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     

Transesterification using the cross-linked enzyme aggregate of Photobacterium lipolyticum lipase M37

Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (~93%). CLEA M37 evidences an optimal temperature of 30oC, and an optimal pH of 9-10. It was stable up to 50°C and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and nbutanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.     

Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media

A novel type of biocatalyst that combines the good properties of cross-linked enzyme aggregates (CLEAs) and hydrophilic microenvironments has been developed. Dextran sulfate- and polyethyleneimine-coated CLEAs of penicillin acylase (CLEA-GDP) were prepared by adding the polymers of different sizes before the precipitation stage of the enzyme. This study presents the development and optimization of a protocol to produce such a biocatalyst using penicillin acylase as a model. Experiments show that CLEA-GDPs have a highly increased stability in organic media. The average half-life of the preparations was much higher than standard CLEA without a microenvironment (CLEA-G), (e.g., more than 25-fold) in the presence of dioxane. However, their thermal stability was not increased, which leads to the conclusion that the stability of CLEA-GDPs in organic media is due to the hydrophilic microenvironment that surrounds the protein enzyme more than to a conformational stiffening effect. This is further supported by solvation experiments that show a preferential hydration of CLEA when polymers are used to coat the enzyme. CLEA-GDPs are clearly better than other biocatalysts in terms of solvent stability.     

交联酶聚集体制备中钙离子的结构调控作用

以葡萄糖氧化酶(GOx)为研究对象,系统地研究了钙离子对交联酶聚集体(CLEA)粒子尺寸和微观结构的调控作用以及酶催化性能和实用性的影响。研究结果表明,GOx酶沉淀过程中引入钙离子可显著降低CLEA粒子尺寸并导致粒子内纳米孔道结构逐步消失。在0.1 mmol/L钙离子浓度下,GOx酶的CLEA仍保有清晰的纳米孔道结构。以葡萄糖为底物的GOx酶CLEA催化结果显示,该CLEA粒子的酶活性为对照CLEA粒子的2.69倍。即便1.0 mmol/L钙离子浓度下制备的CLEA粒子的GOx酶活性仍高出对照CLEA粒子约42%。此外,0.1 mmol/L钙离子浓度下制备的CLEA不仅具有更高的底物转化速率和很好的操作稳定性,而且CLEA中GOx酶的最大反应速度显著提高。这些实验结果明确了钙离子对CLEA粒子尺寸和微观结构的调控作用,为制备具有高效生物催化活性的CLEA粒子奠定了基础。     

Preparation and characterization of cross-linked enzyme aggregates (CLEA) of subtilisin for controlled release applications

Enzyme stabilization is one of the major challenges in the biocatalytic process optimization. Subtilisin was aggregated using ammonium sulphate and polyethylene glycol with surfactants like triton X-100 and tween 20. The resultant aggregates on cross-linking with glutaraldehyde produced insoluble and catalytically active enzyme. The effect of pH, temperature, kinetic parameter, thermal stability and stability in organic solvents were studied. The cross-linked enzyme aggregates (CLEA) exhibited broad pH optima of 9.0 and higher temperature optima of 70 degrees C. Reusability and surface morphology of the CLEA were also studied. CLEA of subtilisin has good stability in nonpolar organic solvents, such as hexane, and cyclohexane and it has high thermal stability up to 60 degrees C and therefore can be used as a catalyst for the biotransformation of compounds which are not soluble in aqueous medium. The CLEAs were entrapped in the hydrogel composite beads of alginate:guar gum (3:1) which were resistant to low pH conditions in the stomach and thus was found to be useful for the oral drug delivery. This process can be used to deliver the protein and peptide drugs which involve high concentrations at the delivery stage, and which usually degrades in the stomach before reaching the jejunum. Application of these pH-sensitive beads for the controlled release of subtilisin in vitro was studied and found to be a feasible strategy.     

Immobilization of formate dehydrogenase from Candida boidinii through cross-linked enzyme aggregates

We employed a cross-linked enzyme aggregate (CLEA) method to immobilize formate dehydrogenase (FDH) from Candida boidinii. The optimal conditions for the preparation of CLEAs were determined by examining effects of various parameters: the nature and amount of cross-linking reagent, additive concentration, cross-linking time, and pH during CLEA preparation. The recovered activities of CLEAs were significantly dependent on the concentration of glutaraldehyde; however, the recovered activity was not severely influenced by the content of dextran polyaldehyde as a mild cross-linker. Bovine serum albumin (BSA) was also used as a proteic feeder and enhanced the activity recovery by 130%. The highest recovered activity of CLEA was 18% for formate oxidation reaction and 25% for CO₂ reduction reaction. The residual activity of CLEA prepared with dextran polyaldehyde (Dex-CLEA) was over 95% after 10 cycles of reuse. The thermal stability of Dex-CLEA was increased by a factor of 3.6 more than that of the free enzyme. CLEAs of FDH could be utilized efficiently for both NADH regeneration and CO₂ reduction.     

