Morphological, biochemical and kinetic properties of lipase from Candida rugosa immobilized in zirconium phosphate

Zirconium phosphate (ZrP), a low-cost inorganic material with well-defined physicochemical properties, was successfully used as support for immobilizing Candida rugosa lipase by covalent bonding. The immobilized derivative showed high catalytic activity in both aqueous and non-aqueous media. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy measurements demonstrated that the ZrP fulfilled the morphological requirements for use as a matrix for immobilizing lipases. The free and immobilized lipases were compared in terms of pH, temperature and thermal stability. The immobilized lipase had a higher pH optimum (7.5) and higher optimum temperature (50°C) than the free lipase. Immobilization also increased the thermal stability. The hydrolysis of p-nitrophenyl palmitate (pNPP) by immobilized lipase, examined at 37°C, followed Michaelis-Menten kinetics. Values for K_m=1.18 µM and V_max=325Umg^-1 indicated that the immobilized system was subject to mass transfer limitations. The immobilized derivative was also tested under repetitive reaction batches in both ester hydrolysis and synthesis.     

Enhancing the catalytic properties of porcine pancreatic lipase by immobilization on SBA-15 modified by functionalized ionic liquid

The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.     

Morphological,biochemical and kinetic properties of lipase from Candida rugosa immobilized in zirconium phosphate

Zirconium phosphate (ZrP), a low-cost inorganic material with well-defined physicochemical properties, was successfully used as support for immobilizing Candida rugosa lipase by covalent bonding. The immobilized derivative showed high catalytic activity in both aqueous and non-aqueous media. Fourier transform infrared spectroscopy, X-ray diffraction, and scanning electron microscopy measurements demonstrated that the ZrP fulfilled the morphological requirements for use as a matrix for immobilizing lipases. The free and immobilized lipases were compared in terms of pH, temperature and thermal stability. The immobilized lipase had a higher pH optimum (7.5) and higher optimum temperature (50°C) than the free lipase. Immobilization also increased the thermal stability. The hydrolysis of p-nitrophenyl palmitate (pNPP) by immobilized lipase, examined at 37°C, followed Michaelis–Menten kinetics. Values for K_m=1.18 µM and V_max=325Umg^?1 indicated that the immobilized system was subject to mass transfer limitations. The immobilized derivative was also tested under repetitive reaction batches in both ester hydrolysis and synthesis.     

Immobilization of lipase onto micron-size magnetic beads

A novel and economical magnetic poly(methacrylate-divinylbenzene) microsphere (less than 8 microm in diameter) was synthesized by the modified suspension polymerization of methacrylate and cross-linker divinylbenzene in the presence of magnetic fluid. Then, surface aminolysis was employed to obtain a high content of surface amino groups (0.40-0.55 mmolg(-1) supports). The morphology and properties of these magnetic supports were characterized with scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy and a vibrating sample magnetometer. These magnetic supports exhibited superparamagnetism with a high specific saturation magnetization (sigma(s)) of 14.6 emicrog(-1). Candida cylindracea lipase was covalently immobilized on the amino-functionalized magnetic supports with the activity recovery up to 72.4% and enzyme loading of 34.0 mgg(-1) support, remarkably higher than the previous studies. The factors involved in the activity recovery and enzymatic properties of the immobilized lipase prepared were studied in comparison with free lipase, for which olive oil was chosen as the substrate. The results show that the immobilized lipase has good stability and reusability after recovery by magnetic separation within 20s.     

Fabrication of an organic–inorganic nanocomposite carrier for enzyme immobilization based on metal–organic coordination

An organic–inorganic nanocomposite which combined mesoporous silica SBA-15 and chitosan using a carboxyl functionalized ionic liquid as the bridging agent (SBA@CS) was successfully fabricated, and was used to immobilize porcine pancreas lipase (PPL) by physical adsorption, cross-linking and metal–organic coordination, respectively. The as-prepared carriers were characterized by scanning electron microscopy, Fourier transform infrared and energy-dispersive X-ray spectroscopy. Compared with immobilization onto the pure mesoporous silicon material SBA-15, all the batches of PPL immobilized onto organic–inorganic nanocomposites showed higher activity, improved stability and reusability as well as better resistance to pH and temperature changes. Among the immobilized PPLs, immobilization based on Co²⁺ coordination (SBA@CS-Co-PPL) produced the best enzymatic properties. The maximum immobilization efficiency and specific activity of 79.6% and 1975.8 U g⁻¹ were obtained with SBA@CS-Co, separately. More importantly, the activity of immobilized enzyme can still maintain 84.0% after 10 times of reuse. These results demonstrated that thus prepared organic–inorganic nanocomposite could be an ideal carrier for enzyme immobilization by metal–organic coordination.     

