A comparison of lipase-catalysed ester and lactone synthesis in low-water systems: analysis of optimum water activity.

We investigated the effects of the lyophilisation medium (enzyme plus buffer salt and additives) and of water activity (a(w)) on the catalytic properties of lipase from Chromobacterium viscosum (lipase CV) in organic solvents; catalysis of ester and lactone synthesis were compared and, despite the similarities of the reactive groups involved in these reactions, some interesting differences were observed. Including 2-[N-morpholino]ethanesulfonic acid (MES) buffer in the lyophilisation medium of lipase CV increased its catalytic activity in transesterification and lactonisation, although the buffer salt requirement for maximal activity differed between the two reactions. Sorbitol, glucose, lactose, 18-crown-6 (crown ether 18-C-6), beta-cyclodextrin and bovine serum albumin were employed as alternative additives in the transesterification reaction, but were not as effective as MES buffer. Salt hydrates were used to investigate the effect of a(w) on esterification and lactonisation reactions catalysed by lipase CV. The maximum rate of hexadecanolide synthesis in toluene occurred at a(w) = 0.48. The optimum a(w) for the transesterification reaction in heptane/alcohol mixtures depended on the alcohol substrate employed (1-heptanol, 2-heptanol, or 3-methyl-3-hexanol) but not on the acyl donor (p-NP acetate or caprylate). The optimum a(w) values for both reactions were unchanged when a common solvent system (toluene/1-heptanol) was employed, indicating that the dependence of enzyme activity on a(w) is an intrinsic property of the enzyme-catalysed reaction and not a function of the solvent or other additives.     

有机溶剂中固定化脂肪酶催化硅醇的酯化反应

固定化Mucor miehei脂肪酶可催化有机硅醇和脂肪酸的酯化反应．对固定化酶用量、脂肪酸链长、不同有机硅醇底物、有机溶剂极性和水含量等影响因素进行了初步研究．     

底物中的硅原子对酶反应的影响

在酶工程学的研究史上,人们一方面不断地研制开发新的酶种;一方面利用固定化、酶分子改造和修饰等技术来提高酶的活性和稳定性;另一方面,则不断地开拓酶的新用途。酶催化非天然化合物的生物合成和转化（正是这一方面研究的新进展）。由于有机硅化合物在有机合成,尤其...     

Statistical Design for Formulation Optimization of Hydrocortisone Butyrate-Loaded PLGA Nanoparticles

The aim of this investigation was to develop hydrocortisone butyrate (HB)-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NP) with ideal encapsulation efficiency (EE), particle size, and drug loading (DL) under emulsion solvent evaporation technique utilizing various experimental statistical design modules. Experimental designs were used to investigate specific effects of independent variables during preparation of HB-loaded PLGA NP and corresponding responses in optimizing the formulation. Plackett–Burman design for independent variables was first conducted to prescreen various formulation and process variables during the development of NP. Selected primary variables were further optimized by central composite design. This process leads to an optimum formulation with desired EE, particle size, and DL. Contour plots and response surface curves display visual diagrammatic relationships between the experimental responses and input variables. The concentration of PLGA, drug, and polyvinyl alcohol and sonication time were the critical factors influencing the responses analyzed. Optimized formulation showed EE of 90.6%, particle size of 164.3 nm, and DL of 64.35%. This study demonstrates that statistical experimental design methodology can optimize the formulation and process variables to achieve favorable responses for HB-loaded NP.     

Optimization of chitinase production by a new <Emphasis Type="Italic">Streptomyces griseorubens</Emphasis> C9 isolate using response surface methodology

The purpose of this article is to use statistical Plackett–Burman and Box–Wilson response surface methodology to optimize the medium components and, thus, improve chitinase production by Streptomyces griseorubens C9. This strain was previously isolated and identified from a semi-arid soil of Laghouat region (Algeria). First, syrup of date, colloidal chitin, yeast extract and K₂HPO₄, KH₂PO₄ were proved to have significant effects on chitinase activity using the Plackett–Burman design. Then, an optimal medium was obtained by a Box–Wilson factorial design of response surface methodology in liquid culture. Maximum chitinase production was predicted in medium containing 2% colloidal chitin, 0.47% syrup of date, 0.25 g/l yeast extract and 1.81 g/l K₂HPO₄, KH₂PO₄ using response surface plots of the STATISTICA software v.12.0.     