Activity and stability of cross-linked tyrosinase aggregates in aqueous and nonaqueous media

Cross-linked tyrosinase aggregates were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Both activity and stability of these cross-linked enzyme aggregates (CLEAs) in aqueous solution, organic solvents, and ionic liquids have been investigated. Immobilization effectively improved the stability of the enzyme in aqueous solution against various deactivating conditions such as pH, temperature, denaturants, inhibitors, and organic solvents. The stability of the CLEAs in various organic solvents such as tert-butanol (t(1/2)=326.7h at 40°C) was significantly enhanced relative to that in aqueous solution (t(1/2)=5.5h). The effect of thermodynamic water activity (a(w)) on the CLEA activity in organic media was examined, demonstrating that the enzyme incorporated into CLEAs required an extensive hydration (with an a(w) approaching 1.0) for optimizing its activity. The impact of ionic liquids on the CLEA activity in aqueous solution was also assessed.     

Chemical amination of lipase B from Candida antarctica is an efficient solution for the preparation of crosslinked enzyme aggregates

Lipase B from Candida antarctica (CALB) is not very adequate to prepare crosslinked enzyme aggregates (CLEAs). Although the precipitation step is easy using different precipitants, the crosslinking step becomes a problem due to the low amount of Lys residues in this enzyme. In this paper, we have enriched the enzyme in amino groups by chemical amination of the enzyme using ethylenediamine and carbodiimide. The modification was performed using a solid phase strategy modifying the enzyme adsorbed on octyl-Sepharose. After desorption from the support, the enzyme was more active at pH 7.0 than the unmodified enzyme. This modified enzyme showed to be suitable to produce CLEAs. Using this modified enzyme, precipitation is also effective but the crosslinking step did not fail in giving an intense intermolecular crosslinking. This way, the CLEA did not release enzyme molecules even if boiled in SDS. Stability of this CLEA was higher in both thermal and cosolvent inactivation experiments than that of the coCLEA produced by coagregation of BSA and CALB; another alternative to produce a CLEA of this interesting enzyme.The strategy may be of high interest for many other enzymes as a way to both permit the production of CLEAs and to improve enzyme stability during CLEA production.     

High Temperature Lactic Acid Production by Bacillus coagulans Immobilized in LentiKats

Bacillus coagulans spores were immobilized in polyvinylalcohol (PVA) hydrogel, lens-shaped capsules known as LentiKats. The immobilized spores were used in an anaerobic, non-sterile process in the repeated batch fermentations at 50 degrees C and produced lactic acid at 7.4 g l(-1) h(-1), which was double that of the free cell system. No mechanical deformation of the capsules and no contamination were observed.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Encapsulation in LentiKats of Dextransucrase from Leuconostoc mesenteroides NRRL B-1299, and its Effect on Product Selectivity

Insoluble (cell-bound) dextransucrase from Leuconostoc mesenteroides B-1299 was encapsulated in highly elastic and stable hydrogels formed by polyvinyl alcohol. The gelation was carried out by controlled partial drying at room temperature, resulting in lens-shaped particles, called LentiKats. A similar recovery of activity (approximately 55%) was achieved when compared with entrapment in calcium alginate gels. Under reaction conditions, the protein leakage in LentiKats was reduced from 18% to 4% by pre-treatment of the dextransucrase with glutaraldehyde. The immobilized dextransucrases were tested in the acceptor reaction with methyl α-D-glucopyranoside. The conversion to oligosaccharides using Lentikat-dextransucrase was higher than that obtained for alginate-dextransucrase, probably due to the reduction of diffusional limitations derived from its lenticular shape. In addition, a shift of selectivity towards the synthesis of oligosaccharides containing α(1→2) bonds was observed for the Lentikat-biocatalysts. These non-digestible compounds are supposed to be specifically fermented by beneficial species of the human microflora (prebiotic effect). The Lentikat-entrapped dextransucrase can be efficiently reused in this process at least for five cycles of 24 h.     