Immobilized lipase on macroporous polystyrene modified by PAMAM-dendrimer and their enzymatic hydrolysis

The novel enzyme carrier, polyamidoamine (PAMAM) dendrimers modified macroporous polystyrene, has been synthesized by Michael addition and firstly used in the immobilization of porcine pancreas lipase (PPL) effectively by covalent attachment. The resulting carrier was characterized with the Fourier transform infrared spectra (FT-IR), scanning electron microscopy (SEM), elemental analysis and thermogravimetric (TG) analysis. Meanwhile, the amount of immobilized lipase was up to 100 mg g⁻¹ support and the factors related with the enzyme activity were investigated. The immobilization of the PPL improved their performance in wider ranges of pH and temperature. Thermal stability of the immobilized lipase also increased dramatically in comparison with the free ones and the immobilized lipase exhibited a favorable denaturant tolerance. As a biocatalyst, the immobilized lipase for batch hydrolysis of olive oil emulsion retained 85% activity after 10 times of recycling. This well-reusability of immobilized lipase was very valuable and meaningful in enzyme technology.     

γ-Fe_2O_3-凹土超顺磁性纳米复合材料固载猪胰脂肪酶

以凹凸棒石黏土为原料,制备γ-Fe2O3-凹土超顺磁性纳米复合材料(γ-Fe2O3-ATP)作为猪胰脂肪酶(PPL)固定化的载体,利用透射电子显微镜(TEM)、N2吸附脱附等温图(BET)、振动试样磁强计(VSM)等对材料进行表征,同时对固定化条件和固定化酶的相关性质进行了研究。结果表明:制备的γ-Fe2O3-ATP是介孔材料,比表面积为102.63 m2/g,平均孔径为10.862 nm,饱和磁化强度为8.915 emu/g,其作为载体能实现固定化酶与反应介质简单、快速分离回收和重复利用。在固定化时间为4 h及pH 6.0时制备的固定化酶效果最佳;经过6 h高温保存后固定化酶可保留初始酶活的52%,而游离酶仅保留初始酶活的19%,同时固定化酶在重复使用5次后酶活仍保留初始酶活的60%。     

Enantioselective enzymatic hydrolysis of racemic glycidyl esters by using immobilized porcine pancreas lipase with improved catalytic properties

The crude porcine pancreas lipase (PPL) extract is a mixture of several proteins (mainly lipases and esterases). In order to develop enzymatic catalysts with good catalytic properties for hydrolytic enantioselective reactions in aqueous homogeneous medium, we studied the immobilization of the different enzymes contained in the crude PPL extracts by selective sequential adsorption on hydrophobic supports bearing octyl, octadecyl and phenyl groups. Some minor proteins were selectively adsorbed on octyl and octadecyl supports while the most abundant lipase was adsorbed on the support bearing phenyl groups. The enantioselectivity of the different lipase derivatives were tested considering the hydrolysis of esters of 1,2-epoxi-1-propanol (glycidol). The most abundant lipase contained in the commercial crude PPL extract resulted almost inactive while some lipases contained in low concentrations displayed high activities and enantioselectivities. The most interesting results were obtained with a 28-kDa protein selectively adsorbed on octyl-agarose. With this enzyme derivative, the residual butyric ester of glycidol was recovered with 96% enantiomeric excess at 55% conversion.     

Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.     

Immobilization of lipase in organic solvent in the presence of fatty acid additives

In this study porcine pancreatic lipase (PPL) was covalently immobilized on cross-linked polyvinyl alcohol (PVA) in organic media in the presence of fatty acid additives in order to improve its immobilized activity. The effects of fatty acid additions to the immobilization media were investigated choosing tributyrin hydrolysis in water and ester synthesis by immobilized PPL in n-hexane. Various fatty acids which are also the substrates of lipases in esterification reactions were used as active site protecting agents during the immobilization process in an organic solvent. The obtained results showed that covalent immobilization carried out in the presence of fatty acids as protective ligands improved the hydrolytic and esterification activity of immobilized enzyme. A remarkable increase in activity of the immobilized PPL was obtained when octanoic acid was used as an additive and the hydrolytic activity was increased from 5.2 to 19.2 μmol min⁻¹ mg⁻¹ as compared to the non-additive immobilization method. With the increase of hydrolytic activity of immobilized lipase in the presence of octanoic acid, in an analogous manner, the rate of esterification for the synthesis of butyl octanoate was also increased from 7.3 to 26.3 μmol min⁻¹ g⁻¹ immobilized protein using controlled thermodynamic water activities with saturated salt solutions. In addition, the immobilized PPL activity was maintained at levels representing 63% of its original activity value after 5 repeated uses. The proposed method could be adopted for a wide variety of other enzymes which have highly soluble substrates in organic solvent such as other lipases and esterases.     

