Showing papers in "Journal of Molecular Catalysis B-enzymatic in 2010"

Lipase from Thermomyces lanuginosus: Uses and prospects as an industrial biocatalyst

Abstract: The lipase from Thermomyces laguginosus (formerly Humicola laguginosa) (TLL) is a basophilic and noticeably thermostable enzyme, commercially available in both soluble and immobilized form. Although initially oriented toward the food industry, the enzyme has found applications in many different industrial areas, from biodiesel production to fine chemicals (mainly in enantio and regioselective or specific processes). This review intends to show some of the most relevant aspects of the use of this interesting enzyme. After checking the enzyme features, some of the most efficient methods of TLL immobilization will be commented. Finally, the main uses of the enzyme will be revised, with special emphasis in the modification of fats and oils, production of biodiesel, resolution of racemic mixtures, enantioselective hydrolysis of prochiral esters and regioselective process involving sugar preparations. In many instances, TLL has been compared to other lipases, the advantages or disadvantages of the enzyme will be discussed.

Biodiesel production through lipase catalyzed transesterification: An overview

Abstract: Recently, with the global shortage of fossil fuels, excessive increase in the price of crude oil and increased environmental concerns have resulted in the rapid growth in biodiesel production. The central reaction in the biodiesel production is the transesterification reaction which could be catalyzed either chemically or enzymatically. Enzymatic transesterification has certain advantages over the chemical catalysis of transesterification, as it is less energy intensive, allows easy recovery of glycerol and the transesterification of glycerides with high free fatty acid contents. Limitations of the enzyme catalyzed reactions include high cost of enzyme, low yield, high reaction time and the amount of water and organic solvents in the reaction mixture. Researchers have been trying to overcome these limitations in the enzyme catalyzed transesterification reaction. This paper is meant to review the latest development in the field of lipase catalyzed transesterification of biologically derived oil to produce biodiesel.

Lipase from Rhizomucor miehei as an industrial biocatalyst in chemical process

Abstract: The lipase from Rhizomucor miehei (formerly Mucor miehei) (RML) is a commercially available enzyme in both soluble and immobilized form with very high activity and good stability under diverse conditions (anhydrous organic solvents, supercritical fluids, etc.). Although this lipase was initially produced to be used in food industry, in this review we will focus our attention on the application of this enzyme in organic chemistry, from biodiesel production to fine chemicals (mainly in enantio or regioselective or specific processes). After showing the enzyme features, some of the most efficient methods of RML immobilization will be commented (entrapping on reverse micelles, preparation of cross-linked RML aggregates or immobilization on pre-existing solids). Finally, the main uses of the enzyme in organic chemistry will be revised. The use of RML in the production of biodiesel will be analyzed, and compared to the performance of other lipases. The synthesis of esters of carboxylic acids as flavors is other example where RML has been successfully employed. Taking advantage of the wide specificity of the enzyme, mainly a high enantiospecificity, many examples of the use of RML in the resolution of racemic mixtures of chiral carboxylic acids, alcohols or esters will be presented. Special mention requires the use of the regioselectivity of RML, mainly the chemistry of sugars. Finally, more unusual uses of RML will be presented (anomalous substrates, novel uses, etc.). In general, this enzyme seems very adequate for esterification reactions due to its high stability in anhydrous media and good esterification activity.

Lipase from Rhizomucor miehei as a biocatalyst in fats and oils modification

Abstract: The lipase from Rhizomucor miehei (RML), formerly Mucor miehei, is a commercially available enzyme in both soluble and immobilized forms with very high activity and good stability under diverse experimental conditions (anhydrous organic solvents, supercritical fluids, etc.). The uses of the enzyme were initially oriented towards food industry, that way the enzyme has found a broad application in this area. This review intends to show the enzyme features and some of the most relevant aspects of the use of this interesting enzyme in oils and fats modification. The enzyme has been the first lipase whose structure was reported and its interfacial activation is very well known, making this enzyme a good template for modeling studies. Finally, the main uses of the enzyme in fatty acids, oils and fats modification will be revised (hydrolysis of glycerides, transesterification, esterification, acidolysis and interesterification). This lipase seems to be advantageous when compared to other lipases mainly in esterificaton reactions in anhydrous media, while other lipases can be preferred in transesterification reactions.

Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases

Abstract: Cross-linked enzyme aggregates (CLEA®) were prepared from laccases from three different sources: Trametes versicolor, Trametes villosa and Agaricus bisporus. The effect of the various parameters – nature of the precipitant, pH, temperature, glutaraldehyde concentration and cross-linking time – on the activity recovery and storage and operational stability of the resulting CLEAs was different. The laccase CLEAs exhibited the expected increased stability compared to the free enzyme but there was no direct correlation with the number of surface lysine residues in the latter. It is clearly not the only parameter influencing the properties of the CLEA. Co-aggregation with albumin did not improve the stability. The laccase CLEAs, in combination with the stable N-oxy radical, TEMPO, were shown to be active and stable catalysts for the aerobic oxidation of linear C5–C10 aliphatic alcohols, to the corresponding aldehydes, in aqueous buffer (pH 4). Rates were an order of magnitude higher than those observed with the corresponding free enzyme and the CLEAs could be recycled several times without appreciable loss of activity. The addition of water immiscible or water miscible solvents showed no further improvement in rate compared with reactions in aqueous buffer alone.

Aspergillus sp. lipase: Potential biocatalyst for industrial use

Abstract: The lipases obtained from the genus Aspergillus present remarkable importance in biotechnological applications, and numerous studies have reported the importance of the fermentation parameters, such as nutrients, temperature and fermentation time. Moreover, many Aspergillus spp. lipases present several properties of immense industrial importance, such as their pH and temperature stability and excellent enantioselectivity. Different strategies have been used in order to immobilize crude or purified Aspergillus spp. lipases. Hence, Aspergillus spp. lipases have been studied for different industrial applications such as in the food and detergent industries, and also in the kinetic resolution of pharmaceuticals and chiral intermediates. This review highlights the production, purification, characterization, applications and immobilization of lipases from Aspergillus spp.

Pore size of macroporous polystyrene microspheres affects lipase immobilization

Abstract: Polystyrene (PST) microspheres are commonly chosen as immobilization carriers due to their unique advantages such as ideal mechanical strength, adjustable particle size, and favorable chemical stability. However, there were few reports on immobilization by using microspheres with large pore sizes (>100 nm). We have successfully prepared the PST microspheres with macropores and gigapores by a novel method. In this study, giga-/macro-/meso-porous PST microspheres (314 nm, 104 nm, and 14.7 nm in pore sizes) were employed to immobilize lipase (from Burkholderia cepacia) by strong hydrophobic interactions, and the effects of pore sizes on lipase distribution, relative activity, kinetic behavior, thermal stability, storage stability, and reusability were also investigated in detail. According to laser scanning confocal microscope (LSCM) observation, lipase penetrated into the center of those giga-/macro-porous microspheres. With regard to the mesoporous microspheres, lipase only adsorbed to the external shell. The relative activities of immobilized lipase were 146%, 126%, and 50.9% for giga-/macro-/meso-porous PST microspheres, respectively. Comparing with the kinetic constants of free lipase (0.441 mM), the K(m) value for mesoporous PST-lipase (0.532 mM) was higher, whereas for giga-/macro-porous PST-lipase (0.402 mM and 0.411 mM), the K(m) values were comparatively lower suggesting the accessibility of substrate to the enzyme active sites was unlimited. The thermal stability, storage stability, and reusability were all improved significantly with the increase of pore sizes. In stimulant system, even after 100 times of recycling, the activity of lipase immobilized on gigaporous and macroporous PST microspheres remained nearly 100% and 93%, respectively, while that of lipase-PST with 14.7 nm pores could only keep 64.1%. In real system, 73.5% and 68.8% activity of gigaporous PST-lipase and macroporous PST-lipase, respectively, were retained after being used 30 times, whereas only 49.4% activity was remained for mesoporous PST-lipase. Therefore, compared with other microspheres, the gigaporous PST microsphere exhibited obvious advantages as a potential enzyme support in industry. (C) 2010 Elsevier B.V. All rights reserved.

