Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Combining the bioimprinting technique with lipase immobilization for interesterification

Lipases were bioimprinted, immobilized and bioimprinted–immobilized (coupled technique), and interesterification activities were compared. Bioimprinted–immobilized enzyme preparations gave 4–6.2-fold activity enhancements compared to solid enzyme preparations supplied by manufacturers. This increase was higher than that of bioimprinting alone and immobilization alone. Solvent-free media were equally effective and water addition caused erasure of bioimprinting. Also, new preparations showed higher thermostability and reusability.     

Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (vol) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% by volume in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased threefold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase by 37%. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.     

Immobilization of lipases for non-aqueous synthesis

Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n-butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.     

Biocatalysis of immobilized chlorophyllase in a ternary micellar system.

The immobilization of chlorophyllase was optimized by physical adsorption on various inorganic supports, including alumina, celite, Dowex-1-chloride, glass beads and silica gel. The enzyme was also immobilized in different media, including water, Tris-HCl buffer solution and a ternary micellar system containing Tris-HCl buffer solution, hexane and surfactant. The highest immobilization efficiency (84.56%) and specific activity (0.34 mumol hydrolyzed chlorophyll mg protein-1 per min) were obtained when chlorophyllase was suspended in Tris-HCl buffer solution and adsorbed onto silica gel. The effect of different ratios of chlorophyllase to the support and the optimum incubation time for the immobilization of chlorophyllase were determined to be 1-4 and 60 min, respectively. The experimental results showed that the optimum pH and temperature for the immobilized chlorophyllase were 8.0 and 35 degrees C, respectively. The use of optimized amounts of selected membrane lipids increased the specific activity of the immobilized chlorophyllase by approximately 50%. The enzyme kinetic studies indicated that the immobilized chlorophyllase showed a higher affinity towards chlorophyll than pheophytin as substrate.     

月桂酸生物印迹对脂肪酶酯化活力的影响

生物印迹是改良酶学特性,扩大脂肪酶工业应用领域的新兴技术。本研究结合溶胶-凝胶脂肪酶固定化工艺,以甲基三甲氧基硅烷（MTMS）和四甲氧基硅烷（TMOS）为前驱体,月桂酸为印迹分子,考察了月桂酸生物印迹对脂肪酶PS酯化活力的影响。脂肪酶酯化活力测定及扫描电镜观察表明生物印迹能显著提高脂肪酶的活性及稳定性。印迹体系经正交试验优化获得的最优条件为：水和硅烷摩尔比（R）为12,聚乙二醇（PEG）加入量为120μl,月桂酸加入量为0.15mmol。在最优反应条件下,印迹酶相对于游离酶比活力提高了44.3倍,相对于未印迹固定化酶提高了2.4倍;印迹酶具有较好的热稳定性,在80℃下处理0.5h后,残余酶活分别为58%,而游离酶未检测到活性。     

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

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

Enzymatic enrichment of polyunsaturated fatty acids using novel lipase preparations modified by combination of immobilization and fish oil treatment

Novel modification methods for lipase biocatalysts effective in hydrolysis of fish oil for enrichment of polyunsaturated fatty acids (PUFAs) were described. Based on conventional immobilization in single aqueous medium, immobilization of lipase in two phase medium composed of buffer and octane was employed. Furthermore, immobilization (in single aqueous or in two phase medium) coupled to fish oil treatment was integrated. Among these, lipase immobilized in two phase medium coupled to fish oil treatment (IMLAOF) had advantages over other modified lipases in initial reaction rate and hydrolysis degree. The hydrolysis degree increased from 12% with the free lipase to 40% with IMLAOF. Strong polar and hydrophobic solvents had negative impact on immobilization-fish oil treatment lipases, while low polar solvents were helpful to maintain the modification effect of immobilization-fish oil treatment. After five cycles of usage, the immobilization-fish oil treatment lipases still maintained more than 80% of relative hydrolysis degree.     

