A stable serine protease, wrightin, from the latex of the plant Wrightia tinctoria (Roxb.) R. Br.: purification and biochemical properties

Today proteases have become an integral part of the food and feed industry, and plant latex could be a potential source of novel proteases with unique substrate specificities and biochemical properties. A new protease named "wrightin" is purified from the latex of the plant Wrightia tinctoria (Family Apocynaceae) by cation-exchange chromatography. The enzyme is a monomer having a molecular mass of 57.9 kDa (MALDI-TOF), an isoelectric point of 6.0, and an extinction coefficient (epsilon1%280) of 36.4. Optimum activity is achieved at a pH of 7.5-10 and a temperature of 70 degrees C. Wrightin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity; for example, the Km value is 50 microM for casein as substrate. Wrightin showed weak amidolytic activity toward L-Ala-Ala-p-nitroanilide but completely failed to hydrolyze N-alpha-benzoyl- DL-arginine-p-nitroanilide (BAPNA), a preferred substrate for trypsin-like enzymes. Complete inhibition of enzyme activity by serine protease inhibitors such as PMSF and DFP indicates that the enzyme belongs to the serine protease class. The enzyme was not inhibited by SBTI and resists autodigestion. Wrightin is remarkably thermostable, retaining complete activity at 70 degrees C after 60 min of incubation and 74% of activity after 30 min of incubation at 80 degrees. Besides, the enzyme is very stable over a broad range of pH from 5.0 to 11.5 and remains active in the presence of various denaturants, surfactants, organic solvents, and metal ions. Thus, wrightin might be a potential candidate for various applications in the food and biotechnological industries, especially in operations requiring high temperatures.     

Purification and characterization of a stable cysteine protease ervatamin B, with two disulfide bridges, from the latex of Ervatamia coronaria

Latex of the medicinal plant Ervatamia coronaria was found to contain at least three cysteine proteases with high proteolytic activity, called ervatamins. One of these proteases, named ervatamin B, has been purified to homogeneity using ion-exchange chromatography and crystallization. The molecular mass of the enzyme was estimated to be 26 000 Da by SDS-PAGE and gel filtration. The extinction coefficient (epsilon(1%)(280 nm)) of the enzyme was 20.5 with 7 tryptophan and 10 tyrosine residues per molecule. The enzyme hydrolyzed denatured natural substrates such as casein, azoalbumin, and azocasein with a high specific activity. In addition, it showed amidolytic activity toward N-succinyl-alanine-alanine-alanine-p-nitroanilide with an apparent K(m) and K(cat) of 6.6 +/- 0.5 mM and 1.87 x 10(2) s(-)(1), respectively. The pH optima was 6.0-6.5 with azocasein as substrate and 7.0-7.5 with azoalbumin as substrate. The temperature optimum was around 50-55 degrees C. The enzyme was basic with an isoelectric point of 9.35 and had no carbohydrate content. Both the proteolytic and amidolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. Interestingly, the enzyme had only two disulfide bridges versus three as in most plant cysteine proteases of the papain superfamily. The enzyme was relatively stable toward pH, denaturants, temperature, and organic solvents. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and typical color in ELISA. Other related proteases do not cross-react with the antisera to ervatamin B showing that the enzyme is immunologically distinct. The N-terminal sequence showed conserved amino acid residues and considerable similarity to typical plant cysteine proteases.     

A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria

A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.     

Carnein, a serine protease from noxious plant weed Ipomoea carnea (morning glory)