A disposable Laccase-Tyrosinase based biosensor for amperometric detection of phenolic compounds in must and wine

Abstract: An amperometric biosensor for the detection of polyphenols in wine has been developed immobilizing the two enzymes Tyrosinase and Laccase on graphite screen printed electrodes modified with ferrocene. Different immobilization procedures have been carried out, the sensor operational parameters have been optimized, determining the best conditions and the analytical method for the analysis of samples. The biosensor has been then tested with real samples, using wines and musts supplied by Astra, experimental winery, in Imola (Italy). The biosensor gave good results when employed for wine analysis, showing a good agreement with the spectrophotometric data obtained with the Folin-Ciocalteu test, the official method for polyphenols’ analysis in wine. On the other hand, the measurements on musts and wines recently bottled, were seriously affected by the presence of an high level of free sulphur dioxide. SO2 is the likely responsible for enzyme activity inhibition on the sensor. Further studies are currently proceeding to find out the most suitable conditions to obtain results not influenced by the presence of sulphur dioxide. © 2009 Elsevier B.V. All rights reserved.

Lipases from the genus Penicillium: Production, purification, characterization and applications

Abstract: Lipases are ubiquitous enzymes with considerable industrial potential since they have not only general advantages of biocatalysts, such as high catalytic activity, mild reaction conditions, environmental friendliness, and exquisite chemical, enantio- and regioselectivity, but also very broad substrate range and excellent stability. A number of Penicillia are good producers of lipases; some of them are already commercialized. In the past two decades, the lipases from the genus Penicillium have been studied extensively by the researchers. In this review, we presented an overview on their production, purification, biochemical properties, specificity, tolerance toward surfactants, inhibitors and organic solvents, and applications.

Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production

Abstract: An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56 kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40 °C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50 °C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production.

Application of lipase from the physic nut (Jatropha curcas L.) to a new hybrid (enzyme/chemical) hydroesterification process for biodiesel production

Abstract: The aim of this study was to characterize a new plant enzyme (with high lipase activity) extracted from germinated physic nut seeds, and to apply this lipase to the production of biodiesel using a new process of enzyme/chemical hydroesterification. The lipase activity was detected only in the vegetable enzyme extract from germinated seeds (VEEG). Similar activities were obtained for substrates with different lengths of fatty-acid chains (111 ± 19 U/g for tributyrin (C4:0), 106 ± 49 U/g for tricaprylin (C8:0), and 96 ± 4 U/g for olive oil (C18:1)). The VEEG, obtained by a controlled processes of seed germination, was able to hydrolyze a wide range of biodiesel raw materials (vegetable oils, tallow, and biodiesel waste); of these, soy and physic nut oil showed especially high hydrolysis conversion (97% FFA). The biodiesel (fatty acid methyl esters) was produced by the hydrolysis of the physic nut oil using the VEEG, and subsequent esterification of the generated fatty acids with methanol by heterogeneous acid catalysis (niobic acid in pellets). The resulting biodiesel was of excellent quality, with the following properties: viscosity (5.5 mm 2 /s), ester content (97.1%), total glycerol (0.09 % w/w), max. methanol (0.05 % w/w), and CFPP (0 °C).

Mixed lipases for efficient enzymatic synthesis of biodiesel from used palm oil and ethanol in a solvent-free system

Abstract: The enzymatic synthesis of biodiesel from used palm oil and ethanol using immobilized lipases in a solvent-free system was attempted. Five immobilized lipases, Lipase AK from Pseudomonas fluorescens, Lipase PS from Pseudomonas cepacia, Lipase AY from Candida rugosa, Lipozyme TL IM from Thermomyces lanuginosa and Novozym 435 from Candida antarctica, were screened based on their catalytic activities on reactions involved in biodiesel synthesis. The combined use of Lipase AY and Lipase AK gave a higher yield of biodiesel than using Lipase AK alone. The optimal conditions for biodiesel synthesis using mixed lipases in a batch system were: 2% water content, 10% enzyme dosage and 3:1 molar ratio of ethanol to oil. The mixed lipases could be used in 15 replicates with retained relative activity higher than 50%. In a continuous system using mixed lipases packed in packed-bed reactor, >67% of biodiesel was achieved.

