Kinetic properties of Penicillium cyclopium lipases studied with vinyl esters

Penicillium cyclopium produces two lipases with different substrate specificities. Lipase I is predominantly active on triacylglycerols whereas lipase II hydrolyzes mono- and diacylglycerols but not triacylglycerols. In this study, we compared the kinetic properties of P. cyclopium lipases and human pancreatic lipase, a classical triacylglycerol lipase, by using vinyl esters as substrates. Results indicate that P. cyclopium lipases I and II and human pancreatic lipase hydrolyze solutions of vinyl propionate or vinyl butyrate at high relative rates compared with emulsions of the same esters, although, in all cases, maximal activity is reached in the presence of emulsified particles, at substrate concentrations above the solubility limit. It appears that partially water-soluble short-chain vinyl esters are suitable substrates for comparing the activity of lipolytic enzymes of different origin and specificity toward esters in solution and in emulsion.     

Cloning of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles. The cloned gene was 1,480 nucleotides in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides. The open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide, with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases. However, this protein had a low homology with lipases of P. camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level. The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation. A distinct band with a M.W. of 33 kDa was detected on SDS-PAGE. Results of a Western blot analysis and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase. Its catalytic properties were not changed when compared with its natural counterpart. Zhikang Qian and Peihong Jiang are equal contributors as first author of this paper.     

Biochemical Properties of a New Cold-Active Mono- and Diacylglycerol Lipase from Marine Member Janibacter sp. Strain HTCC2649

Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1) from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1), Penicillium camembertii lipase U-150 (PCL), and Aspergillus oryzae lipase (AOL). Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.     

Synthesis of triacylglycerol containing conjugated linoleic acid by esterification using two blended lipases

We have developed an efficient esterification for the synthesis of triacylglycerol (TAG) containing conjugated linoleic acids (CLA) using a blend of two powdered lipases. Two pairs of blended lipases promoted the esterification. Rhizomucor miehei lipase, plus Alcaligenes sp. lipase and Penicillium cammembertii MAG and DAG lipase plus Alcaligenes sp. lipase were used. At the optmal ratio of two lipases, the content of TAG containing CLA (TAG-CLA) in all glycerols reached 82–83% after 47 h using 1 wt% of lipases. With R. miehei lipase plus Alcaligenes sp. lipase, the reaction time to obtain ca. 60% of TAG-CLA was one-third of that needed with R. miehei lipase alone. The optimal ratio of two lipases differed between these two pairs. The optimal ratio was 70–80 wt% of R. miehei lipase in the last stage of the reaction, whereas it was over a wide range of 10–90 wt% for P. camembertii lipase. In the blend of R. miehei lipase plus Alcaligenes sp. lipase, activity remained very high after 10 cycles of esterification (every 47 h) and could be used in the industrial production of TAG-CLA.     

Esterification of n‐butyric acid with n‐butyl alcohol and transesterification of (R, S)‐phenylethanol by lipase immobilized on cellulose acetate–TiO2 gel fibre

Lipase (EC 3.1.1.3) was immobilized on cellulose acetate–TiO₂ gel fibre by the sol–gel method. The immobilized lipases were used for esterification of n‐butyric acid with n‐butyl alcohol and enantioselective acylation of (R, S)‐phenylethanol using vinyl acetate as an acyl donor. Compared with native lipase, the activity of the immobilized lipase was stable and relatively unaffected by the water content of the solvent and the substrate concentration. The data indicate that the lipases are immobilized on the fibre surface and that enzyme activity is influenced by bound water. However, the thermal reactivity and enantioselectivity of the immobilized lipase were less than those of native lipase. This may not reflect thermal inactivation of the enzyme but rather significant thermal contraction of the gel fibre by cellulose crystallization, resulting in liberation of bound water and a decrease in the amount of enzyme which is available for the reaction. Copyright © 2001 Society of Chemical Industry     

Further improvements in the yield of monoglycerides during enzymatic glycerolysis of fats and oils

Three approaches were used in an effort to increase the yield of monoglycerides (MG) during the lipase catalyzed reaction of glycerol with triglyceride fats and oils: i) various commercially available lipases were screened for ability to catalyze MG synthesis; ii) mixtures of lipases were compared with single lipases; and iii) two-step temperature programming was applied during the reaction. Of these, temperature programming was found to be the most effective. With an initial temperature of 42°C for 8–16 hr followed by incubation at 5°C for up to 4 days, a yield of approximately 90 wt% MG was obtained from beef tallow, palm oil and palm stearin. When the second incubation temperature was greater than 5°C, the yield of MG was progressively lower with increasing temperature. In the case of screening of newly available commercial lipase preparations, lipases fromPseudomonas sp. were found to be most effective, giving a yield of approximately 70 wt% MG at 42°C from tallow. Lipases fromGeotrichum candidum, Penicillium camembertii (lipase G) andCandida rugosa were inactive. A mixture of lipases fromPenicillium camembertii andHumicola lanuginosa was found to be more effective than either enzyme alone, giving a yield of approximately 70 wt% MG using beef tallow or palm oil. A mixture ofPenicillium camembertii lipase with eitherPseudomonas fluorescens lipase orMucor miehei lipase was not more effective thanPseudomonas fluorescens orMucor miehei lipase alone.     