Versatility of glutaraldehyde to immobilize lipases: Effect of the immobilization protocol on the properties of lipase B from Candida antarctica

Glutaraldehyde chemistry has been used to immobilize lipase B from Candida antarctica (CALB) under different situations. Using high ionic strength, ionic adsorption is avoided, but CALB is adsorbed on the support via interfacial activation. Using non-ionic detergents (e.g., Triton X-100), the enzyme becomes ionically adsorbed on the activated support. If detergent and salt are simultaneously present during immobilization, a covalent attachment to the support is first produced. In absence of detergent or high ionic strength, a mixture of all of the previous immobilization reasons should coexist. Thus, 5 different CALB biocatalysts were prepared following the previous described protocols, and its stability and activity, pH/activity profile and specificity versus R and S methyl mandelate were analyzed. The existence of covalent attachment of more than 95% of the enzyme molecules was confirmed by washing the biocatalysts in salt and detergent solutions. The glutaraldehyde treatment of the enzyme adsorbed on aminated supports did not produce a significant improvement on the activity of the enzyme versus p-nitrophenylpropinate (pNPB) nor a high stabilization of the enzyme. This differed from the effects of a similar treatment of CAL adsorbed on octyl agarose. However, they were similar to the effects of this treatment on covalently immobilized CALB, suggesting that the immobilization protocol may greatly affect the final effect of a chemical modification on the enzyme properties.Dramatic changes in the enzyme features were observed comparing the different preparations, mainly in the specificity of CALB versus p-NPB and R-methyl mandelate (from 2.5 to 20), or in the enantiospecificity versus R/S methyl mandelate (from 1.8 to 16), confirming that these different immobilization protocols produced biocatalysts with different features. Moreover, changes in experimental conditions produced very different effects on the properties of the different CALB preparations.     

Combination of bioimprinting and silane precursor alkyls improved the activity of sol–gel-encapsulated lipase

Bioimprinting and sol–gel encapsulation of lipases by silane precursors are efficient methods of enhancing lipase performance in non-aqueous medium. The correlation between bioimprinting, the alkyl-chain length of silane precursors, and the catalytic activity of gel-encapsulated lipase was investigated using a series of silane precursors: methyltrimethoxysilane (MTMS), vinyltrimethoxysilane (VTMOS), vinyltriethoxysilane (VTEOS), and n-octyltrimethoxysilane (OTMOS). The optimal parameters for lipase immobilization were also determined. Both bioimprinting and increasing the chain-length of alkyl groups, apparently by increasing hydrophobicity, significantly improved the specific activity and the total activity of the immobilized lipase. Compared to a non-imprinted MTMS/TMOS gel, the specific activity of an imprinted OTMOS/TMOS gel improved 14.4-fold, and the total activity improved 6.8-fold. Nitrogen adsorption–desorption assays and gel matrix surface characterization showed that the bioimprinting molecule and the hydrophobic alkyl groups of silane triggered lipase to change from the closed to the open conformation, and contributed to creating sol–gel matrices that were more porous and with less mass transfer resistance structure, apparently improving the activity of encapsulated lipase.     

Enhanced Performance of Rhizopus oryzae Lipase Immobilized on Hydrophobic Carriers and Its Application in Biorefinery of Rapeseed Oil Deodorizer Distillate

In this study, hydrophobic macroporous resin NKA was employed as matrix for immobilization of free Rhizopus oryzae lipase (ROL). The performance of the immobilized ROL was significantly enhanced. The recovery activity was up to 1,293.78 % and the specific activity increased to 152,914 U/g-protein, which was 46-fold higher than that of the free lipase. Moreover, the immobilized lipase showed higher thermostability and better pH-resistance than its free counterpart. Additionally, three different nonaqueous modification strategies (including bioimprinting, lecithin coating, and lyophilization protection) were further utilized to improve the performance of the immobilized lipase. The corresponding enhancements were 33.68 %, 31.98 %, and 99.86 %. When these modifications were combined together, the activity improved 209.51 %. In order to confirm its practical application, the modified ROL was used to biorefine rapeseed oil deodorizer distillate (RODD) for biodiesel production. The highest conversion yield reached 98.23 %, much close to that (97.46 %) of Novozym 435. The results suggest that the prepared lipase in this study is a promising biocatalyst with high stability, efficiency and operational reusability.     

