A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols.

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.     

Ethyl oleate synthesis using Candida rugosa lipase in a solvent-free system. Role of hydrophobic interactions

The solvent-free esterification reaction of a commercial oleic acid and ethanol was selected as the test reaction for Candida rugosa lipase immobilized on polypropylene (PP) at 318 K (initial molar ratio 1:1). Adding of water from 0 to 30 wt. % (in gram per gram of fatty acid x 100) and the pretreatment of Candida rugosa lipase with polyethylenglycol (PEG), octane, and acetone increases the conversion to ethyl esters. The role of hydrophobic interactions of the lipase with PP and PEG was studied using molecular mechanics (MM2) for calculation of steric energies and the parametrized model (PM3) for calculation of enthalpy changes upon interaction. The nonpolar lateral groups of amino acids interact strongly with PP, whereas polar groups interact more strongly with PEG. Both interactions stabilize the open, active conformation of the lipase from Candida rugosa. Activities ranged from 5 x 10(-5) to 2.0 x 10(-4) mol ethyl oleate/h/mg enzyme, depending on reaction conditions. Steric energy changes vary between +30 and -10 kcal/mol, whereas the enthalpy changes ranged from +10 to -10 kcal/mol.     

Enzymatic resolution to (-)-ormeloxifene intermediates from their racemates using immobilized Candida rugosa lipase

In the synthesis of (-)-ormeloxifene, a drug candidate recently under development, enzymatic resolution of potential intermediates can be carried out using a simple, practical method. Five commercially available lipases, Candida rugosa lipase, Candida antarctica lipase B, Mucor miehei lipase, Pseudomonas cepacia lipase, and Humicola lanuginosa lipase, all immobilized on Accurel(R), were initially screened in combination with four different substrates belonging to the class of phenyl esters. Excellent stereoselectivity was observed using C. rugosa lipase with an acetate as substrate, but low reaction rates were observed in scale-up experiments. However, by changing the acyl part of the ester into a hexanoyl moiety and subjecting this substrate to enzymatic hydrolysis in aqueous acetonitrile at room temperature by C. rugosa lipase, it became possible to run the reaction to a 50% conversion on a 10 g scale within a period of 4 h, obtaining a phenolic product of more than 95% ee that could be converted to the target molecule, (-)-ormeloxifene, in two synthetic steps. Simple recovery of the immobilized enzyme by filtration allowed multiple recycling of the catalyst without significant loss of enzymatic activity. Capillary electrophoresis with sulfobutyl ether beta-cyclodextrin as a chiral buffer additive and acetonitrile as an organic modifier was demonstrated to provide an excellent chiral analytical tool for monitoring the enzymatic reactions.     

Esterification of fatty acids using Candida antarctica lipase A in water-abundant systems

The feasibility of using native lipase A from Candida antarctica (CAL-A) to esterify fatty acids with water-insoluble alcohols in the presence of excess water was investigated in stirred-tank reactors. For high reaction rates, a ratio of water:substrates of 0.6-1.4:1 (v/v) was required. CAL-A showed higher substrate selectivity for the esterification of saturated palmitic acid with branched-chain 2-ethyl-1-hexanol than for unsaturated oleic acid with linear alcohol (1-decanol). After 18 h at 70 °C in a 1.5 l bulk stirred-tank reactor, an 2-ethyl-1-hexyl palmitic acid ester was obtained near 100 % yield [molar ratio palmitic acid:2-ethyl-1-hexanol ~1:1.25, with 1.11 % (w/w) Novocor ADL (based on palmitic acid weight)].     

D301树脂固定化假丝酵母脂肪酶

本研究选择7种吸附和离子交换树脂进行了假丝酵母脂肪酶(Candida sp.lipase)的固定化试验,通过测定固定化后各脂肪酶的酶活,筛选出固定化效果较好的弱碱性阴离子交换树脂D301;并通过扫描电镜将D301与脂肪酶Novozym 435的表面形貌做比较,进一步选定D301树脂作为载体,并对其采用戊二醛交联固定化,研究并优化了其固定化条件。结果表明,5%戊二醛溶液的加入量为8mL,处理时间为5h,酶液浓度为1.0g/L,磷酸缓冲盐溶液pH6.0,固定化处理10h效果最好,获得的固定化酶活力可达35U/mg,酶的固定化效率约为3.5U/(mg·h)。     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

Lysophosphatidylcholine synthesis with Candida antarctica lipase B (Novozym 435)

Immobilized lipase from Candida antarctica lipase B (Novozym 435) was effective in the synthesis of lysophosphatidylcholine (LPC). The transesterification of L-alpha-glycerophosphorylcholine (GPC) and vinyl laurate was carried out in a solvent free system or in the presence of 50% (v/v) t95%) were easily achieved. The lipase was selective for the sn10 times). High purity products could be produced by a decrease of the reaction temperature to induce precipitation of the product. The temperature needed depended on the fatty acid chain length. Thus, only lysophosphatidylcholine was produced with palmitic acid vinyl ester at 45 degrees C, whereas for the vinyl esters of lauric acid, capric acid, and caprylic acid, a lower reaction temperature (25 degrees C) was necessary to obtain solely the lysophospholipid products.     