离子液体修饰的SBA-16材料在猪胰脂肪酶固定化中的应用

合成了功能化的甲基咪唑类离子液体,并将功能化离子液体修饰介孔材料SBA-16。以三乙酸甘油酯的水解为探针反应,考察离子液体修饰的SBA-16固定化猪胰脂肪酶(PPL)的酶活、最适反应条件及重复稳定性等酶学性质。结果表明:固定化酶对温度的敏感度降低,酶活力及稳定性均显著提高,比酶活是原粉SBA-16固定化酶的1.75倍,重复使用6次后仍然保持最初活性的57%;与原粉SBA-16固定化酶保留的38%相比,有明显的提高。同时通过N2吸附-脱附、红外光谱和热重等方法分析了离子液体修饰对SBA-16结构的影响,结果发现,离子液体修饰后材料保持了原有的介孔结构,修饰后载体表面性质和结构性质导致了PPL酶学性质的变化。     

Interface-mediated inactivation of pancreatic lipase by a water-reactive compound: 2-sulfobenzoic cyclic anhydride

2-Sulfobenzoic cyclic anhydride (SBA) rapidly and selectively inactivates porcine pancreatic lipase (PPL) only when added during the hydrolysis of an emulsified ester such as tributyrin or dodecyl acetate. The present data suggest that the inactivation of PPL occurs preferentially at the oil/water interface and not in the aqueous phase, since colipase and bile salt were found to adversely affect the inhibition process. Moreover, it is shown that at a molar ratio of SBA to pure PPL of 1, 40% of the lipase activity was already irreversibly lost. Complete inactivation was observed at SBA to pure PPL molar ratios of 120. A 60% inactivation occurred when 0.5 mol of 3H-labeled SBA was attached per mole of PPL. The SBA-inactivated PPL competes for binding to the dodecyl acetate/water interface as efficiently as the native enzyme. Larger SBA concentrations are required when crude lipase preparations are used as well as with pure PPL in the presence of bile salts and colipase. Lipases were found to have variable sensitivities to SBA inactivation, depending on their origin. In the presence of bile salts and tributyrin at pH 6.0, human gastric lipase activity was not affected by the presence of a 10(6) molar excess of SBA.     

Production of Ricinoleic Acid from Castor Oil by Immobilised Lipases

Abstract

Porcine pancreatic lipase (PPL), Candida rugosa lipase (CRL), and Castor bean lipase (CBL) were immobilized on celite by deposition from aqueous solution by the addition of hexane. Lipolytic performance of free and immobilized lipases were compared and optimizations of lipolytic enzymatic reactions conditions were performed by free and immobilized derivatives using olive oil as substrate. Afterwards, the influence on lipolysis of castor oil of free lipases and immobilized lipase derivatives have been studied in the case of production of ricinoleic acid. All of the lipases performances were compared and enzyme derivative was selected to be very effective on the production of ricinoleic acid by lipolysis reaction. Various reaction parameters affecting the production of ricinoleic acid were investigated with selected the enzyme derivative.

The maximum ricinoleic acid yield was observed at pH 7–8, 50°C, for 3 hours of reaction period with immobilized 1,3-specific PPL on celite. The kinetic constants K_m and V_max were calculated as 1.6 × 10^?4 mM and 22.2 mM from a Lineweaver–Burk plot with the same enzyme derivative. To investigate the operational stability of the lipase, the three step lipolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentange of conversion after usage decreased markedly.     

海泡石吸附固定化猪胰脂肪酶

以海泡石作为猪胰脂肪酶(PPL)的固定化载体,考察采用物理吸附的方法制备固定化脂肪酶的条件。结果表明:在固载时间4 h、反应磷酸盐(PBS)溶液pH 6.0、反应温度25℃时,可达最大比酶活309 U/g,固定化酶的化学稳定性和热稳定性均较高。同时利用红外谱仪(FT-IR)和扫描电子显微镜(SEM)的分析手段对固定化猪胰脂肪酶试样进行分析,进一步确定了海泡石材料在固定化酶中的作用。     

1,3‐Regiospecific ethanolysis of soybean oil catalyzed by crosslinked porcine pancreas lipase aggregates