A new serine protease from the latex of Ipomoea carnea spp. fistulosa (Morning glory), belonging to the Convolvulaceae family, was purified to homogeneity by ammonium sulfate fractionation followed by cation exchange chromatography. The enzyme, named carnein, has a molecular mass of 80.24 kDa (matrix-assisted laser desorption/ionization time-of-flight) and an isoelectric point of pH 5.6. The pH and temperature optima for proteolytic activity were 6.5 and 65 degrees C, respectively. The extinction coefficient (epsilon2801%) of the enzyme was estimated as 37.12, and the protein molecule consists of 35 tryptophan, 76 tyrosine, and seven cysteine residues. The effect of several inhibitors such as iodoacetic acid, diisopropylfluorophosphate, phenyl-methanesulfonyl fluoride, chymostatin, soybean trypsin inhibitor, HgCl2, 3S-3-(N-{(S)-1-[N-(4-guanidinobutyl)carbamoyl]3-ethylbutyl}carbamoyl)oxirane-2-carboxylic acid, N-ethyl maleimide, ethylene glycol-bis(alpha-amino ethyl ether)tetraacetic acid, ethylenediamminetetraacetic acid, and o-phenonthroline indicates that carnein belongs to the family of serine proteases. The enzyme is not prone to autolysis even at very low concentrations. The N-terminal sequence of carnein (T-T-H-S-P-E-F-L-G-L-A-E-S-S-G-L-X-P-N-S) exhibited considerable similarity to those of other plant serine proteases; the highest similarity was with alnus AG12, one of the subtilase family endopepetidases.     

Proteolytic activities in dormant rye (Secale cereale L.) grain.

Endoproteolytic, exoproteolytic, carboxypeptidase, aminopeptidase, and N-alpha-benzoyl-arginine-p-nitroanilide hydrolyzing activities were detected in 0.05 M sodium acetate buffer (pH 5.0) extracts of whole meal of the rye (Secale cereale L.) varieties Amando, Halo, and Humbolt. The proteolytic enzymes of Humbolt, the variety with the highest proteolytic activity, optimally hydrolyzed hemoglobin around pH 3.5 and 40-45 degrees C. In the different milling fractions of Humbolt, azocasein and hemoglobin hydrolytic activities were especially found in the bran and shorts. Proteolytic enzymes in the bran extract were concentrated in the 35-60% ammonium sulfate precipitate. Pepstatin A, an inhibitor of aspartic proteases, reduced approximately 88 and approximately 75% of the hemoglobin and azocasein hydrolyzing activities of this precipitate, respectively. Phenylmethanesulfonyl fluoride, an inhibitor of serine proteases, inhibited approximately 33% of both cited activities. Both rye and wheat storage proteins were degraded by Humbolt rye whole meal enzyme extract and the above-mentioned ammonium sulfate rye bran fraction in vitro. With the latter fraction digestion was more pronounced.     

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous)

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.     

Characterization of the proteases involved in gel weakening of beef heart surimi.

The goal of this study was to elucidate the nature and characteristics of the proteases involved in gel weakening of beef heart surimi. Acidic (E1) and neutral (E2) protease extracts were prepared from the surimi. The major active components in E1 were found to be cathepsins B and L. E1 exhibited optimum activity to hydrolyze substrates specific to cathepsins B and B+L at 50 degrees C and pH 5.5. At pH 6.0, it retained approximately 50% of its maximum activity. The catheptic activity of E1 was inhibited almost completely by E-64 and leupeptin. The active component in E2 was unidentified and was not inhibited by cysteine or serine protease inhibitors. However, beef plasma powder effectively inhibited the hydrolysis of FITC-casein and myosin heavy chain by E2.     

Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.     

Kinetic study of the activation process of a latent mushroom (Agaricus bisporus) tyrosinase by serine proteases.

Latent mushroom tyrosinase can be considered as a zymogen when activated by proteases because the activation process fulfilled all of the kinetic dependencies predicted by a theoretical zymogen activation model previously reported. The activation was studied under two assay conditions: high and low ratio of latent tyrosinase/serine protease (trypsin and subtilisin Carlsberg) concentrations, in the presence and in the absence of a serine protease inhibitor (aprotinin). The size of the latent enzyme was 67 kDa, determined by denaturing SDS-PAGE electrophoresis and Western blot assays. After proteolytic activation, the size was 43 kDa, with an intermediate band of 58 kDa. The values of the catalytic () and Michaelis () constants for the active forms of tyrosinase resulting from the activation by subtilisin, trypsin, or sodium dodecyl sulfate on the substrate tert-butylcatechol were slightly different, which could support the idea of "one activator-one different active tyrosinase". Vacuum infiltration experiments tried to reproduce in vivo the role of mushroom serine proteases in the activation of latent tyrosinase. The use of serine protease inhibitors is proposed as a new alternative tool to prevent melanin formation.     

Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.     

