Lipases: introduction

Lipases differ greatly with regard to their origins and their properties. Lipases can be obtained from microorganism, plant and animal. They can catalyze the hydrolysis or synthesis of a wide range of different substances. Microbial lipases are widely diversified in their enzymatic properties and substrates specify which make them attractive for many industrial applications including detergents, food, flavor industry, ester and amino acid derivatives, baking, fine chemical, bioremediation, hard surface cleaning, leather andpaper industry. Lipases stand amongst the most important biocatalysts due to their ability to utilize a wide spectrum of substrates, high stability towards extremes of temperature, pH and organic solvents, and chemo-, regio- and enantioselectivity. The enantioselectiveand regionselective nature of lipases have been utilized for the resolution of chirl drugs, fat modification, synthesis of cocoa butter substituents, biofuels, and for the synthesis of personal care products and flavor enhancers

Lipase catalysed synthesis of adipate wax esters in ionic liquid

Adipate wax ester shows excellent properties such as its low toxicity, good thermal stability, low volatility and high biodegradability, which made it as a very useful compound and significant to various industrial applications including pharmaceutical, food and coating. Green route organic synthesis via enzymatic synthesis, offers clean and mild reaction conditions provides an opportunity to increase productivity, efficiency and quality output. Considering the industrial importance of the ester, the optimal conditions for upscaling process was performed using Response Surface Methodology (RSM). Ionic liquid of [bmim]PF6 was used as a solvent in the esterification of different chain length of alcohol with adipic acid, and Novozyme as a biocatalyst. Higher percentage of yield (>80%) was obtained for longer alcohol chain (dioleyl adipate) but slightly lower yields were found for medium alcohol chain than in hexane. Ionic liquid become the major interest as it can recycled and remained the efficiency once compared to conventional organic solvent

Conformational destabilization of Bacillus licheniformis α-amylase induced by lysine modification and calcium depletion

Bacillus licheniformis α-amylase (BLA) was chemically modified using 100-fold molar excess of succinic anhydride over protein or 0.66 M potassium cyanate to obtain 42 % succinylated and 81 % carbamylated BLAs. Size and charge homogeneity of modified preparations was established by Sephacryl S-200 HR gel chromatography and polyacrylamide gel electrophoresis. Conformational alteration in these preparations was evident by the larger Stokes radii (3.40 nm for carbamylated and 3.34 nm for succinylated BLAs) compared to 2.43 nm obtained for native BLA. Urea denaturation results using mean residue ellipticity (MRE) as a probe also showed conformational destabilization based on the early start of transition as well as ΔGDH2O values obtained for both modified derivatives and Ca-depleted BLA. Decrease in ΔGDH2O value from 5,930 cal/mol (for native BLA) to 3,957 cal/mol (for succinylated BLA), 3,336 cal/mol (for carbamylated BLA) and 3,430 cal/mol for Ca-depleted BLA suggested reduced conformational stability upon modification of amino groups of BLA or depletion of calcium. Since both succinylation and carbamylation reactions abolish the positive charge on amino groups (both α- and ε- amino), the decrease in conformational stability can be ascribed to the disruption of salt bridges present in the protein which might have released the intrinsic calcium from its binding site

High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM

The mature ARM lipase gene was cloned into the pTrcHis expression vector and over-expressed in Escherichia coli TOP10 host. The optimum lipase expression was obtained after 18h post induction incubation with 1.0mM IPTG, where the lipase activity was approximately 1623-fold higher than wild type. A rapid, high efficient, one-step purification of the His-tagged recombinant lipase was achieved using immobilized metal affinity chromatography with 63.2% recovery and purification factor of 14.6. The purified lipase was characterized as a high active (7092Umg -1), serine-hydrolase, thermostable, organic solvent tolerant, 1,3-specific lipase with a molecular weight of about 44kDa. The enzyme was a monomer with disulfide bond(s) in its structure, but was not a metalloenzyme. ARM lipase was active in a broad range of temperature and pH with optimum lipolytic activity at pH 8.0 and 65°C. The enzyme retained 50% residual activity at pH 6.0-7.0, 50°C for more than 150min

A unique thermostable and organic solvent tolerant lipase from newly isolated Aneurinibacillus thermoaerophilus strain HZ: physical studies

A newly isolated thermophilic bacterium, Aneurinibacillus thermoaerophilus strain HZ, from a hot spring recreational area (Sungai Kelah, Malaysia), showed an extracellular lipase activity. It was identified based on 16S rRNA sequencing, where phylogenetic analysis revealed its homology to Aneurinibacillus thermoaerophilus. The strain produced a lipase that was stable in various organic solvents such as dimethyl sulfoxide, toluene, p-xylene, and hexane. In order to increase lipase production, optimization of physical factors which affected the growth and lipase production was studied. The optimal growth was obtained at 50°C and pH 8.0; while the maximal lipase production was achieved in the logarithmic decline phase at 60°C and pH 7.5 with 7% starting inoculum and 150 rev/min shaking rate for 48 h incubation