The preparation of crosslinked aggregates of pancreatic porcine lipase (PPL‐CLEA) was systematically studied, evaluating the influence of three precipitants and two crosslinking agents, as well as the use of soy protein as an alternative feeder protein on the catalytic properties and stability of the immobilized PPL. Standard CLEAs showed a global yield (CLEA’ observed activity/offered total activity) of less than 4%, whereas with the addition of soy protein (PPL:soy protein mass ratio of 1:3) the global yield was approximately fivefold higher. The CLEA of PPL prepared with soy protein as feeder (PPL:soy protein mass ratio of 1:3) and glutaraldehyde as crosslinking reagent (10 μmol of aldehyde groups/mg of total protein) was more active mainly because of the reduced enzyme leaching in the washing step. This CLEA, named PPL‐SOY‐CLEA, had an immobilization yield around 60% and an expressed activity around 40%. In the ethanolysis of soybean oil, the PPL‐SOY‐CLEA yielded maximum fatty acid ethyl ester (FAEE) concentration around 12‐fold higher than that achieved using soluble PPL (34 h reaction at 30°C, 300 rpm stirring, soybean oil/ethanol molar ratio of 1:5) with an enzyme load around 2‐fold lower (very likely due to free enzyme inactivation). The operational stability of the PPL‐SOY‐CLEA in the ethanolysis of soybean oil in a vortex flow type reactor showed that FAEE yield was higher than 50% during ten reaction cycles of 24 h. This reactor configuration may be an attractive alternative to the conventional stirred reactors for biotransformations in industrial plants using carrier‐free biocatalysts. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:910–920, 2018     

硅藻土固定中性脂肪酶的制备及其性质

以硅藻土为载体,采用吸附法,对脂肪酶进行固定化,研究了固定化条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件：给酶量为33374U/g,固定化温度为35℃,pH值为7.5,时间为4h,此时固定化酶的活力约为5833U/g载体。固定化酶的热稳定性较游离酶有了很大的提高,其在80℃以下能保持80%以上的酶活,而游离酶60℃残余酶活仅为5%。最适反应温度和最适pH值也分别由游离酶的40℃上升至50℃和由7上升到7.5。对固定化中的中性脂肪酶在生物柴油合成中的应用也进行了初步研究。     

纳米银复合介孔碳材料制备固定化猪胰脂肪酶

以稻壳为原料制备介孔碳材料,对其表面进行纳米银修饰后作为猪胰脂肪酶(PPL)的固定化载体.初步研究并优化该载体固定猪胰脂肪酶的条件.结果表明:在AgNO3浓度为0.01 mol/L、固载时间6h、反应磷酸盐溶液pH为6.0、反应温度25℃时,可达最大酶活,酶活提高3.2倍.同时利用红外图谱(FT-IR)、N2等温吸附-脱附曲线(BET)和扫描电子显微镜(SEM)的分析手段对固定化猪胰脂肪酶试样进行分析,进一步确定了纳米银复合介孔性碳材料在固定化酶实验中的作用.     

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester [(+/-)-1] to produce the corresponding acid [2], the octyl-25KDa preparation showed the best enantioselectivity (E) value (E = 7) at pH 5 and 25 degrees C, whereas the phenyl-PPL was the most enantioselective (E = 10) at pH 5, 4 degrees C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (+/-)-2-O-butyryl-2-phenylacetic acid [(+/-)-3] with a high E value (E > 100); for example, with octadecyl-33 KDa enzyme at pH 8.     

Rice straw as a support for immobilization of microbial lipase.

Candida rugosa lipase was covalently immobilized on rice straw activated with glutaraldehyde using poly(ethylene glycol) (PEG) as the stabilizing agent. The effects of PEG molecular weight and enzyme loading were studied according to a full 2(2) factorial design. Higher immobilization yields (>70%) were attained when the lipase loading was 95 units/mg of dry support, independent of PEG molecular weight. All derivatives showed high hydrolytic and synthetic activities. This work provides preliminary results on the use of agricultural residues as a support matrix for immobilizing lipase and on the application of the resulting derivatives to butyl butyrate synthesis as a study model.     

Bacterial cellulose-chitosan composite hydrogel beads for enzyme immobilization

In this work, we report the preparation of bacterial cellulose (BC)-chitosan composite hydrogel beads by co-dissolution of BC and chitosan in 1-ethyl-3-methylimidazolium acetate and subsequent reconstitution with distilled water. The BC-chitosan hydrogel beads were used as enzyme supports for immobilizing Candida rugosa lipase by physical adsorption and covalent cross-linking. BC-chitosan hydrogel beads immobilized lipase more efficiently than microcrystalline cellulose (MCC)-chitosan hydrogel beads. The amount of protein adsorbed onto BCchitosan beads was 3.9 times higher than that adsorbed onto MCC-chitosan beads, and the catalytic activity of lipase was 1.9 times higher on the BC-chitosan beads. The lipase showed the highest thermal and operational stability when covalently cross-linked on BC-chitosan hydrogel beads. The half-life time of the lipase cross-linked on BC-chitosan bead at 60°C was 22.7 times higher than that of free lipase. Owing to their inherent biocompatibility and biodegradability, the BC-chitosan composite hydrogel beads described here could be used to immobilize proteins for various biomedical, environmental, and biocatalytic applications.