lipase production and activity characteristics were determined using different substrates. Sixteen, out of the 75 fungi isolated, exhibited lipolytic characteristics. Yeast isolate PL2 gave the highest zone of hydrolysis (44.5 mm) and was identified as Kodamaea ohmeri. This yeast was cultured under diverse media conditions for lipase production and the enzyme produced under each physicochemical condition was determined spectrophotometrically. The optimum parameters for lipase production (1% Tween-80, Ca2+, peptone, 37°C and pH 6) were used to produce lipase which was further purified through (NH4)2SO4 precipitation, membrane dialysis and gel chromatography. The activity of the partially purified lipase increased progressively with each purification stage. The lipase had optimum activity at 50oC and was alkalophilic with peak pH for enzymatic hydrolysis at 8.0. The lipase was supported by 0.5% concentrations of SDS and Triton X-100 (129.9 and 129.4%, respectively), and a 100-fold activity increase with 0.5% Tween-80. Lipase activity was augmented by Manganese and calcium ions (10 mM) but inhibited by NH4+, Fe3+ and Na+. The use of Tween-80 resulted in increased enzyme yield as well as better enzyme characteristics compared to other substrates. The knowledge obtained in this study may be harnessed for commercialization of Kodamaea ohmeri lipase.

Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot.

Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217–245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.

English

Lipases catalyze the hydrolysis of long chain triglycerides. Microbial lipases are receiving much attention because of their industrial potential in the chemical, pharmaceutical, medical, cosmetic, biosurfactant synthesis, leather industries, mutation, agrochemicals and paper manufacturing industries. This article presents the isolation of maximum lipase producing bacteria and the optimization of different conditions for the maximum production of lipase. Bacillus subtilis PCSIRNL-39 shows maximum production of lipase at 45°C with pH 7 using nitrogen source peptone and carbon source sucrose. B. subtilis PCSIRNL-39 showed the best production at 5% inoculum size, while Ca2+ and Mg2+ were found best stimulator for enzyme production during the study. Tween 20 and 80 enhanced better lipase production than other surfactant. The kinetic parameters of Vmax and Km for the lipase were measured to be 101 µM/min.mL and 7.6 mg, respectively. Key words: Bacillus subtilis, microbial lipase, production, optimization.

Optimization of Alkaline Lipase Production from Burkholderia cepacia through Submerged Fermentation

The hydrolysis of long chain triglycerides can be catalysed by a class of enzymes known as lipases. Currently, microbial lipases are receiving much attention because of their actual and potential application in industry. In this paper, the production of lipase from Burkholderia cepacia through submerged fermentation (SmF) has been optimized. Various parameters which include pH (4.4 – 9.0), agitation speed (100 – 300 rpm), incubation time (12 – 72 h) and mode of autoclave (mix or separate) were optimized. It was observed that the fermentation medium should first be mixed before autoclaved instead of mixing the fermentation medium after autoclave as the former has higher lipase production. The optimum operating condition for the fermentation to obtain maximum production of lipase (11.18 U/mL) was at pH 9.0, 300 rpm and incubation time of 72 h.

Cloning and expression of lipase gene from Ralstonia eutropha in Escherichia coli

Background Lipases are enzymes belonging to the class of hydrolases, acting in the aqueous-organic interface demonstrating considerable levels of activity and stability in aqueous environments and non-aqueous [1]. Its biological function is to catalyze the hydrolysis of long chain triglycerides to form fatty acids and glycerol, in addition to performing esterification [2]. They have wide industrial applicability and can be used in the composition of detergents, in food industry, in wastewater treatment and fine chemistry [3]. The goal this work was clone and express lipase gene from Rasltonia eutropha in Escherichia coli BL21DE3.

Isolation and characterization of some moderately halophilic bacteria with lipase activity

Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group ofbiocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from Maharlu salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 +/- 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from Maharlu lake.