Production of aroma esters by immobilized Candida rugosa and porcine pancreatic lipase into calcium alginate gel

Abstract: Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were immobilized into calcium alginate (Ca-Alg) gel beads by means of entrapment and were used to produce three industrially important flavour esters, namely isoamyl acetate (banana flavour), ethyl valerate (green apple flavour) and butyl acetate (pineapple flavour). Immobilization conditions were optimized in terms of sodium alginate (Na-Alg) and CaCl 2 concentrations by determination of the entrapped enzyme amount as well as by esterification of 4-nitrophenol and acetic acid. The best results were obtained at 2.5% Na-Alg and 2.5 M CaCl 2 for CRL while at 2.5% Na-Alg and 2.0 M CaCl 2 for PPL. On carrying out flavour syntheses in solvent-free medium and also in hexane medium, higher ester yields were obtained in hexane medium for all esters and both types of lipases. Ester esterification efficiency increased in parallel with both enzyme concentrations at immobilization medium and the immobilized lipase amount in esterification medium. Maximum ester production was observed between 40 and 50 °C for CRL and PPL. Besides, the effect of substrate concentrations on ester conversion was remarkable. The best ester yield was obtained for isoamyl acetate when immobilized PPL was used.

Lipases for use in industrial biocatalysis : Specificity of selected structural groups of lipases

Abstract: Lipases for biocatalysis The substrate specificity of a selected group of lipases was investigated. The enzymes selected were from four structural groups. Group 1: lipases having wide alcohol bindi ...

Immobilization of ω-transaminases by encapsulation in a sol–gel/celite matrix

Abstract: Commercially available ω-transaminases ω-TA-117, -113, and Vibrio fluvialis (Vf-AT) have been immobilized in a sol–gel matrix. Improved results were obtained by employing Celite 545 as additive. The immobilized ω-transaminases ω-TA-117, -113, and V. fluvialis (Vf-AT) were tested in the kinetic resolution of α-chiral primary amines. In contrast to the free enzyme ω-TA-117, the sol–gel/celite immobilized enzyme showed activity even at pH 11. Recycling of the sol–gel/Celite 545 immobilized ω-transaminase ω-TA-117 was performed over five reaction cycles without any substantial loss in enantioselectivity and conversion. Finally, the immobilized ω-TA 117 was employed in a one-pot two-step deracemization of rac-mexiletine and rac-4-phenyl-2-butylamine, two pharmacologically relevant amines. The corresponding optically pure (S)-amines were obtained in up to 95% isolated yield (>99% ee).

On the nature of mutual inactivation between [Cp*Rh(bpy)(H2O)]2+ and enzymes – analysis and potential remedies

Abstract: Pentamethylcyclopentadienyl rhodium bipyridine ([Cp*Rh(bpy)(H2O)]2+) is a versatile catalyst to promote biocatalytic redox reactions. However, its major drawback lies in the mutual inactivation of [Cp*Rh(bpy)(H2O)]2+ and the biocatalyst. This interaction was investigated using the alcohol dehydrogenase from Thermus sp. ATN1 (TADH) as model enzyme. TADH binds 4 equiv. of [Cp*Rh(bpy)(H2O)]2+ without detectable decrease in catalytic activity and stability. Higher molar ratios lead to time-, temperature-, and concentration-dependent inactivation of the enzyme suggesting [Cp*Rh(bpy)(H2O)]2+ to function as an ‘unfolding catalyst’. This detrimental activity can be circumvented using strongly coordinating buffers (e.g. (NH4)2SO4) while preserving its activity as NAD(P)H regeneration catalyst under electrochemical reaction conditions.

Potential biocatalysts originating from sea environments

Abstract: This review is intended to give an account of the knowledge about known enzymes of marine origin described in literature thus stimulating future applications in biocatalysis that these biocatalysts can offer to a large spectra of end-users. The uniqueness of marine biocatalysts is not only based on habitat-related properties such as salt tolerance, hyperthermostability, barophilicity, cold adaptivity, etc. A marine enzyme in fact may carry more, e.g. novel chemical and stereochemical properties. This “chemical biodiversity” increases interest in this field; substrate specificity and affinity are evolved properties linked to the metabolic functions of the enzymes and to ecological asset related to the natural source and this is an important aspect in the bioprospecting for new biocatalysts. The importance of all examples reported should be sufficient to trigger the attention of the biocatalytically oriented scientific community towards marine environment as source of biocatalysts, and this could in turn enhance both new discovery and improvement of marine enzymes.