Hydrolysis of fish oils containing polymers of triacylglycerols by pancreatic lipasein vitro

Fish oils containing different levels of polymers of triacylglycerols formed during autoxidation were incubated with pancreatic lipase to establish whether these polymers are substrates for lipase hydrolysis. With oils containing low amounts (less than 4%) of triacylglycerol polymers as substrates, both triacylglycerols and polymers of triacylglycerols were almost completely hydrolyzed, and fatty acid monomers and monoacylglycerols were the major lipid products. Under the same incubation conditions, some triacylglycerols remained intact when highly oxidized oils containing 20 or 30% triacylglycerol polymers were the substrate. The fatty acid composition of these residual triacylglycerols was almost identical to that of triacylglycerols present at the start of the assay. When fish oil containing 30% triacylglycerol polymers was incubated with the lipase, the component triacylglycerols and polymers of triacylglycerols were hydrolyzed at similar rates, and fatty acid dimers were detected as a product. It is concluded that the high molecular weight polymers of triacylglycerols present in oxidized fish oils can be hydrolyzed by pancreatic lipasein vitro.     

Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution ¹³C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the ¹³C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.     

Single step recovery of lipase from Penicillium cyclopium by aqueous two-phase extraction

Recovery of lipase from Penicillium cyclopium by aqueous two-phase extraction was studied with maximal possible crude enzyme loads. In polyethylene glycol/dextran and polyethylene glycol/salt systems the influences of molecular weight and concentration of polyethylene glycol, phase-forming salt and phase volume ratio were evaluated. Lipase partition coefficient 9 followed by the top phase yield 95.7% and purification factor 3.4 were achieved in 15% (w w^?1) polyethylene glycol 4000/15% (w w^?1) KH₂PO₄/70% (w w^?1) crude enzyme. Efficient single-step recovery of lipase followed by partial enzyme purification indicated possible integration of production and primary bioseparation step by aqueous two-phase extraction. By varying phase volume ratio, the concentration of phosphate was reduced without decrease in lipase recovery.     

Esterification of glycerol with conjugated linoleic acid and long-chain fatty acids from fish oil

Free fatty acids from fish oil were prepared by saponification of menhaden oil. The resulting mixture of fatty acids contained ca. 15% eicosapentaenoic acid (EPA) and 10% docosahexaenoic acid (DHA), together with other saturated and monounsaturated fatty acids. Four commercial lipases (PS from Pseudomonas cepacia, G from Penicillium camemberti, L2 from Candida antarctica fraction B, and L9 from Mucor miehei) were tested for their ability to catalyze the esterification of glycerol with a mixture of free fatty acids derived from saponified menhaden oil, to which 20% (w/w) conjugated linoleic acid had been added. The mixtures were incubated at 40°C for 48h. The ultimate extent of the esterification reaction (60%) was similar for three of the four lipases studied. Lipase PS produced triacylglycerols at the fastest rate. Lipase G differed from the other three lipases in terms of effecting a much slower reaction rate. In addition, the rate of incorporation of omega-3 fatty acids when mediated by lipase G was slower than the rates of incorporation of other fatty acids present in the reaction mixture. With respect to fatty acid specificities, lipases PS and L9 showed appreciable discrimination against esterification of EPA and DHA, respectively, while lipase L2 exhibited similar activity for all fatty acids present in the reaction mixture. The positional distribution of the various fatty acids between the sn-1,3 and sn-2 positions on the glycerol backbone was also determined.     