Regioselective acylation of disaccharides in tert-butyl alcohol catalyzed by Candida antarctica lipase

The acylation of several disaccharides by ethyl butanoate and ethyl dodecanoate was catalyzed by Candida antarctica lipase in tert-butyl alcohol, at temperatures ranging from 40 degrees to 82 degrees C (reflux temperature). The relative reaction rates of the various disaccharides were directly related to their solubility. The primary products were the monoesters derived from acylation of the primary alcohol groups. At higher conversions diesters were formed, and the ratio of diester to monoester was markedly dependent on the structure of the disaccharide. Thus, reaction of maltose with ethyl dodecanoate in refluxing tert-butyl alcohol afforded the 6'-monododecanoate even at high conversions. Trehalose, in contrast, afforded the 6,6'-diester. Acylation of the less soluble sucrose and lactose was much slower, but a moderate (37%) conversion of sucrose was observed after a prolonged reaction time (7 days). A number of other lipases and proteases were tested but C. antarctica lipase was unique in catalyzing the acylation of sucrose in refluxing tert-butyl alcohol. (c) 1996 John Wiley & Sons, Inc.     

Alcohol inhibition and specificity studies of lipase B from candida antarctica in organic solvents

Alcohol inhibition of the lipase B from Candida antarctica has been studied through two different approaches: using the same inhibitor (1-butanol) in different organic solvents and using different inhibitors (differing in chain length) in the same solvent. The competitive inhibition constant values obtained in each case correlate with the calculated activity coefficients of the substrate, suggesting that desolvation of the alcohol is the major force changed. Data dispersion observed using the second approach has been interpreted to come from contributions of enzyme-inhibitor interactions to the binding energy. On the other hand, deacylation has been found to be much less influenced by the solvent variation than the acylation step, despite of the fact that solvation of the substrate involved in this step (the alcohol) is expected to change more than for the ester. Concerning the specificity behavior of the enzyme, a bimodal pattern was observed for the deacylation rate dependence on the alcohol chain length, with the highest values for hexanol (C6) and decanol (C10). With regard to the ester specificity, ethyl caproate (C6) is the preferred one. These results have been confronted with those reported for the lipase from Candida rugosa. Copyright 1998 John Wiley & Sons, Inc.     

One-step enzyme extraction and immobilization for biocatalysis applications

An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.     

Lipase-catalyzed esterification of unusual substrates: Synthesis of glucuronic acid and ascorbic acid (vitamin C) esters

The synthesis of n-butanol and cinnamic alcohol esters of glucuronic acid and the esterification of ascorbic acid (vitamin C) with phenylbutyric acid was performed with lipase from Candida antarctica B (CAL-B, SP435) in a mainly solid-phase system. Products were obtained in 15 to 22 % yield. Computer modelling based on the structure of CAL-B was used to elucidate the access of glucuronic acid to the catalytic site of the lipase. A fixation of glucuronic acid via H-bonds to Q157, D134 and H224 during the transition state was observed. © Rapid Science Ltd. 1998     

New differences between isoenzymes A and B from Candida rugosa lipase

Water adsorption isotherms of pure lipases A and B from Candida rugosa are different and can be used to distinguish between the isoenzymes. The maximum esterification yield (50%, 20h) can be achieved at initial 0.9<1.0. Lipase B is more stereoselective (49% yield, 98% enantiomeri excess) than lipase A (47% yield, 72% enantiomerci excess) but both isoenzymes mainly esterify the (S) 2(4-isobutylphenyl)propionic acid (Ibuprofen).     

Effects of acid concentration and solvent choice on enzymatic acrylation by Candida antarctica lipase B

Lipase-mediated acrylation is an attractive alternative to more traditional chemical processes, since it provides specific catalysis under mild conditions. A detailed study of the effects of solvent choice and substrate concentrations on the acrylation of octanol by Candida antarctica lipase B (Novozym 435) is presented. Acrylic acid was found to have a pronounced inhibitory effect. Partial neutralisation of the acid substrate by addition of an organo-soluble base markedly altered the activity profile, indicating the inhibitory mechanism to be related to acid-base interactions. The concentration of acrylic acid to be employed was found to be important in the choice of an appropriate solvent. At low acrylic acid concentrations, the highest rates and conversions were obtained using hydrophobic solvents, whereas at higher acrylic acid concentrations more polar solvents were advantageous.     

