Immobilization of lipase on a new inorganic ceramics support, toyonite, and the reactivity and enantioselectivity of the immobilized lipase

A porous ceramics support, Toyonite 200-M (TN-M), for the immobilization of lipases was prepared hydrothermally from the minerals of kaolinite. Compared with some other commercial solid supports, the TN-M one exhibited better stability and higher selectivity for lipase proteins, and lipase PS (Pseudomonas cepacia) immobilized on the ceramics support showed higher reactivity for organic substrates than the free crude enzyme.     

Commercial lipase immobilization on Accurel MP 1004 porous polypropylene

Three commercial lipases (CLs), A Amano 6 (from Aspergillus niger), M Amano 10 (from Mucor javanicus), and R Amano (from Penicillium roqueforti) - called lipase A, M and R respectively - were characterized in terms of carbohydrate content, protein content and enzymatic activity (p-nitrophenylacetate assay). All the CL preparations contained different proteins as observed from electrophoresis. Lipases were immobilized on Accurel MP1004 porous polypropylene by physical adsorption.The Immobilization process caused a loss of enzymatic activity. The retained activity was similar for lipase M and R (about 15%). In contrast, lipase A retained only the 1.3% of the specific activity of the free lipase. The retained activity of lipases M and R seems to be due to a feature of the support, while the lower activity a of lipase A may be attributed to a strong structure distortion caused by lipase-support interaction.     

Commercial lipase immobilization on Accurel MP 1004 porous polypropylene

Three commercial lipases (CLs), A Amano 6 (from Aspergillus niger), M Amano 10 (from Mucor javanicus), and R Amano (from Penicillium roqueforti) – called lipase A, M and R respectively – were characterized in terms of carbohydrate content, protein content and enzymatic activity (p-nitrophenylacetate assay). All the CL preparations contained different proteins as observed from electrophoresis. Lipases were immobilized on Accurel MP1004 porous polypropylene by physical adsorption.The Immobilization process caused a loss of enzymatic activity. The retained activity was similar for lipase M and R (about 15%). In contrast, lipase A retained only the 1.3% of the specific activity of the free lipase. The retained activity of lipases M and R seems to be due to a feature of the support, while the lower activity a of lipase A may be attributed to a strong structure distortion caused by lipase–support interaction.     

Miniemulsion as efficient system for enzymatic synthesis of acid alkyl esters

The aim of this work is to devise an efficient enzymatic process for the production of linear alkyl esters in aqueous miniemulsion systems. The esterification reactions of linear alcohols and carboxylic acids were performed with three different enzymes, commercial Amano lipase PS from Pseudomonas cepacia, Lipase type VII from Candida rugosa, and lyophilized Fusarium solani pisi cutinase expressed in Saccharomyces cerevisiae SU50. The miniemulsion system shows a high potential for the synthesis of linear alkyl esters, for example, hexyl octanoate, which could be synthesized with an ester yield of 94% using Amano lipase PS. Even with hydrophilic alcohols as ethanol, ethyl decanoate could be obtained with a concentration of 0.45 M and a yield of 62% using F. s. pisi cutinase as catalyst. High esterification rates for ethyl‐ and hexyloleate in miniemulsion showed a significant shift in cutinase selectivity towards longer chain length carboxylic acids. The stepwise addition of the alcohol led to an increase of the esterification yield. Moreover, increasing the amount of dispersed organic phase, mainly consisting of the substrates, led to a significant increase of the final ester concentration (e.g., concentration of 1.4 M for ethyl decanoate for the esterification with Amano Lipase PS). Biotechnol. Bioeng. 2010;106: 507–515. © 2010 Wiley Periodicals, Inc.     

Kinetic resolution of amino acid esters catalyzed by lipases

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D ,L -homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids. © 1996 Wiley-Liss, Inc.     