Influence of culture conditions on lipase production byBacillus sp. FH5

Lipases are a class of enzymes, which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases have industrial potential in the chemical, pharmaceutical, medical, cosmetic, leather and paper manufacturing industries, biosurfactant synthesis, and agrochemicals. ABacillus strain isolated from soil was tested for the production of extracellular lipase, by batch culturing in shake flask. The growth conditions were optimised for the maximum production of enzyme. Various parameters for the production of lipase, such as temperature, incubation period, pH, carbon source, nitrogen source and lipids were studied. Maximum lipase production was found in 48-h-old culture filtrate at 37 °C, pH 8.0. Among all the carbon sources, salicin gave the maximum activity and among all the nitrogen sources yeast extract gave maximum production/activity. Tween (20 and 80) does not stimulate the growth much but assisted in enzyme production.

Industrial applications of microbial lipases

Lipases are a class of enzymes which catalyse the hydrolysis of long chain triglycerides. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases constitute the most important group of biocatalysts for biotechnological applications. This review describes various industrial applications of microbial lipases in the detergent, food, flavour industry, biocatalytic resolution of pharmaceuticals, esters and amino acid derivatives, making of fine chemicals, agrochemicals, use as biosensor, bioremediation and cosmetics and perfumery.

Colipase enhances hydrolysis of dietary triglycerides in the absence of bile salts.

This study explores how dietary lipids are digested when intraduodenal bile salts are low or absent. Long-chain triglycerides emulsified with phosphatidylcholine were found to be hydrolyzed very slowly by pancreatic lipase alone, as if the surface layer of phospholipids enveloping the triglycerides impeded the action of the enzyme. Colipase enhanced triglyceride hydrolysis severalfold, both when added before or after the lipase. Hydrolysis became even more rapid when the emulsion was first incubated with pancreatic phospholipase. Hydrolysis of long-chain triglycerides was also severely impeded when other proteins were added to the system, probably because they adsorbed to the oil-water interface of the emulsion droplets. It was previously known that bile salts can relieve such inhibition, presumably by desorbing the adsorbed proteins. Colipase was found to enhance hydrolysis severalfold in a dose-dependent manner even in the absence of bile salts, i.e., it could partially or completely relieve the inhibition depending upon the amount and the type of inhibitory protein added to the system. Prior exposure of a protein-coated triglyceride emulsion to another lipase also enhanced the rate at which pancreatic lipase could then hydrolyze the lipids. Most dietary triglycerides are probably presented for intestinal digestion in emulsions covered by proteins and/or phospholipids. These emulsions would be hydrolyzed slowly by pancreatic lipase alone. However, through the action of the lipase in stomach contents and of pancreatic phospholipase and through the lipolysis-promoting effects of collipase, these triglycerices can be rather efficiently hydrolyzed, even in the absence of bile salts.

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.

Action of some effectors on the hydrolysis of long-chain triglycerides by pancreatic lipase

In order to study the action of various effectors on lipolysis of emulsified longchain triglycerides by pancreatic lipase a simple system was used. It was composed of the two reactants, triglyceride and water, with NaCl and a trace of oleate for the stabilization of the emulsions. The initial effect of NaCl is to favor the normally poor ionization of long chain acids at pH 9.0. In the system described above, lipase was very active at the beginning of the reaction. However, early inhibition was observed. This inhibition is caused by the long-chain soaps formed during the reaction. Ca 2+ and deoxycholate were not found to appreciably increase the initial rate of lipolysis, but rather to prevent to some extent the inhibitory effect of long-chain soaps. Higher concentrations of deoxycholate and of other bile salts were inhibitory. Lipolysis did not appear to take place in this system unless some NaCl was added. In the presence of increasing NaCl concentrations, the initial rate soon reached a maximum and then decreased. Various mechanisms are discussed for the inhibition of lipolysis by soaps and for the observed effect of Ca 2+, bile salts and NaCl.