Esterification activities of non‐commercial lipases after pre‐treatment in pressurized propane

BACKGROUND: The objective of this work was to assess the influence of propane pre‐treatment on the esterification activities of two non‐commercial lipases from Penicillium simplicissimum and Aspergillus parasiticus in the lyophilized and immobilized forms, evaluating the effect of pressure, exposure time and depressurization rate on the lipase activity by a full 2³ experimental design. RESULTS: For both lyophilized and immobilized lipases from all sources evaluated, enhancement in residual activities after incubation in pressurized propane was observed, under several experimental conditions. The highest increment (427%) occurred with the lyophilized enzyme from P. simplicissimum was maintained pressurized at 30 bar for 1 h and then depressurized at the fastest rate (20 bar min^?1). CONCLUSION: The enzyme activity changes significantly depending on the enzyme source, its presentation form, and the experimental pre‐treatment conditions investigated, such as exposure time, depressurization rate and pressure. The results obtained here can contribute as a basis for the selection of appropriate operational conditions, so that these catalysts can be applied in biotransformation reactions. Copyright © 2010 Society of Chemical Industry     

Studies on the substrate specificity of purified human milk lipoprotein lipase

The fatty acid specificity of purified human milk lipoprotein lipase was studied using the C₁₈ to C₅₄ (total acyl carbon number) saturated and the C₅₄ mono-, di- and triunsaturated monoacid triacylglycerols. Kinetic determinations indicated that the medium-chain triacylglycerols were better substrates than long- or very short-chain saturated triacylglycerols. The unsaturated triacylglycerols were hydrolyzed at rates comparable to that of tricaprylin with triolein having the highest rate of hydrolysis of the unsaturated species tested. The enzyme attacked the primary ester bond much more readily than the secondary ester bond. The purified human milk lipoprotein lipase showed a preferential stereospecific lipolysis of thesn-1-position of the triacylglycerol molecule.     

Wax ester-synthesizing activity of lipases

The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, was ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium.     

Enzymatische Herstellung von festen Fettsäuremonoglyceriden

Enzymatic Preparation of Solid Fatty Acid Monoglycerides Lipases can be used to synthesize monoglycerides from solid fatty acids and glycerol. We have examined the conditions of such reaction systems with a view to developing a simple technical process. The selectivity of the Penicillium cyclopium and Rhizopus sp. lipases were studied at high rates of fatty acid turnover. By the correct choice of lipase, temperature and water content, the reaction may be steered in the direction of either monoglycerides of diglycerides. Using the Penicillium lipase under narrowly defined reaction conditions a highly selective monoglyceride synthesis is possible. Di glycerides are almost the sole products when using the Rhizopus lipase at 40°C. At 20°C, but under otherwise identical conditions, the main products are monoglycerides. The Penicillium lipase catalyzes the synthesis of monoglycerides at both 20°C and 40°C, provided that the water content of the glycerine is less than 10%. At a glycerol concentration of 80% the selectivity changes such that more diglycerides are formed. The enzymatic synthesis of glycerides can be so regulated that more than 95% of the available fatty acid is incorporated into monoglyceride. After melting the reaction mixture and allowing it to stand for less than an hour, the phases separate and excess glycerol can be separated very simply. A product conforming to industry specifications can then be produced by distilling off the trace amounts of remaining glycerol from the lipid phase.     

Immobilization of lipases on porous polypropylene: Reduction in esterification efficiency at low loading

Rhizomucor miehei, Humicola sp., Rhizopus niveus, and Candida antarctica B lipases were immobilized by physical adsorption onto a macroporous polypropylene support. In an esterification reaction, the enzyme efficiency, and therefore cost-effectiveness, is greatly affected by enzyme loading, with an apparent suppression of efficiency at low lipase loadings for both R. miehei and Humicola sp. lipases. This results in the appearance of a pronounced maximum in the efficiency-loading relationship at approximately 100,000 lipase units (LU)/g for R. miehei lipase (10% of its saturation loading) and at approximately 200,000 LU/g for Humicola sp. lipase (50% of its saturation loading). The other lipases studied do not show similar trends. At low loadings, only a small portion of the surface area is occupied and gives the lipase the opportunity to spread; it is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinityfor the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted.     

Interactions between Lipases and Amphiphiles at Interfaces

This review deals with interactions at interfaces between lipases and low molecular weight amphiphiles, such as polar lipids and synthetic surfactants. The interaction between polar lipids and lipases is particularly important in the gastrointestinal tract, where fat is digested by gastric lipases in the stomach and by pancreatic lipases in the duodenum. Polar lipids have been found to influence lipase activity in numerous ways. For example, it has been found that Sn-2 monoacylglycerols, which are the main degradation products from fat metabolism, take over at the triacylglycerol oil–water interface and prevent further access of the lipase to its substrate, i.e., triacylglycerols and diacylglycerols. Additionally, different types of surfactants interact differently with lipases and the interaction can result in loss of enzymatic activity. As both lipases and the surfactants are strongly surface active, this type of interaction preferentially takes place at an interface. Lipase-surfactant interactions have been systematically studied at the air–water, the solid–water, and the oil–water interfaces. In general, it is found that cationic surfactants interact stronger than anionic or nonionic surfactants at all interfaces but not in bulk water. However, somewhat contradictory results have been reported in the literature and it is likely that the inconsistency is due to the fact that lipases of different origins are used in the different studies.     