Application of artificial neural network for yield prediction of lipase-catalyzed synthesis of dioctyl adipate

In this study, an artificial neural network (ANN) trained by backpropagation algorithm, Levenberg–Marquadart, was applied to predict the yield of enzymatic synthesis of dioctyl adipate. Immobilized Candida antarctica lipase B was used as a biocatalyst for the reaction. Temperature, time, amount of enzyme, and substrate molar ratio were the four input variables. After evaluating various ANN configurations, the best network was composed of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The correlation coefficient (R 2) and mean absolute error (MAE) values between the actual and predicted responses were determined as 0.9998 and 0.0966 for training set and 0.9241 and 1.9439 for validating dataset. A simulation test with a testing dataset showed that the MAE was low and R 2 was close to 1. These results imply the good generalization of the developed model and its capability to predict the reaction yield. Comparison of the performance of radial basis network with the developed models showed that radial basis function was more accurate but its performance was poor when tested with unseen data. In further part of the study, the feed forward back propagation model was used for prediction of the ester yield within the given range of the main parameters

A new thermostable and organic solvent-tolerant lipase from Aneurinibacillus thermoaerophilus strain HZ

A thermostable and organic solvent-tolerant lipase produced by Aneurinibacillus thermoaerophilus strain HZ was purified and characterised. The lipase was purified to apparent homogeneity with two steps: anion exchange chromatography on Q-Sepharose and gel filtration on Sephadex-G75. A final specific activity of 43.5 U/mg was obtained with an overall recovery of 19.7% and 15.6 purification fold. The molecular mass of the HZ lipase was estimated to be 50 kDa. The optimum pH for the activity of the purified HZ lipase was 7.0. The stability showed a broad range of pH values between pH 4.0 and 9.0 at 30°C. The purified HZ lipase exhibited an optimum temperature of 65°C with a half-life of 3 h and 10 min at 65°C. The activity of the purified HZ lipase was stimulated in the presence of Ca2+. Organic solvents such as dimethyl sulfoxide (DMSO), methanol, n-tetradecane and n-hexadecane enhanced the lipase activity. Studies on the effect of oil showed that the lipase preferred natural oil, such as sunflower oil, over synthetic substrates

Effect of alcohol chain length on the optimum conditions for lipase-catalysed synthesis of adipate esters

Immobilized Candida antarctica lipase B, Novozym® 435, was used in the esterification of adipic acid and alcohols with different chain lengths (C1–C18). Optimum conditions for the synthesis of adipate esters were obtained using response surface methodology (RSM) with respect to important reaction parameters including time, temperature, substrate molar ratio and amount of enzyme. Alcohol chain length specificity of the enzyme in the synthesis of adipate esters was also determined. Minimum reaction time (215 min) for achieving maximum ester yield was obtained for butyl alcohol. Methanol required an increased time (358 min) and enzyme amount (10.2%, w/w) for attaining maximum yield. The maximum required temperature and time of 65°C and 523 min, respectively, were obtained for the synthesis of dioctadecyl adipate. The results demonstrate that alcohol chain length is a determining parameter in optimization of the lipase-catalyzed synthesis of adipate esters. Reactions under optimized conditions yielded a high percentage of esterification (>97%). The optimum conditions can be used to scale up the process

Specialty oleochemicals from palm oil via enzymatic syntheses

Palm-based spe­cialty oleo­chem­i­cals are spe­cial indus­trial chem­i­cals from palm oil which are highly priced with high profit mar­gins. These oleo­chem­i­cals exhibit inter­est­ing prop­er­ties such as excel­lent emol­liency, sur­face activ­ity, emul­si­fy­ing prop­er­ties as well as ben­e­fi­cial bio­log­i­cal prop­er­ties. As such, these com­pounds find many appli­ca­tions in the cos­metic, phar­ma­ceu­ti­cal and food indus­tries. Enzyme catal­ysed syn­the­ses of these chem­i­cals are prefer­able as com­pared to their usual petro-chemical coun­ter­part as these processes are nature iden­ti­cal and ‘green’. Lipase-catalysed syn­the­ses of spe­cialty oleo­chem­i­cals were car­ried out. The oleo­chem­i­cals include amino acid esters, fatty alka­nolamides, fatty esteramines, var­i­ous wax esters, medium chain triglyc­erides and bio­log­i­cally active esters. The opti­mi­sa­tion of the reac­tion con­di­tions was dis­cussed. The effects of the para­me­ters influ­enc­ing the reac­tions includ­ing tem­per­a­ture, reac­tion time, sub­strate molar ratio, amount of enzyme and sol­vents used were pre­sented. The char­ac­ter­is­tics of the reac­tion sys­tem and the prod­ucts were determined