Optimization and kinetic study of immobilized lipase-catalyzed synthesis of ethyl lactate

Abstract

Enzymatic synthesis of ethyl lactate catalyzed by immobilized lipase has been investigated. The reaction variables (including the molar ratio of ethanol to acid, total substrate amount, temperature, reaction time and rotation speed) were selected in accordance with the Plackett–Burman design and were further optimized via response surface methodology. The molar ratio of ethanol to acid, total substrate amount and reaction time were screened out as significant variables for the optimization study. A 20-run, full-factorial, central composite design was used to construct the statistical model and the optimal conditions obtained were as follows: molar ratio of ethanol to acid of 8.3:1, total substrate amount of 0.4 g, reaction time of 26.87 h with temperature of 55°C and rotation speed of 150 rpm. Under the optimal conditions, the yield of ethyl lactate was up to 24.32%; close to the 25.13% obtained using the commercial lipase, Novozym 435. Due to the low cost and simple immobilization process, the lipase prepared in the present work could have great potential in enzymatic applications. Additionally, a kinetic model with inhibition by both ethanol and lactic acid following a ping-pong bi-bi mechanism was proposed.     

Statistical optimization of critical medium components for lipase production from Yarrowia lipolytica (MTCC 35)

Statistical optimization is an effective technique for the investigation of complex processes with minimal number of experimental runs. In this study, statistical approach was used to study the optimization of media components for lipase production from Yarrowia lipolytica MTCC 35. Mahua cake, glucose, MnCl₂ and KH₂PO₄ were screened to be the most significant variables among the nine medium variables that were tested to determine influence on lipase production by Plackett–Burman design. Central Composite Design was used for further optimization of these screened variables for enhanced lipase production. The determination coefficient (R²) value of 0.922 showed that the regression models adequately explain the data variation and represent the actual relationships between the variables and response. The optimum values of investigated variables for the maximum lipase production were 6.0% Mahua cake, 2.0% glucose, 0.2% MnCl₂ and 0.2% KH₂PO₄. The maximum lipase production (9.40 U mL^?1) was obtained under optimal condition.     

Optimization of isoamyl acetate production by using immobilized lipase from Mucor miehei by response surface methodology

Immobilized lipase from Mucor miehei was employed for the esterification of isoamyl alcohol with acetic acid in n-heptane solvent. The important process variables studied were enzyme/substrate (E/S) ratio, alcohol (acid) concentration, and incubation period. Based on Box-Behnken design of experiments, a second order response function was developed. The percentage esterification increased with both E/S ratio and time and decreased with alcohol (acid) concentration. The model indicated optimum conditions for maximum esterification ranging from 20 to 99.6% in the alcohol (acid) concentration range of 0.031 to 0.3 M for a range of E/S ratios 8.33 to 50 g/mol, which were in good agreement with the experimental yields.     

Esterification of lauric acid with lauryl alcohol using cross-linked enzyme crystals: Solvent effect and kinetic study

In this paper, we report a comprehensive kinetic study on esterification of lauric acid with lauryl alcohol catalysed by commercial porcine pancreatic lipase (PPL) in the form of cross-linked enzyme crystals (CLEC) using glutaraldehyde as the cross linker. The stability of the CLEC was better than the immobilized enzyme for practical applications. Comparative studies using six different solvents having hydrophobicity (log p) values ranging from 0.70 to 3.50 revealed that the esterification reaction was favoured in hydrophobic solvents. The kinetics of the esterification reaction conformed with the so-called Ping-Pong-Bi-Bi mechanism with alcohol inhibition.     

Esterification of lauric acid with lauryl alcohol using cross-linked enzyme crystals: Solvent effect and kinetic study

In this paper, we report a comprehensive kinetic study on esterification of lauric acid with lauryl alcohol catalysed by commercial porcine pancreatic lipase (PPL) in the form of cross-linked enzyme crystals (CLEC) using glutaraldehyde as the cross linker. The stability of the CLEC was better than the immobilized enzyme for practical applications. Comparative studies using six different solvents having hydrophobicity (log p) values ranging from 0.70 to 3.50 revealed that the esterification reaction was favoured in hydrophobic solvents. The kinetics of the esterification reaction conformed with the so-called Ping-Pong–Bi-Bi mechanism with alcohol inhibition.     

Optimization of laccase production from a novel strain-Streptomyces psammoticus using response surface methodology

Response surface methodology was employed for the optimization of different nutritional and physical parameters for the production of laccase by the filamentous bacteria Streptomyces psammoticus MTCC 7334 in submerged fermentation. Initial screening of production parameters was performed using a Plackett - Burman design and the variables with statistically significant effects on laccase production were identified. Incubation temperature, incubation period, agitation rate, concentrations of yeast extract, MgSO(4)7H(2)O, and trace elements were found to influence laccase production significantly. These variables were selected for further optimization studies using a Box-Behnken design. The statistical optimization by response surface methodology resulted in a three-fold increase in the production of laccase by S. psammoticus MTCC 7334.     