Enhanced biomimetic sequestration of CO2 into CaCO3 using purified carbonic anhydrase from indigenous bacterial strains

Abstract: Phenomenal rise in CO2 concentration have led to disastrous consequences. The present study endeavors the biomimetic sequestration of CO2 into CaCO3 using biological catalyst; carbonic anhydrase (CA) purified from Pseudomonas fragi, Micrococcus lylae and Micrococcus luteus 2 along with a comparative evaluation of their efficiency against commercial bovine carbonic anhydrase (BCA). At pH range 8.0–9.0 and temperature range 35–45 °C, maximum stability was observed for CA from M. luteus 2 followed by P. fragi CA, M. lylae CA and BCA. P. fragi CA demonstrated maximum stability as function of time with respect to pH and temperature. The anionic inhibitors, Cl−, SO42−, NO3−, HCO3 and toxic metal ions viz., lead, arsenic and mercury showed varied inhibitory profile against the four different CAs. The level of inhibition was significantly higher for BCA and M. luteus 2 CA compared to P. fragi CA and M. lylae CA. Calcium estimation was found to be a reliable method for the determination of sequestration efficiency. Indigenous CAs and their consortia exhibited enhanced CO2 sequestration competence compared to commercial BCA. Sequestration efficiency at 45 °C, under process parameters was found to be maximum for CA consortia (61%) and minimum for commercial BCA (17.8%) indicating its potential application in an onsite scrubber.

Lactose hydrolysis by β galactosidase immobilized on concanavalin A-cellulose in batch and continuous mode

Abstract: β Galactosidase from Aspergillus oryzae was immobilized on an inexpensive bioaffinity support, (concanavalin A) Con A-cellulose. The mode of interaction between Con A-cellulose and β galactosidase is shown by Fourier transform infrared spectroscopy. Con A-cellulose adsorbed and crosslinked β galactosidase preparation retained 78% of the initial activity. Soluble and immobilized β galactosidase showed the same pH-optimum at pH 4.6. The temperature-optimum was increased from 50 to 60 °C for the immobilized β galactosidase. The immobilized enzyme had higher thermal stability at 60 °C. The crosslinked adsorbed enzyme retained 80 and 70% of the original enzyme activity in the presence of 3% calcium chloride and 3% galactose, respectively. Moreover, the adsorbed crosslinked and adsorbed β galactosidase exhibited 84 and 75% enzyme activity even after their sixth repeated use, respectively. The crosslinked adsorbed enzyme retained 93% activity after 1 month storage while the native enzyme showed only 63% activity under similar incubation conditions. Immobilized β galactosidase showed higher lactose hydrolysis from solution in batch process at 60 °C as compared to its hydrolysis at 50 °C. The continuous hydrolysis of lactose was appreciably different at various flow rates. Thus, the reactor filled with crosslinked Con A-cellulose adsorbed β galactosidase could be successfully employed for the continuous hydrolysis of lactose from milk and whey.

Biochemical characterization of the cutinases from Thermobifida fusca

Abstract: Thermobifida fusca produces two cutinases which share 93% identity in amino acid sequence. In the present study, we investigated the detailed biochemical properties of T. fusca cutinases for the first time. For a better comparison between bacterial and fungal cutinases, recombinant Fusarium solani pisi cutinase was subjected to the similar analysis. The results showed that both bacterial and fungal cutinases are monomeric proteins in solution. The bacterial cutinases exhibited a broad substrate specificity against plant cutin, synthetic polyesters, insoluble triglycerides, and soluble esters. In addition, the two isoenzymes of T. fusca and the F. solani pisi cutinase are similar in substrate kinetics, the lack of interfacial activation, and metal ion requirements. However, the T. fusca cutinases showed higher stability in the presence of surfactants and organic solvents. Considering the versatile hydrolytic activity, good tolerance to surfactants, superior stability in organic solvents, and thermostability demonstrated by T. fusca cutinases, they may have promising applications in related industries.