Preparation of cross-linked enzyme aggregates by using bovine serum albumin as a proteic feeder

Addition of bovine serum albumin (BSA) as a proteic feeder facilitates obtaining cross-linked enzyme aggregates (CLEAs) in cases where the protein concentration in the enzyme preparation is low and/or the enzyme activity is vulnerable to the high concentration of glutaraldehyde required to obtain aggregates. CLEAs of Pseudomonas cepacia lipase and penicillin acylase were prepared. CLEA of lipase prepared in the presence of BSA retained 100% activity whereas CLEA prepared without BSA retained only 0.4% activity of the starting enzyme preparation. Lipase CLEA showed 12-fold increase in activity over free enzyme powder when the CLEA was used in transesterification of tributyrin. For the transesterification of Jatropha oil, while free enzyme powder required 8 h and 50 mg lipase to obtain 77% conversion, CLEA required only 6 h and 6.25 mg lipase to obtain 90% conversion. In the case of penicillin acylase, 86% activity could be retained in CLEA prepared with BSA whereas CLEA made without BSA retained only 50% activity. CLEA prepared without BSA lost 20% activity after 8 h at 45 degrees C whereas CLEA with BSA retained full activity. CLEA prepared with BSA showed Vmax/Km of 36.3 min-1 whereas CLEA prepared without BSA had Vmax/Km of 17.4 min-1 only. Scanning electron microscopy analysis showed that CLEAs prepared in the presence of BSA were less amorphous and closer in morphology to CLEAs of other enzymes described in the literature.     

Advances in enzyme immobilisation

Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.     

外消旋薄荷醇对映选择性酶促酯化中酸酐与羧酸的比较研究

利用脂肪酶在有机溶剂中催化对映选择性酯化反应对外消旋薄荷醇进行了有效的光学拆分。对分别使用酸酐和相应的游离羧酸作酰基给体时的反应性能进行了比较。发现酸酐的反应性远高于对应的游离羧酸,但在酶的催化作用下酸酐易水解成为游离羧酸;在微水系统中使用过高浓度的酸酐会导致酶缺水而失活,同时会促进手性醇的非选择性酯化,从而降低产物的光学纯度。然而,在连续流加丙酸酐的半批式反应系统中,所有这些缺点均可有效地克服。与使用游离丙酸的批式反应系统相比,ｄｌ-薄荷醇的反应时间缩短了一半,酶的稳定性大幅度提高,而产物ｌ薄荷醇酯的光学纯度不相上下（＞９８％ｅ）。     

Continuous malolactic fermentation by Oenococcus Oeni entrapped in LentiKats

A continuous process to deacidify apple juices and cider was developed by entrapping Oenococcus oeni in LentiKats, a new polyvinyl alcohol hydrogel. For a residence time of 0.55 h, malic acid was completely converted into lactic acid when the LentiKats bioreactor was fed with apple juice at pH 4.46 and 3.95 and thirty three percent of initial malic acid (6.7 g l^–1) was converted when the initial apple juice pH was 2.30. The optimal malolactic activity of this bioreactor was obtained at 30°C and a 50% reduction in malic acid conversion was measured between 15°C and 20°C, at a residence time of 0.3 h. The LentiKats bioreactor gave better performance than continuous reactor with Oenococcus oeni immobilised in alginate beads (specific malic acid consumption increased by a factor of 4.6) due to the increase of the ratio external surface to volume, allowing better mass transfer.     

Remarkable stability of Candida antarctica lipase B immobilized via cross-linking aggregates (CLEA) in deep eutectic solvents

The combination of Deep-eutectic-solvents (DES) with water as “co-solvent” enables a low-viscous reaction medium that keeps its “non-conventional” nature and thus enables synthetic lyophilization reactions (e.g. esterification) catalyzed by hydrolases. Substrates with different polarity may be employed. This paper shows how the enzyme immobilization with cross-linking aggregates (CLEA) leads to highly stable and active immobilized catalysts in different DES. As a remarkable case, when choline chloride-glycerol DES is used, CLEA derivatives of Candida antarctica lipase B (CLEA-CALB) are stable for at least 14?days without any loss of activity. The immobilized biocatalysts are applied in non-viscous DES-water blends (8% v/v) to catalyze the esterification of benzoic acid and glycerol to furnish glyceryl monobenzoate (α-MBG) in productivities of ～35?g α-MBG L^?1d^?1. Compared to other commercial immobilized CALB, the CLEA-CALB derivatives rendered more product (higher conversions by 30%). Moreover, CLEA derivatives were successfully reused for six times without any loss of activity. Given the ease of immobilization (CLEA), their excellent performance in DES and the low viscosity of the DES-water blends, the reported approach may be useful for many synthetic procedures and even for continuous processes with largely optimized outcomes.     