Purification of balansain I, an endopeptidase from unripe fruits of Bromelia balansae Mez (Bromeliaceae)

A new plant endopeptidase was obtained from unripe fruits of Bromelia balansae Mez (Bromeliaceae). Crude extracts were partially purified by ethanol fractionation. This preparation (redissolved ethanol precipitate, REP) showed maximum activity at pH 8.8-9.2, was very stable even at high ionic strength values (no appreciable decrease in proteolytic activity could be detected after 24 h in 1 M sodium chloride solution at 37 degrees C), and exhibited high thermal stability (inactivation required heating for 60 min at 75 degrees C). Anion exchange chromatography allowed the isolation of a fraction purified to mass spectroscopy, SDS-PAGE, and IEF homogeneity, named balansain I, with pI = 5.45 and molecular mass = 23192 (mass spectrometry). The purification factor is low (2.9-fold), but the yield is high (48.3%), a common occurrence in plant organs with high proteolytic activity, where proteases represent the bulk of protein content of crude extracts. Balansain I exhibits a similar but narrower pH profile than that obtained for REP, with a maximum pH value approximately 9.0 and was inhibited by E-64 and other cysteine peptidases inhibitors but not affected by inhibitors of the other catalytic types of peptidases. The alanine and glutamine derivatives of N-alpha-carbobenzoxy-L-amino acid p-nitrophenyl esters was strongly preferred by the enzyme.The N-terminal sequence of balansain I showed a very high homology (85-90%) with other known Bromeliaceae endopeptidases.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Steady-state kinetics and thermodynamics of the hydrolysis of beta-lactoglobulin by trypsin

Hydrolysis of beta-lactoglobulin (in an equimolar mixture of the A and B variant) by trypsin in neutral aqueous solution [pH 7.7 at 25 degrees C, ionic strength 0.08 (NaCl)] was followed by capillary electrophoresis and thermodynamic parameters derived from a Michaelis-Menten analysis of rate data obtained at 10, 20, 30, and 40 degrees C for disappearance of beta-lactoglobulin. Enthalpy of substrate binding to the enzyme and the energy of activation for the catalytic process were found to have the values, DeltaH(bind) = -28 +/- 4 kJ mol(-)(1) and E(a) = 51 +/- 18 kJ mol(-)(1), respectively. Thus, beta-lactoglobulin shows an enthalpy of activation for free substrate reacting with free enzyme of about 21 kJ mol(-)(1), corresponding to a transition state stabilization of 60 kJ mol(-)(1) when compared to acid-catalyzed hydrolysis. The catalytic efficiency of trypsin in hydrolysis of beta-lactoglobulin is increased significantly by temperature; however, this effect is partly counteracted by a weaker substrate binding resulting in an increase by only 25%/10 degrees C in overall catalytic efficiency.     

Inactivation of heat-resistant pectinmethylesterase from orange by manothermosonication

Pectinmethylesterase of navel oranges shows two fractions greatly differing in thermostability. The most thermostable fraction accounts for approximately 10% of total activity. The thermal inactivation of this fraction follows first-order kinetics both in 5 mM, pH 3.5, citrate buffer and in orange juice at the same pH, showing a z value of 5.1 degrees C and an activation energy (E(a)) of 435 kJ mol(-)(1) K(-)(1). The heat resistance of the enzyme is approximately 25-fold higher in the juice than in citrate buffer. When ascorbic acid, sucrose, glucose, and fructose are added to the citrate buffer at the concentrations found in orange juice, the heat resistance of the enzyme increases 3-fold. The addition of pectin at 0.01% concentration multiplies it by a factor of 50. Manothermosonication (MTS), the simultaneous application of heat and ultrasound under moderate pressure (200 kPa), at 72 degrees C, increases the inactivation rate 25 times in buffer and >400 times in orange juice. MTS inactivation shows a higher z value (35.7 degrees C) and lower E(a) (56.9 kJ mol(-)(1) K(-)(1)) than simple heating.     