Technical methods to improve yield, activity and stability in the development of microbial lipases

Abstract: Lipases are ubiquitous biocatalysts that catalyze various reactions in organic solvents or in solvent-free systems and are increasingly applied in various industrial fields. In view of the excellent catalytic activities and the huge application potential, more than 20 microbial lipases have been realized in large-scale commercial production. The potential for commercial exploitation of a microbial lipase is determined by its yield, activity, stability and other characteristics. This review will survey the various technical methods that have been developed to enhance yield, activity and stability of microbial lipases from four aspects, including improvements in lipase-producing strains, modification of lipase genes, fermentation engineering of lipases and downstream processing technology of lipase products.

Immobilization of catalase onto Eupergit C and its characterization

Abstract: Bovine liver catalase was covalently immobilized onto Eupergit C. Optimum conditions of immobilization: pH, buffer concentration, temperature, coupling time and initial catalase amount per gram of carrier were determined as 7.5, 1.0 M, 25 °C, 24 h and 4.0 mg/g, respectively. V max and K m were determined as 1.4(±0.2) × 10 5 U/mg protein and 28.6 ± 3.6 mM, respectively, for free catalase, and as 3.7(±0.4) × 10 3 U/mg protein and 95.9 ± 0.6 mM, respectively, for immobilized catalase. The thermal stability of the immobilized catalase in terms of half-life time (29.1 h) was comparably higher than that of the free catalase (9.0 h) at 40 °C. Comparison of storage stabilities showed that the free catalase completely lost its activity at the end of 11 days both at room temperature and 5 °C. However, immobilized catalase retained 68% of its initial activity when stored at room temperature and 79% of its initial activity when stored at 5 °C at the end of 28 days. The highest reuse number of immobilized catalase was 22 cycles of batch operation when 40 mg of immobilized catalase loaded into the reactor retaining about 50% of its original activity. In the plug flow type reactor, the longest operation time was found as 82 min at a substrate flow rate of 2.3 mL/min when the remaining activity of 40 mg immobilized catalase was about 50% of its original activity. The resulting immobilized catalase onto Eupergit C has good reusability, thermal stability and long-term storage stability.

Optimization of immobilization conditions of Thermomyces lanuginosus lipase on styrene–divinylbenzene copolymer using response surface methodology

Abstract: Microbial lipase from Thermomyces lanuginosus (formerly Humicola lanuginosa ) was immobilized by covalent binding on a novel microporous styrene–divinylbenzene polyglutaraldehyde copolymer (STY–DVB–PGA). The response surface methodology (RSM) was used to optimize the conditions for the maximum activity and to understand the significance and interaction of the factors affecting the specific activity of immobilized lipase. The central composite design was employed to evaluate the effects of enzyme concentration (4–16%, v/v), pH (6.0–8.0), buffer concentration (20–100 mM) and immobilization time (8–40 h) on the specific activity. The results indicated that enzyme concentration, pH and buffer concentration were the significant factors on the specific activity of immobilized lipase and quadratic polynomial equation was obtained for specific activity. The predicted specific activity was 8.78 μmol p -NP/mg enzyme min under the optimal conditions and the subsequent verification experiment with the specific activity of 8.41 μmol p -NP/mg enzyme min confirmed the validity of the predicted model. The lipase loading capacity was obtained as 5.71 mg/g support at the optimum conditions. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation (12%) occurred after being used repeatedly for 10 consecutive batches with each of 24 h. The effect of methanol and tert -butanol on the specific activity of immobilized lipase was investigated. The immobilized lipase was almost stable in tert -butanol (92%) whereas it lost most of its activity in methanol (80%) after 15 min incubation.