Lipase-catalyzed enantioselective acylation of 3-benzyloxypropane-1,2-diol in supercritical carbon dioxide

Lipase-catalyzed acylation of 3-benzyloxypropane-1,2-diol with vinyl acetate as acyl donor using different lipases [porcine pancreas lipase (PPL), Lipase AK “Amano”, Lipase PS “Amano”, and crude enzymes from Trichoderma reesei RUT-C30, Thermoascus thermophilus (NRRL5208), Talaromyces emersonii (NRLL3221)] was studied in supercritical carbon dioxide (scCO₂). In the reactions catalyzed by different lipases different amounts of monoacetate and diacetate products along with minor amounts of cyclic acetals forming from the diol and acetaldehyde were obtained.Application of Lipase AK led to the highest conversion (84.7%) and the highest enantiomeric excess values (ee_monoacetates = 38%, ee_diacetate = 85%). Effect of water content of scCO₂ on the productivity and the enantiomer selectivity of the reactions with Lipase AK was also investigated.     

Effects of growth surface on differentiation of cultures of human tracheal epithelium

Optical measurements from epithelial cells grown on clear solid surfaces (e.g., coverslips, petri dishes) are often compared with other measurements (e.g., short-circuit current; I(sc)) obtained from cells grown on opaque porous surfaces (inserts). However, the relative levels of differentiation of cells grown under the two conditions are usually unknown. To address this issue, we grew primary cultures of human tracheal epithelium on solid surfaces or on porous inserts and compared their total levels of protein and deoxyribonucleic acid, electrical properties in Ussing chambers, and ultrastructure. To measure ion transport across cells grown on solid supports, cells were grown on inserts placed on parafilm. Later, separation of insert from parafilm allowed the cells' I(sc) to be measured in Ussing chambers. Four different media were used. Cells grown in one medium showed very low levels of differentiation on all growth supports. In the other media, growth on inserts markedly enhanced differentiation as compared with solid supports. Baseline I(sc) of cells grown on either clear or opaque inserts was at least 30 times greater than that of cells grown on solid supports, though I(sc) with clear inserts averaged approximately 30% lower than that with opaque inserts. We conclude that though differentiation of cells may vary slightly depending on the insert used, cells on any type of insert are much better differentiated than cells grown on solid surfaces. Thus, it is both possible and desirable to make all functional measurements on cells grown on clear porous supports.     

Production of diacylglycerols from glycerol monooleate and ethyl oleate through free and immobilized lipase-catalyzed consecutive reactions

The ability of free and immobilized lipase on the production of diacylglycerols (DAG) by transesterification of glycerol monooleate (GMO) and ethyl oleate was investigated. Among three free lipases such as lipase G (Penicillium cyclopium), lipase AK (Pseudomonas fluorescens) and lipase PS (Pseudomonas cepacia), lipase PS exhibited the highest DAG productivity, and the DAG content gradually increased up to 24 hours reaction and then remained steady. The comparative result for DAG productivity between free lipase PS and immobilized lipases (lipase PS-D and Lipozyme RM IM) during nine times of 24 hours reaction indicated that total DAG production was higher in immobilized lipase PS-D (183.5mM) and Lipozyme RM IM (309.5mM) than free lipase PS (122.0mM) at the first reaction, and that the DAG production rate was reduced by consecutive reactions, in which more sn-1,3-DAG was synthesized than sn-1,2-DAG. During the consecutive reactions, the activity of lipase PS was relatively steady by showing similar DAG content, whereas DAG production of lipase PS-D and Lipozyme RM IM was gradually decreased to 69.9 and 167.1mM at 9th reaction, respectively, resulting in 62% and 46% reduced production when compared with 1st reaction. Interestingly, from 7th reaction lipase PS produced more DAG than immobilized lipase PS-D, and exhibited a stable activity for DAG production. Therefore, the present study suggested that DAG productivity between GMO and ethyl oleate was higher in immobilized lipases than free lipases, but the activity was reduced with repeated uses.     