Protease-assisted clarification of black currant juice: synergy with other clarifying agents and effects on the phenol content

Conventional clarification with gelatin and silica sol removes a considerable amount of antioxidant phenolics from berry juices. This study examined the clarification and haze-diminishing effects of alternative clarification strategies on black currant juice including centrifugation and addition of acidic protease and pectinolytic enzyme preparations and gallic acid. Centrifugation of freshly pressed juice (10,000 g for 15 min) resulted in a approximately 95% reduction of immediate turbidity and had a decreasing effect on haze development in the juice during cold storage without significantly compromising the total phenols levels. The extent of clarification and haze diminishment varied after individual treatments with five different acidic proteases, but one of the protease preparations, Enzeco, derived from Aspergillus niger, consistently tended to perform best. The individual and interactive effects on juice turbidity, total phenols, and total anthocyanin contents of clarification treatments involving the use of two selected acid proteases (Enzeco and Novozyme 89L), a pectinase (Pectinex BE 3-L), and gallic acid were evaluated in a full factorial 2(4) experimental design. Haze development during cold storage decreased when gallic acid or any of the enzyme preparations were employed individually, but negative interaction effects resulted when the pectinase was employed in combination with any of the proteases. After 28 storage days at 2 degrees C, the lowest levels of haze formation were achieved when the Enzeco protease preparation, added at 0.025 g/L, was added with 0.050 g/L of gallic acid and allowed to react in the juice for 90 min at 50 degrees C. The corresponding anthocyanin reduction was approximately 12% (compared to approximately 30% with gelatin silica sol treatment). The data support the hypothesis that phenol-protein interactions are involved in juice turbidity development during cold storage of berry juices and demonstrate that precentrifugation and protease-assisted clarification show promise as an alternative, phenolics-retaining clarification strategy in black currant juice processing.     

Tentative assignment of the potato serine protease inhibitor group as beta-II proteins based on their spectroscopic characteristics

Potato serine protease inhibitor (PSPI) is the most abundant protease inhibitor group in potato tuber. The investigated PSPI isoforms have a highly similar structure at both the secondary and the tertiary level. From the results described, PSPI is classified as a beta-II protein based on (1) the presence in the near-UV spectra of sharp peaks, indicating a rigid and compact protein; (2) the sharp transition from the native to the unfolded state upon heating (only 6 degrees C) monitored by a circular dichroism signal at 222 nm; and (3) the similarity in secondary structure to soybean trypsin inhibitor, a known beta-II protein, as indicated by a similar far-UV CD spectrum and a similar amide I band in the IR spectrum. The conformation of PSPI was shown also to be stable at ambient temperature in the pH range 4-7.5. Upon lowering the pH to 3.0, some minor changes in the protein core occur, as observed from the increase of the intensity of the phenylalanine peak in the near-UV CD spectrum.     

Enzymatic digestion-high-pressure homogenization prior to slurry introduction graphite furnace atomic absorption spectrometry for selenium determination in plant and animal tissues.

Homogenization using a new flat valve homogenizer in combination with enzymatic digestion with a crude protease was investigated as a means of releasing Se compounds from zoological and botanical matrixes prior to slurry introduction GFAAS. Timed trials with four zoological certified reference materials (CRMs), three botanical reference materials (RMs), and a food crop indicated that Se release into 5% (v/v) ethanol-0.03 M TRIS containing 20 mg of protease was quantitative after homogenization or became quantitative within 1 h of digestion at 60 degrees C. For each of the zoological RMs (whole egg powder, dogfish muscle, and dogfish liver), three passes through the homogenizer in the presence of protease provided a quantitative release of selenium, and incubation with the enzyme was not necessary. No separation of the Se between the liquid phase and the particulate phase was evident even after several days of subsequent storage at 4 degrees C. The botanical matrixes (three milled wheat RMs and a rapeseed sample) were more resistant to selenium release and required up to 1 h of digestion with protease at 60 degrees C. Alternatively, 10 passes through the homogenizing valve (in the presence of the enzyme) resulted in the quantitative release of analyte.     