Novel lipase-catalysed highly selective acetylation studies on D-arabino- and D-threo-polyhydroxyalkyltriazoles

Capabilities of lipases from Candida antarctica, Candida rugosa and porcine pancreas have been evaluated for regioselective acetylation of 2-phenyl-4-(D-arabino-tetrahydroxybutyl)-2H-1,2,3-triazole, 2-phenyl-4-(D-arabino-O-1',2'-isopropylidene-3',4'-dihydroxybutyl)-2H-1,2,3-triazole and 2-phenyl-4-(D-threo-trihydroxypropyl)-2H-1,2,3-triazole, precursors for the synthesis of triazolylacyclonucleosides. C. antarctica lipase and porcine pancreatic lipase exhibited exclusive selectivity for the acetylation of primary hydroxyl group over secondary hydroxyl group(s) in all the three cases.     

Enzymatic procedures in the preparation of regioprotected D-fructose derivatives

A combination of different lipases from Pseudomonas cepacia, Candida antarctica B, Candida rugosa and Mucor miehei, aided the regioesterification of the free fructose allowing the synthesis of 1,6-di-O-acetyl-D-fructofuranose, 1,4,6-tri-O-acetyl-D-fructofuranose, 1,6-di-O-acetyl-4-O-benzoyl-D-fructofuranose and 1,6-di-O-benzoyl-D-fructofuranose. Using C. antarctica B and C. rugosa lipases the alcoholysis of fructose peracetate (alpha, beta-form) has furnished 1,2,3,4-tetra-O-acetyl-alpha-D-fructofuranose and 2,3,4,6-tetra-O-acetyl-beta-D-fructofuranose. 1,4,6-Tri-O-acetyl-D-fructofuranose was successfully employed to produce a rare ketohexose, namely D-psicose.     

Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a cellulose-binding domain

Candida antarctica lipase B (CALB) and C. antarctica lipase B fused to a cellulose-binding domain (CBD-CALB) were expressed functionally in the methylotrophic yeast Pichia pastoris. The cellulose-binding domain originates from cellulase A of the anaerobic rumen fungus Neocallimastix patriciarum. The genes were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. The recombinant proteins were secreted into the culture medium reaching levels of approximately 25 mg/L. The proteins were purified using hydrophobic interaction chromatography and gel filtration with an overall yield of 69%. Results from endoglycosidase H digestion of the proteins showed that CALB and CBD-CALB were N-glycosylated. The specific hydrolytic activities of recombinant CALB and CBD-CALB were identical to that reported for CALB isolated from its native source. The fusion of the CBD to the lipase resulted in a greatly enhanced binding toward cellulose for CBD-CALB compared with that for CALB.     

硬脂酸和月桂酸在Novozym 435催化下对芦丁酯化及分子筛对酯化率的影响

为了增加芦丁的脂溶性从而使其具有更优秀的抗氧化活性,以硬脂酸和月桂酸为酰基供体,在脂肪酶Novozym 435催化下对芦丁选择性酯化.经色谱柱提纯,得到两种带不同长度烃基的芦丁脂肪酸酯.用红外光谱和核磁共振波谱对芦丁硬脂酸进行了结构鉴定,表明该类酯化物的酯化反应位为鼠李糖的C4′″位羟基.以高效液相色谱监测酯化反应进程,分子筛添加时间对酯化率的研究结果显示,分子筛对酯化率和反应速率有提高的作用.分子筛添加时间对酯化率有影响.对于硬脂酸为酰基供体的情况,反应24h后添加分子筛的酯化反应可以得到最大的酯化转化率46%.以月桂酸为酰基供体的酯化反应,反应11 h后添加分子筛可以得到最大的酯化转化率64.5%.     

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity

The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.     

Treatment of Candida Rugosa Lipase with Short-Chain Polar Organic Solvents Enhances its Hydrolytic and Synthetic Activities

Following a simple and quick treatment based on dissolving the crude lipase from Candida rugosa in different percentages (v/v) of several polar organic solvents (methanol, ethanol, 1 and 2-propanol, 1 and 2-butanol and acetone) followed by dialysis, different preparations with enhanced activities were obtained. The opening of the lid covering the active site is proposed as the reason for explaining the activity enhancement, both in aqueous and anhydrous organic media.     

Enzymatic synthesis of unsaturated fatty acid glucoside esters for dermo-cosmetic applications.

Unsaturated fatty acid alpha-butylglucoside esters were prepared by enzymatic esterification of alpha-butylglucoside in nonaqueous media. Conditions were firstly optimized using oleic acid as acyl group. Synthesis was possible in several solvents but the presence of water co-product in the medium limited the reaction to a thermodynamic equilibrium corresponding to a maximal conversion yield of 62%. In pure molten substrates, the removal of water under reduced pressure enabled yields superior to 95% to be obtained. Product profiles depended on enzyme origin : whatever the support, immobilized lipase B from Candida antarctica proved to be far more regioselective for the primary hydroxyl group of glucose than immobilized lipase from Rhizomucor miehei. Results obtained could be easily transposed to the acylation of alpha-butylglucoside with a commercial mixture of unsaturated fatty acids containing more than 60% of linoleic acid. The biocatalyst could be recycled more than ten times without any significant activity loss.