Study on acyl migration kinetics of partial glycerides: Dependence on temperature and water activity

Abstract: Acyl migration phenomenon was often observed during 1,3-positional specificity lipase-catalyzed reactions from triglycerides and partial glycerides, including acyl migration of 1,2-diglyceride (1,2-DG) to 1,3-diglyceride (1,3-DG) and 2-monoglyceride (2-MG) to 1-monoglyceride (1-MG). However, the acyl migration mechanism and kinetics were seldom studied despite of numerous researches on process optimization of 1,3-positional specificity lipase-catalyzed reaction. In this paper, the influence of related factors on acyl migration process as well as their influencing mechanism was further studied. It was found that temperature and water activity were two crucial factors that would influence acyl migration kinetics. Determination of the kinetic parameters under different temperatures revealed that the acyl migration reaction rates were greatly promoted by the increasing of temperature. The acyl migration rates of 1,2-diglyceride and 2-monoglyceride were quite different from each other, which was found to be due to the different activation energies. Further study of how would water influence the acyl migration process showed that water activity rather than water content was a key factor that influenced acyl migration and the acyl migration rate would decrease with the increase of water activity. It was further revealed that water activity influenced the charge dispersion of the transition state, which ultimately influenced the reaction activation energy and then influenced the acyl migration rate.

Both hydrolytic and transesterification activities of Penicillium expansum lipase are significantly enhanced in ionic liquid [BMIm][PF6]

Abstract: Both hydrolytic and transesterification activities of Penicillium expansum lipase (PEL) were investigated in the ionic liquid [BMIm][PF 6 ] as well as in organic solvents such as hexane. The initial rate of PEL-catalyzed hydrolysis of p -nitrophenyl palmitate (pNPP) was 12-fold enhanced in the ionic liquid. The optimal water content required by PEL in [BMIm][PF 6 ] was 10 times higher relative to that in hexane due to the greater polarity of the ionic liquid. Direct addition of salt hydrates into the two nonaqueous reaction media showed different impacts on the enzyme activity, which could be related to the dual functions of the salt hydrates, i.e., the water buffering effect, and the specific ion effect (Hofmeister effect). The transesterification activity of PEL was reflected by the yield of producing fatty acid methyl esters (FAMEs) in the methanolysis of corn oil for 25 h, which was 69.7% in [BMIm][PF 6 ], as compared to 19.4%, 14.0%, and 1.0% obtained in tert -butanol, solvent-free system, and hexane, respectively. The high production yield of FAMEs obtained in [BMIm][PF 6 ] demonstrates the potential use of ionic liquids as the reaction media for PEL-catalyzed biodiesel production.

Immobilization of Candida rugosa lipase on glutaraldehyde-activated polyester fiber and its application for hydrolysis of some vegetable oils

Abstract: Candida rugosa lipase was effectively immobilized on the poly(ethylene terephthalate) grafted acrylamide (PET-g-AAm) fiber which was prepared through Hofmann reaction. The activities of the immobilized enzyme and the free enzyme were investigated in the hydrolysis reaction of olive oil in isooctane–water biphasic medium. The activities of the free and the immobilized lipases were measured at different pH values, and temperatures. The thermal stability and storage stability of them were also determined. The kinetic parameters of the free and the immobilized lipases, Km and Vmax were calculated, as well. Moreover, the application of immobilized lipase to the hydrolysis of different vegetable oils was realized. The effect of organic solvents on olive oil hydrolysis was examined and time course of the oil hydrolysis was studied. The optimum pH was shifted from 6 to 7 by immobilization of the enzyme. The maximum activity of the free and the immobilized enzymes occurred at 40 °C. It was found that the immobilized lipase stored at 4 °C retained 90% of its original activity after 60 days, whereas the free lipase stored at 4 °C retained 75% of its activity after the same period. In addition, the immobilized lipase exhibited as 0.06 U of the remaining activity even after 10 times reuses. Kinetics studies show that the corresponding values of Km and Vmax were 47.2 mg ml−1 and 48.1 U mg−1 protein for free lipase and 151.6 mg ml−1 and 10.9 U mg−1 protein for immobilized lipase. The immobilized and free enzymes showed similar behavior with respect to the different oil hydrolysis. The time course of the immobilized lipase on canola and olive oils hydrolysis used as substrate was much better than that of the free lipase. A high rate of oil hydrolysis was obtained when isooctane was used as solvent.