Optimization of fermentation parameters for the biomass and DHA production of Schizochytrium limacinum OUC88 using response surface methodology

Fermentation parameters for biomass and DHA production of Schizochytrium limacinum OUC88 in a fermenter (working volume 7 L) were optimized using Plackett–Burman and central composite rotatable design. Out of 10 factors studied by Plackett–Burman design, 4 influenced the biomass production significantly. Central composite rotatable design was used to optimize the significant factors and response surface plots were generated. Using these response surface plots and point prediction, optimized values of the factors were determined as follows temperature (°C) 23 °C, aeration rate 1.48 L min⁻¹ L⁻¹, agitation 250 rpm and inoculum cells in mid-exponential phase, the maximum yield of DCW and DHA were 24.1 and 4.7 g L⁻¹, respectively. These predicted values were also verified by validation experiments.     

Characteristics of immobilized lipase on hydrophobic superparamagnetic microspheres to catalyze esterification

A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity (a(w)) 0.5-0.8 or higher. At a(w) 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C(8)-C(12)). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Activities of Candida rugosa lipase and other esterolytic enzymes coated on glass beads and suspended in substrate and water vapor: enzymes in thin liquid films.

Candida rugosa lipase was enzymatically active when coated on glass beads and exposed to mixtures of substrate and water vapor over a range of relative humidities up to 100%. Evidence was obtained for operation of the enzyme in a thin liquid film of concentrated buffer on the surface of the glass beads. Formation of the thin film was associated with hygroscopicity of the buffer used to suspend the enzyme in preparation of the enzyme-coated beads. At some buffer concentrations estimated to be on the bead surface, the enzyme was partially soluble and both soluble and insoluble forms were enzymatically active. The vapor mode of operation over a range of relative humidities had comparatively small effects on kinetic constants for hydrolysis of ethyl acetate, which were also similar to those in phosphate buffer. The extent of reaction occurred in the order hydrolysis greater than alcoholysis greater than ester interchange greater than esterification. Reaction preference between alcoholysis and hydrolysis changed as acyl chain length of substrate increased with C. rugosa lipase, as well as with Rhizopus arrhizus lipase and porcine liver esterase, with details depending on the enzyme. The vapor mode approach has the potential of being used with a wide variety of substrates, as shown by the ability to obtain hydrolysis at 30 degrees C with substrate vapor pressures as low as 0.08 mm Hg and with substrates with boiling points as high as 206 degrees C.     

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728?U?ml^?1), which was followed by gram husk (714?U?ml^?1), mustard cake (680?U?ml^?1), and soybean meal (653?U?ml^?1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020?U?ml^?1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.     

Esterification reactions of lipase in reverse micelles

The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w(0), had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w(0) values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.     

Scanning electron microscopic studies of lipase-catalysed esterification catalysis for the synthesis of stearoyl lactate and p-cresyl laurate

The state of three lipases, two from Rhizomucor miehei and one from porcine pancreas, employed in the esterification reactions leading to the preparation of food additive esters were investigated by scanning electron microscopy (SEM). The lipases employed in the synthesis of stearoyl lactic acid and p-cresyl laurate in 10 ml solvent at 40–60 °C in shake-flask experiments and 150 ml in non-polar solvents at 50–60 °C in bench-scale level experiments were compared. All three lipases, which were subjected to high temperatures and non-polar solvents for a prolonged period of incubation of 72–120 h, showed decrease in the compactness when compared to unused lipase. The presence of buffer preserved the activity and compactness and the absence of the same reduced the amount of enzyme per unit area on the support. R. miehei lipase samples subjected to reaction in presence of 0.0004 ml of 0.1 M buffer/mg enzyme preparation at different pH values (4.0–9.0) showed a decrease in compactness of the enzyme on the surface which correlated to an increase in esterification activity. An increase in volume of buffer (0.0002–0.003 ml/mg enzyme preparation) in the reaction mixture at pH 7.0 showed a decrease in compactness and also a reduction in activity. The studies indicate that a compromise between pH and volume of buffer can lead to variation in the extent of adsorption, distribution and activity, enabling the achievement of maximum conversions in the esterification reactions.     

Statistical Optimization of Culture Conditions for Milk-Clotting Enzyme Production by Bacillus Amyloliquefaciens Using Wheat Bran-An Agro-Industry Waste

In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett–Burman experimental design. Subsequently, optimal conditions were obtained using the Box–Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett–Burman design and Box–Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.