Critical temperature for production of MAG by esterification of different FA with glycerol using <Emphasis Type="Italic">Penicillium camembertii</Emphasis> lipase

Production of MAG by a lipase-catalyzed reaction is known to be effective at low temperature. This phenomenon can be explained by assuming that synthesized MAG are excluded from the reaction system because MAG, which have low m.p., are solidified at low temperatures. Consequently, MAG are efficiently accumulated and do not serve as the precursor of DAG. If this hypothesis is correct, the critical temperature for MAG production, defined as the highest temperature at which DAG synthesis is repressed, should depend on the m.p. of the MAG. Esterification of FFA with glycerol using Candida rugosa, Rhizopus oryzae, and Penicillium camembertii lipases produced MAG efficiently at low temperatures. However, Candida lipase showed very low esterification activity at high temperatures (>20°C), and Rhizopus lipase produced not only MAG but also DAG even at low temperatures. Meanwhile, P. camembertii lipase catalyzed synthesis of MAG only from FFA and glycerol at low temperatures, although the enzyme catalyzed synthesis of DAG from MAG in addition to synthesis of MAG at high temperatures. We thus studied the effect of temperature on esterification of C₁₀−C₁₈ FFA with glycerol using Penicillium lipase as a catalyst and determined the critical temperatures for production of MAG. The critical temperature for production of each MAG showed a linear correlation with m.p. of the MAG, which supported the hypothesis. In addition, because the m.p. of MAG are estimated from that of the constituent FA, the optimal temperature for production of MAG can be predicted from the m.p. of the FFA used as a substrate.     

Determining the regioselectivity of immobilized lipases in triacylglycerol acidolysis reactions

Lipase regioselectivity is the ability to distinguish between primary (i.e., sn-1,3) and secondary (sn-2) ester functionalities in a triacylglycerol molecule, which is of importance in the manufacture of structured lipids. Unlike existing methods of assessment, which utilize hydrolysis reactions, an alternative technique to assess the regioselectivity of lipases in triacylglycerol transesterification reactions has been developed. An acidolysis reaction is performed between triolein and decanoic, lauric, or stearic acids under conditions that minimize acyl migration, and products are analyzed by silver-ion complexation liquid chromatography, enabling detection of specific triacylglycerol positional isomers. From the rate of formation of these isomers the relative selectivity of the lipase for sn-2 and sn-1,3 ester bonds is determined. With lipases known to lack regioselectivity, the rate of reaction at sn-2 was similar to that at sn-1,3 from the start of the reaction. With sn-1,3 selective lipases, the formation of triacylglycerol isomers with decanoic acid in the secondary position was not detected at any point in the reaction. Regioselectivity as a function of reaction progress was monitored. Two lipases from the genus Pseudomonas exhibited activity toward all positions, but the rate at sn-2 was much reduced, and no incorporation of decanoic acid into this position was detectable until a high degree of conversion had been achieved.     

Conversion of a Mono‐ and Diacylglycerol Lipase into a Triacylglycerol Lipase by Protein Engineering

Despite the fact that most lipases are believed to be active against triacylglycerides, there is a small group of lipases that are active only on mono‐ and diacylglycerides. The reason for this difference in substrate scope is not clear. We tried to identify the reasons for this in the lipase from Malassezia globosa. By protein engineering, and with only one mutation, we managed to convert this enzyme into a typical triacylglycerol lipase (the wild‐type lipase does not accept triacylglycerides). The variant Q282L accepts a broad spectrum of triacylglycerides, although the catalytic behavior is altered to some extent. From in silico analysis it seems that specific hydrophobic interactions are key to the altered substrate specificity.     

Purification and characterization of an extracellular lipase from Penicillium candidum

Penicillium candidum produces and secretes a single extracellular lipase with a monomer molecular weight of 29 kDa. However, this enzyme forms dimers and higher molecular weight aggregates under nondenaturing conditions. The lipase from P. candidum was purified 37-fold using Octyl-Sepharose CL-4B and DEAE-Sephadex columns. The optimal assay conditions for lipase activity were 35°C and pH 9. The lipase was stable in the pH range of 5–6 with a pl of 5.5, but rapid loss of the enzyme activity was observed above 25°C. Tributyrin was found to be the best substrate for the P. candidum lipase, among those tested. Metal ions such as Fe²⁺ and Cu²⁺ inhibited enzymatic activity and only Ca²⁺ was able to slightly enhance lipase activity. Ionic detergents inhibited the activity of the enzyme, whereas nonionic detergents stimulated lipase activity.