Improving esterification activity of Burkholderia cepacia lipase encapsulated in silica by bioimprinting with substrate analogues

Bioimprinting is a promising, though relatively unexplored, approach to improving the performance of enzymes. In this study, bioimprinting with substrate analogues of fatty acids was systematically conducted to improve the esterification activity of Burkholderia cepacia lipase that had undergone a sol–gel immobilization procedure with methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS) as the precursors. The specific activity of the bioimprinted lipases was 3682.0 μmol h^?1 mg protein, which was a 47.9- and 2.5-fold increase over the free and non-imprinted immobilized lipases, respectively. Compared to the free and non-imprinted immobilized lipases, bioimprinted lipases exhibited better thermal stability, and their activity did not change after being incubated at 60 °C for 12 h. Bioimprinted lipases were more easily affected by alcohol than the non-imprinted ones, whose specific activity could be markedly enhanced by ethanol, isopropanol and n-butanol by factors of 1.23-, 1.28- and 1.12-fold, respectively. The reasons for the improvement of imprinted enzyme activity are also discussed based on the surface structure, specific surface area and average pore diameter of the silane particles.     

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO₃ (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K₂HPO₄ and 0.06% (w/v) MgSO₄. It was possible to increase the productivity of dextranase to 41.8 units ml⁻¹ in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.     

Optimizing Immobilization and Stabilization of Feruloyl Esterase from Humicola Insolens and its Application for the Feruloylation of Oligosaccharides

Feruloyl esterase (FAE)-catalyzed esterification reaction is as a potential route for the biosynthesis of feruloylated oligosaccharides as functional ingredients. Immobilization of FAE from Humicola insolens on metal chelate-epoxy supports was investigated. The study of effects of immobilization parameters using response surface methodology revealed the significance of enzyme/support ratio (3.25-29.25 mg/g support), immobilization time (14-38 h), buffer molarity (0.27-1.25 M) and pH (4.0-8.0). The interactions between enzyme-to-support ratio/buffer molarity and enzyme-to-support ratio/pH were found to be critical for the modulation of the immobilization activity yield and the retention of specific activity, respectively. Optimum conditions for FAE-immobilization on metal chelate Sepabeads® EC-EP R were identified to be 22.75 mg FAE/g support, pH of 5.0, 27.7 h and buffer molarity of 0.86 M. At these conditions, an activity yield of 82.4%, a specific activity retention of 143.4%, and an enzyme activity of 395.4 μmol/min. g support were achieved. Further incubation of the immobilized FAE at pH 10.0 improved its thermostability. Increasing the pore size of the epoxy support improved the retention of FAE hydrolytic activity and the esterifying efficiency of the immobilized biocatalyst. Optimally immobilized and stabilized FAE on metal chelate-epoxy support retained up to 92.9% of the free enzyme feruloylation efficiency to xylooligosaccharides..     

Properties of an immobilized lipase of Bacillus coagulans BTS-1

Lipase (EC 3.1.1.3) is a tri-acylglycerol ester hydrolase, catalysing the hydrolysis of tri-, di-, and mono-acylglycerols to glycerol and fatty acids. To study the effect of adsorption of a lipase obtained from Bacillus coagulans BTS-1, its lipase was immobilized on native and activated (alkylated) matrices, i.e. silica and celite. The effect of pH, temperature, detergents, substrates, alcohols, organic solvent etc. on the stability of the immobilized enzyme was evaluated. The gluteraldahyde or formaldehyde (at 1% and 2% concentration, v/v) activated matrix was exposed to the Tris buffered lipase. The enzyme was adsorbed/entrapped more rapidly on to the activated silica than on the activated celite. The immobilized lipase showed optimal activity at 50 degrees C following one-hour incubation. The lipase was specifically more hydrolytic to the medium C-length ester (p-nitro phenyl caprylate than p-nitro phenyl laurate). The immobilization/entrapment enhanced the stability of the lipase at a relatively higher temperature (50 degrees C) and also promoted enzyme activity at an acidic pH (pH 5.5). Moreover, the immobilized lipase was quite resistant to the denaturing effect of SDS.     