Enzymatic Resolution of (±)-Epoxy-β-cyclogeraniol,a Synthetic Precursor for Abscisic Acid Analogs

Lipase-catalyzed optical resolution of (±)-epoxy-β-cyclogeraniol (1), a key synthetic intermediate for epoxy-β-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (-)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E=1600).     

Enzymatic resolution of (+/-)-epoxy-beta-cyclogeraniol, a synthetic precursor for abscisic acid analogs

Lipase-catalyzed optical resolution of (+/-)-epoxy-beta-cyclogeraniol (1), a key synthetic intermediate for epoxy-beta-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (-)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E = 1600).     

New cationic exchanger support for reversible immobilization of proteins

New tailor-made cationic exchange resins have been prepared by covalently binding aspartic-dextran polymers (e.g. MW 15 000-20 000) to porous supports (aminated agarose and Sepabeads). More than 80% of the proteins contained in crude extracts from Escherichia coli and Acetobacter turbidans have been strongly adsorbed on these porous materials at pH 5. This interaction was stronger than in conventional carboxymethyl cellulose (e.g., at pH 7 and 25 degrees C, all proteins previously adsorbed at pH 5 were released from carboxymethyl cellulose, whereas no protein was released from the new supports under similar conditions). Ionic exchange properties of such composites were strongly dependent on the size of the aspartic-dextran polymers as well as on the exact conditions of the covalent coating of the solids with the polymer (optimal conditions: 100 mg aspartic-dextran 20 000/(mL of support); room temperature). Finally, some industrially relevant enzymes (Kluyveromices lactis, Aspergillus oryzae, and Thermus sp. beta-galactosidases, Candida antarctica B lipase, and bovine pancreas trypsin and chymotrypsin) have been immobilized on these supports with very high activity recovery and immobilization rates. After enzyme inactivation, the enzyme can be fully desorbed from the support and the support could be reused for several cycles.     

Surface loops of extracellular phospholipase A(1) determine both substrate specificity and preference for lysophospholipids

Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A(1) (PLA(1)) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA(1) activities. Two such enzymes, phosphatidylserine (PS)-specific PLA(1) (PS-PLA(1)) and phosphatidic acid (PA)-selective PLA(1)α (PA-PLA(1)α, also known as LIPH) specifically hydrolyze PS and PA, respectively. However, little is known about the mechanisms that determine their substrate specificities. Crystal structures of lipases and mutagenesis studies have suggested that three surface loops, namely, β5, β9, and lid, have roles in determining substrate specificity. To determine roles of these loop structures in the substrate recognition of these PLA(1) enzymes, we constructed a number of PS-PLA(1) mutants in which the three surface loops are replaced with those of PA-PLA(1)α. The results indicate that the surface loops, especially the β5 loop, of PA-PLA(1)α play important roles in the recognition of PA, whereas other structure(s) in PS-PLA(1) is responsible for PS preference. In addition, β5 loop of PS-PLA(1) has a crucial role in lysophospholipase activity toward lysophosphatidylserine. The present study revealed the critical role of lipase surface loops, especially the β5 loop, in determining substrate specificities of PLA(1) enzymes.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

Chemoenzymatic synthesis of enantiomerically enriched α-hydroxyamides

A study on a chemoenzymatic synthesis of model α-hydroxyamide was performed. Special attention was paid to the optimization of the enzymatic process, both on the selection of enzyme and cosolvent. An intriguing influence of cosolvent on the enantioselectivity of Wheat Germ Lipase and Amano PS Lipase catalyzed hydrolysis was observed, as the results obtained proved that enzyme's enantioselectivity is directly correlated with cosolvent's hydrophobicity. In the best example (Wheat Germ lipase, Et₂O used as a cosolvent), the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7%ee.     

Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres were used for immobilization of amyloglucosidase and the properties of immobilized enzyme was studied and compared with those of free enzyme. Sulfonated groups on the PS/PNaSS microspheres present a very simple, mild, and time-saving process for enzyme immobilization. Nonporous microspheres provide their surface for immobilization of enzyme and prevent the diffusion limitation problem in the pore. Despite the high concentration of bound enzyme the influence of immobilization on kinematic parameters, K(m) and V(max), is relatively low compare to other porous supports. Simple and time-saving immobilization procedure as well as the effects of pH and temperature on immobilized enzyme also showed that the PS/PNaSS microspheres could be good support.     