Application potency of engineered G159 mutants on P1 substrate pocket of subtilisin YaB as improved meat tenderizers

A serine protease, subtilisin YaB, produced by alkalophilic Bacillus YaB, shows promises as a potent meat tenderizer, because its substrate specificity is for small amino acids, which are found at high levels in meat connective tissue proteins. Substrate specificity engineering of the substrate binding pockets was used to generate more suitable meat-tenderizing mutants, G124A, G124V, G159A, and G159S, derived from recombinant wild subtilisin YaB and expressed in Bacillus subtilis DB104. The characteristics of these recombinant enzymes were studied to evaluate their usefulness as improved meat tenderizers. The proteolytic activities of recombinant subtilisin YaB, engineered subtilisin YaBs, and commercially available papain, bromelain, collagenase, and elastase were compared using elastin, collagen, casein, and myofibrillar proteins as substrates. Hydrolysis of beef proteins was evaluated using the myofibrillar fragmentation index and collagen solubility. The results demonstrated that recombinant mutant G159A was the most improved meat tenderizer and can be used in the meat pH range of 5.5-6.0 and the temperature range of 10-50 degrees C. Contrary to the result obtained from artificial substrate, mutant enzymes engineered on G124 residues did not exhibit better tenderizing ability when elastin, collagen, or meat was used as substrate, suggesting the necessity of evaluation by real substrate before protein-engineered enzymes are applied commercially.     

Kinetics and hydrolysis of fenamiphos, fipronil, and trifluralin in aqueous buffer solutions.

Hydrolyses of fenamiphos, fipronil, and trifluralin were studied in aqueous buffer solutions of pH 4.1, 7.1, and 9.1 at different temperatures, 5, 22 +/- 1, 32 +/- 1, and 50 +/- 1 degrees C. Fenamiphos, fipronil, and trifluralin were found to be more stable in acidic and neutral buffer solutions at temperatures of 5 and 22 +/- 1, and dissipation is rapid at 50 +/- 1 degrees C. In basic buffer and at higher temperature, degradation of fenamiphos was found to be very rapid when compared with fipronil and trifluralin. The rate constants calculated at 32 degrees C for fenamiphos were 2349.4 x 10(-)(8) (pH 4.1), 225.2 x 10(-)(8) (pH 7.1), and 30476.0 x 10(-)(8) (pH 9.1); for fipronil 1750.0 x 10(-)(8) (pH 4.1), 3103.0 x 10(-)(8) (pH 7.1), and 3883.0 x 10(-)(8) (pH 9.1); and for trifluralin 2331.0 x 10(-)(8) (pH 4.1), 2360.0 x 10(-)(8) (pH 7.1), and 3188.0 x 10(-)(8) (pH 9.1). On the basis of rate constant values, these pesticides appeared to be more susceptible to hydrolysis than synthetic organophosphorus compounds such as chlorpyriphos, diazinon, malathion, and ronnel. DT(50) values calculated at 32 degrees C were 228 (pH 4.1), 5310.24 (pH 7.1), and 37.68 (pH 9.1) h for fenamiphos; 608.6 (pH 4.1), 373.9 (pH 7.1), and 270.2 (pH 9.1) h for fipronil; and 502.1 (pH 4.1), 496.8 (pH 7.1), and 355.7 (pH 9.1) h for trifluralin.     

Optimal formation of hexyl laurate by Lipozyme IM-77 in solvent-free system

A medium-chain ester, hexyl laurate, with fruity flavor is primarily used in personal care formulations as an important emollient for cosmetic applications. To conform to the "natural" interests of consumers, the ability of immobilized lipase from Rhizomucor miehei (Lipozyme IM-77) to catalyze the direct esterification of hexanol and lauric acid by using a solvent-free system was investigated in this study. Response surface methodology (RSM) and four-factor-five-level central composite rotatable design (CCRD) were employed to evaluate the effects of synthesis parameters, such as reaction time (10-50 min), temperature (45-85 degrees C), lipase amount (10-30 mg/volume; 0.077-0.231 batch acidolysis units of Novo (BAUN), and pH memory (5-9), on percentage molar conversion of hexyl laurate by lipase-catalyzed direct esterification. Reaction time, temperature, and enzyme amount had significant effects on percent molar conversion. On the basis of ridge maximum analysis, the optimum synthesis conditions for hexyl laurate were a reaction time of 40.6 min, a temperature of 58.2 degrees C, an enzyme amount of 25.4 mg/volume (0.196 BAUN), and a pH memory of 5.9. The predicted percentage molar conversion of hexyl laurate was 69.7 +/- 1.4%.