An improved method of lipase preparation incorporating both solvent treatment and immobilization onto matrix

A simple and effective preparation of lipases for use in organic solvents is hereby proposed. Lipases in aqueous solution were treated with isopropanol, immediately followed by immobilization onto a commercially available macroporous resin CRBO2 (crosslinked polystyrene with N-methylglucamine as a functional group). The dual modification of lipases by (1) isopropanol treatment and (2) immobilization improved the activity and stability of lipases more significantly than either of the two treatments alone. The degree of lipase activation was dependent on isopropanol–buffer (v/v) ratio and the source of lipase used. Among the lipases tested, Rhizopus oryzae lipase was more significantly activated. The maximum specific activity of R. oryzae lipase after dual modification was 94.9 mmol h⁻¹ g⁻¹, which was, respectively, 3.3-, 2.5- and 1.5-fold of untreated free, untreated immobilized and treated free lipases. The conformations of the treated and untreated free lipases were investigated by circular dichroism (CD) measurement. Changes in the far- and near-UV CD spectra of lipase indicate that lipase activation is accompanied by changes in secondary and tertiary structures of lipases. The increase in negative molar elipticity at 222 nm suggests that the α-helical content of lipase increase after pretreatment.     

Amino silicones finished fabrics for lipase immobilization: Fabrics finishing and catalytic performance of immobilized lipase

Finishing of silk fabric was achieved by using amino-functional polydimethylsiloxane (PDMS) and lipase from Candida sp. 99-125 was immobilized on the treated silk fabrics. Hydrophobic fabrics were obtained by dipping the native fabric in 0.125–0.25% (w/v) PDMS solution and dried at 70 °C. The direct adsorption on PDMS-treated fabric was verified to be a better strategy for lipase immobilization than that by covalent binding. Compared to unfinished fabrics, the hydrolytic activity of immobilized enzyme on the finished fabric was improved by 1.6 times. Moreover, the activity of immobilized enzymes on hydrophobic fabrics was significantly improved in different concentrations of strong polar solvents such as methanol and ethanol, and in common organic solvents with different octanol–water partition coefficients (Log P). Enzymatic activity and stability in 15% water content system (added water accounted for the total reaction mixtures, v/v) showed more than 30% improvement in each batch. The amino–silicone finished fabric surface was investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. The hydrophobic fabric immobilized enzyme could be recycled for more than 80 times with no significant decrease in esterification activity. PDMS-treated woven silk fabrics could be a potential support for lipase immobilization in catalytic esterification processes.     

Biodiesel production from pomace oil by using lipase immobilized onto olive pomace

In the present work, microbial lipase from Thermomyces lanuginosus was immobilized by covalent binding onto olive pomace. Immobilized support material used to produce biodiesel with pomace oil and methanol. The properties of the support and immobilized derivative were evaluated by scanning electron microscopy (SEM). The maximum immobilization of T. lanuginosus was obtained as 18.67 mg/g support and the highest specific activity was 10.31 U/mg protein. The properties of immobilized lipase were studied. The effects of protein concentration, pH and buffer concentration on the immobilization and lipase activity were investigated. Biodiesel production using the immobilized lipase was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 93% at 25 °C in 24 h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.     

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.     

大孔树脂固定化脂肪酶及在微水相中催化合成生物柴油的研究

以不同大孔树脂吸附法固定化假丝酵母99_125脂肪酶,在微水有机相中的应用表明非极性树脂NKA是最佳的固定化载体。分别以正庚烷及磷酸盐缓冲液作为固定化介质,发现在正庚烷介质中树脂NKA的固定化效率能够达到98.98%,与采用磷酸盐缓冲液作为介质相比,固定化酶的水解活力和表观酶活回收率分别提高了4.07和3.43倍。考察了在微水相中固定化酶催化合成生物柴油的催化性能,结果表明,在给酶量为1.92∶1(初始酶粉与树脂的质量比),pH值为7.4,体系水含量为15%(水与油的质量比),反应温度为40℃条件下,固定化酶具有最佳的催化能力;以正庚烷为介质固定化脂肪酶催化合成生物柴油,采用三次流加甲醇的方式,单批转化率最高达到97.3%,连续反应19批以后转化率仍保持为70.2%。