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。     

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.     

Structure and function of extracellular phospholipase A1 belonging to the pancreatic lipase gene family

Phospholipase A1 (PLA1) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids and is conserved in a wide range of organisms. Mammals have several enzymes that exhibit PLA1 activity in vitro. The extracellular PLA1s include phosphatidylserine (PS)-specific PLA1 (PS-PLA1), membrane-associated phosphatidic acid (PA)-selective PLA1s (mPA-PLA1alpha and mPA-PLA1beta), hepatic lipase (HL), endothelial lipase (EL) and pancreatic lipase-related protein 2 (PLRP2), all of which belong to the pancreatic lipase gene family. The former three PLA1s differ from other members in their substrate specificities, structural features and gene organizations, and form a subfamily in the pancreatic lipase gene family. PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta exhibit only PLA1 activity, while HL, EL and PLRP2 show triacylglycerol-hydrolyzing activity in addition to PLA1 activity. The tertiary structures of lipases have two surface loops, the lid and the beta9 loop. The lid and the beta9 loop cover the active site in its closed conformation. An alignment of amino acid sequences of the pancreatic lipase gene family members revealed two molecular characteristics of PLA1s in the two surface loops. First, lipase members exhibiting PLA1 activity (PS-PLA1, mPA-PLA1alpha and mPA-PLA1beta, EL, guinea pig PLRP2 and PLA1 from hornet venom (DolmI)) have short lids. Second, PS-PLA1, mPA-PLA1alpha, mPA-PLA1beta and DolmI, which exhibit only PLA(1) activity, have short beta9 loops. Thus, the two surface loops appear to be involved in the ligand recognition. PS-PLA1 and mPA-PLA1s specifically hydrolyze PS and PA, respectively, producing their corresponding lysophospholipids. Lysophosphatidylserine and lysophosphatidic acid have been defined as lipid mediators with multiple biological functions. Thus, these PLA1s have a role in the production of these lysophospholipid mediators.     

Synthetic p-tetrasulphonatocalix[4]arene as novel excipient for lipase-complex

The present article describes formation of excipient-CRL complex from water soluble calix[4]arene derivative (3 as excipient) and Candida rugosa lipase (CRL), which is proposed as a reusable form of enzyme that is free from steric and diffusion limitations associated with those enzymes immobilized onto porous solid supports. The excipient-CRL could completely hydrolyze 50 mM p-nitrophenyl palmitate (p-NPP) in Tris–HCl buffer at a wide range of temperatures, i.e. 30–80 °C. It is stable under stirred conditions and could be reused multiple times without loss of enzyme activity. It was observed that excipient-CRL complex shows a significant effect on the enzyme activity with an enhancement in thermal stability, while pH and temperature affect the activity of excipient-CRL as well as free CRL. Consequently, the excipient-CRL was found more active than free CRL for the hydrolysis of p-NPP in respect of its reusability.     

Short chain fatty acid esters synthesis by commercial lipases in low-water systems and by resting microbial cells in aqueous medium

Thirteen commercial lipases in hexane and seventeen bacterial cell suspensions in aqueous media were screened for the production of ethyl valerate and ethyl butyrate. The highest esterifying activity was obtained with commercial Pancrealipase (Biozymes Inc.) and Candida rugosa lipase (Amano Enzyme Ltd) and with bacterial cell suspension from Pseudomonas fragi CRDA 446. Commercial enzymes gave molar conversion yield of 68% over 24 h as compared to 17% with whole cells in aqueous medium. However, a comparison of both sources of biocatalyst i.e. whole microbial cells and commercial lipases, based on the amount of ester produced per g of protein for a complete reaction, indicated similar activities. © Rapid Science Ltd. 1998