Chemical modification of stem bromelain with anhydride groups to enhance its stability and catalytic activity

Abstract: Chemical modification of lysine residues in stem bromelain was carried out using two novel reagents pyromellitic anhydride acid and poly(maleic anhydride). About 60% and 57% of the residues in bromelain were found to be modified by pyromellitic anhydride and poly(maleic anhydride), respectively. The modification brought about enhancement of thermal stability and the resistance to alkali and the surfactant. The optimum pH shifted from 7 to 9. Thermodynamic parameters, Δ H *, Δ G * and Δ S *, were determined as a function of temperature. The kinetic constants K m of the modified enzymes were determined as 0.4092 × 10 −2 and 0.2825 × 10 −2 mol l −1 , respectively. SDS-PAGE profiling revealed a major bands of native and modified enzyme with molecular weights of 26 and 28.5 kDa. The results of FT-IR studies suggested that the modification caused only local structural changes. These results provide guidance for future development of stable protein formulations.

A novel thermostable cellulase from Fervidobacterium nodosum

Abstract: A novel cellulase gene encoding a thermostable endoglucanase from the thermophilic eubacterium Fervidobacterium nodosum Rt17-B1 was cloned and expressed, which is the first cellulase cloned from the organisms of genus Fervidobacterium and designated as FnCel5A for being a member of glycoside hydrolase family 5, and the enzymatic properties were characterized. The cellulase was overexpressed in Escherichia coli with a high protein content and good solubility in water, and could be easily purified. The purified recombinant cellulase shows high hydrolytic activities on carboxylmethyl cellulose, regenerated amorphous cellulose, β- d -glucan from barley and galactomannan, with the optimum temperature of 80–83 °C and the optimum pH of 5.0–5.5. Furthermore, this enzyme is highly thermostable and has a half-life of 48 h at 80 °C. With such a combination of thermostability and high activities, this cellulase is expected to be useful for hydrolysis of cellulosic and hemicellulosic substrates at high temperatures, and for industrial hydrolysis of plant cellulose during long-time processing at the elevated temperatures, particularly in converting biomass into biofuels.

Purification and characterization of a solvent, detergent and oxidizing agent tolerant protease from Bacillus cereus isolated from the Gulf of Khambhat

Abstract: Twenty-eight organic solvent tolerant bacteria were isolated from crude oil contaminated samples, out of which, AK1871 isolate produced a solvent, detergent and oxidizing agent tolerant serine alkaline protease. Based on the morphological and biochemical characteristics, FAME analysis as well as 16S rRNA gene sequence, the isolate is identified as Bacillus cereus . A 58-fold purification of protease was achieved by a three-step purification procedure. This protease is active over a broad range of pH (6.0–9.0, optimum at 8.0); and temperature from 40 °C to 70 °C (optimum at 60 °C). Li + , Ba 2+ , K + , Mg 2+ and Mn 2+ did not affect, while heavy metals like Cr 3+ , Hg 2+ and Cu 2+ inactivated the enzyme. It is stable in the presence of non-ionic detergents (Triton X-100 and Tween 80), and oxidizing and bleaching agents (hydrogen peroxide). The protease exhibited noteworthy stability and activation in the presence of organic solvents with log P values equal to or more than 2.0. This protease could be used in detergent formulations, enzymatic peptide synthesis, biotransformation reactions and in the formulation of antifouling agent. This is the first report on the study of organic solvent tolerant protease from a marine organic solvent tolerant bacterium isolated from the Gulf of Khambhat.

Lipase catalysis in ionic liquids/supercritical carbon dioxide and its applications

Abstract: Ionic liquids (ILs) and supercritical carbon dioxide (SC-CO2) have been accepted as solvents facilitating green processing by lots of scholars and researchers in recent decades. The combination of these two solvents owns advantages, such as improving the stability of the enzymes, less or no pollutants. Enzymatic catalysis has been thought as one of the potent methods to replace the chemical catalysis, which has some drawbacks, such as using chemical catalysts caused hard to recycle and to pollute the environment, because the enzymes are easy to be degraded. In the enzymes, lipases are important and potent in industrial application. Therefore, lipase catalysis in IL/SC-CO2 is green and benign to the environment. In this paper, advantages, mass transfer, characteristics and applications of lipase catalysis in IL/SC-CO2 are reviewed.