Ligand-decorated nanogels: fast one-pot synthesis and cellular targeting

Nanoscale vehicles for delivery have been of interest and extensively studied for two decades. However, the encapsulation stability of hydrophobic drug molecules in delivery vehicles and selective targeting these vehicles into disease cells are potential hurdles for efficient delivery systems. Here we demonstrate a simple and fast synthetic protocol of nanogels that shows high encapsulation stabilities. These nanogels can also be modified with various targeting ligands for active targeting. We show that the targeting nanogels (T-NGs), which are prepared within 2 h by a one-pot synthesis, exhibit very narrow size distributions and have the versatility of surface modification with cysteine-modified ligands including folic acid, cyclic arginine-glycine-aspartic acid (cRGD) peptide, and cell-penetrating peptide. T-NGs hold their payloads, undergo facilitated cell internalization by receptor-mediated uptake, and release their drug content inside cells due to the reducing intracellular environment. Selective cytotoxicity to cells, which have complementary receptors, is also demonstrated.     

Immobilized l-aspartate ammonia-lyase from Bacillus sp. YM55-1 as biocatalyst for highly concentrated l-aspartate synthesis

L: -Aspartate ammonia-lyase from Bacillus sp. YM55-1 (AspB, EC 4.3.1.1) catalyzes the reversible conversion of L: -aspartate (Asp) into fumarate and ammonia with a high specific activity toward the substrate. AspB was expressed in Escherichia coli and partially purified by heat precipitation and saturation with ammonium sulfate reaching purification factor of 7.7 and specific activity of 334?U/mg of protein. AspB was immobilized by covalent attachment on Eupergit(?) C (epoxy support) and MANA-agarose (amino support), and entrapment in LentiKats(?) (polyvinyl alcohol) with retained activities of 24, 85 and 63?%, respectively. Diffusional limitations were only observed for the enzyme immobilized in LentiKats(?) and were overcome by increasing substrate concentration. Free and immobilized AspB were used for the synthesis of aspartate achieving high product concentration (≥450?mM) after 24?h of reaction. Immobilized biocatalysts were efficiently reused in 5 cycles of Asp synthesis, keeping over 90?% of activity and reaching over 90?% of conversion in all the cases.     

Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions

A novel cross-linked enzyme aggregates (CLEA) concept called combi-CLEA has been described. It is based upon the fact that CLEA can be made from heterogeneous populations of proteins/enzymes. Porcine pancreatic acetone powder crude extract was used for preparing CLEA in such a way that lipase, -amylase, phospholipase A₂ activities were retained upto 100%. The lipase present in the CLEA showed greater thermal stability at 50 °C as compared to free enzyme. For lipase and phospholipase A₂, V_max/K_m showed no significant change upon combi-CLEA formation but decreased significantly for -amylase activity from 190 to 114 min⁻¹. The lipase activity and -amylase activity in CLEA were completely retained upto three cycles of use. The scanning electron microscopic (SEM) studies showed that morphology of CLEA changed upon inactivation by reuses.     

Thermodynamically controlled synthesis of amide bonds catalyzed by highly organic solvent-resistant penicillin acylase derivatives

A study of various direct condensations between different amines, having very high pK values, and unmodified acyl donors has been performed. This has been possible by the use of a very stable PGA derivative. First, it has been found that the higher the cosolvent concentration, the higher the pK of the acyl donor and thus the higher the yield. Therefore, these high concentrations of cosolvents seem to be a requisite for certain enzymatic condensations. Using ethanolamine and 2-hydroxy-2-phenylethyl-amine as nucleophiles and phenyl acetic acid as the acyl donor, the increase in the diglyme concentration from 50 to 90% (v/v) permitted improvement of not only the yield (reaching values higher than 99% in both cases) but also the reaction rates (by 360- or 3-fold, respectively). However, even when using PGA preparations stabilized by multipoint covalent attachment, it was not possible to obtain these results by inactivation of the enzyme derivative. Thus, in the protection of the octylamine with phenylacetic acid in 90% diglyme, the enzymatic activity was more than 20-fold higher using the hydrophilized derivative than the glyoxyl PGA, which allowed us to obtain a yield higher than 99%. Thus, the use of hydrophilized derivatives that are very stable even in the presence of high concentrations of organic solvents opens new opportunities in the use of PGA in organic chemistry.