Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^-1 h^-1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg^?1 h^?1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.     

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.     

Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80‐pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)‐1‐phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R–S)‐1‐phenylethanol with vinylacetate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 937–945, 2012 This article was published online on June 26, 2012. An edit was subsequently requested. This notice is included in the online and print versions to indicate that both have been corrected [27 June 2012].     

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。     

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.     

Characterization of cross-linked immobilized lipase from thermophilic mould Thermomyces lanuginosa using glutaraldehyde

Cross-linked enzyme aggregates (CLEAs) have emerged as an interesting biocatalyst design for immobilization. Using this approach, a 1,3 regiospecific, alkaline and thermostable lipase from Thermomyces lanuginosa was immobilized. Efficient cross-linking was observed when ammonium sulphate was used as precipitant along with a two fold increase in activity in presence of SDS. The TEM and SEM microphotographs of the CLEAs formed reveal that the enzyme aggregates are larger in size as compared to the free lipase due to the cross-linking of enzyme aggregates with glutaraldehyde. The stability and reusability of the CLEA with respect to olive oil hydrolysis was evaluated. The CLEA showed more than 90% residual activity even after 10 cycles of repeated use.     

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.     

A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates

Cross-linked enzyme aggregates (CLEAs) were prepared from several enzymes (penicillin G acylase, hydroxynitrile lyase, alcohol dehydrogenase, and two different nitrilases) by precipitation and subsequent cross-linking using dextran polyaldehyde. In most cases, higher immobilization yields were obtained using the latter cross-linker as compared with the commonly used glutaraldehyde. Active site titration of penicillin acylase CLEAs showed that the higher activity originated from a significantly lower loss in active sites using dextran polyaldehyde as a cross-linking agent. It is proposed that macromolecular cross-linkers are too large to penetrate the protein active site and react with catalytically essential amino acid residues.     

Adding an appropriate amino acid during crosslinking results in more stable crosslinked enzyme aggregates

Carrier free immobilization, especially crosslinked enzyme aggregates (CLEAs), has become an important design for biocatalysis in several areas. Adding amino acids during formation of CLEAs was found to give biocatalysts more stable at 55 °C and in the presence of 60% acetonitrile. The half-lives of CLEAs prepared with and without Arg addition were 21 and 15 h (subtilisin) and 4 and 1.6 h (α-chymotrypsin) at 55 °C, respectively. The corresponding half-lives during acetonitrile presence were 4.1 and 3.0 h (subtilisin) and 39 and 22 min (α-chymotrypsin), respectively. CLEAs made with Arg had higher percentages of alpha helix. CLEAs made by adding Lys, Ala, or Asp also were more stable. In the case of Thermomyces lanuginosus lipase (TLL), CLEA with Ala was even more stable than CLEA with Arg. The addition of a suitable amino acid, thus, enhances CLEA stabilities. The results are discussed in the light of earlier results on chemical modification of proteins and the observation that the Arg/Lys ratio is invariably high in the case of enzymes from thermophiles.     

A new method for spectrophotometric assay of activity of cross-linked penicillin acylase aggregates

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability of immobilized enzyme preparations.     

Crosslinked enzyme aggregates in hierarchically-ordered mesoporous silica: a simple and effective method for enzyme stabilization

alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for 2 weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.     

Effect of the degree of cross-linking on the properties of different CLEAs of penicillin acylase

Cross-linked enzyme aggregates (CLEAs) are novel type biocatalysts well suited to catalyze reactions of organic synthesis. Penicillin acylase is a versatile enzyme that can both hydrolyze and synthesize β-lactam antibiotics. CLEAs and CLEAs covered with polyionic polymers (polyethyleneimine and dextran sulfate at two different enzyme to polymer ratios) were prepared at varying cross-linking agent to enzyme ratio: 0.15 and 0.25. Results are presented on the effect of such variables on immobilization yield, specific activity, stability and performance of penicillin acylase CLEAs in the kinetically controlled synthesis of cephalexin. The cross-linking agent to enzyme ratio had no significant effect on the specific activity of the CLEAs, but affected immobilization yield, stability in ethylene glycol medium and conversion yield and productivity in the synthesis of cephalexin, being always higher at the lower cross-linking agent to enzyme ratio. Best results were obtained with CLEAs at 0.15 glutaraldehyde to enzyme protein ratio: specific activity of hydrolysis and synthesis was 708 and 325 UI/g_CLEA respectively, conversion yield was 87%, specific productivity was 5.4 mmol cephalexin/(g_CLEA·h) and 90% of the enzyme remained active after 170 h at operating conditions.     

Highly efficient biosynthesis of sucrose-6-acetate with cross-linked aggregates of Lipozyme TL 100 L

As a short chain monoester, sucrose-6-acetate (S-6-a) is a key intermediate in the preparation of an eminent sweetener (sucralose). To replace the traditional multi-step chemical route for sucralose biosynthesis, enzymatic synthesis of S-6-a was investigated, using cross-linked enzyme aggregates (CLEAs) of Lipozyme TL 100 L. The optimal CLEA preparation conditions was obtained as follows: using 33.3% (v/v) PEG600 co-precipitated with additive of D-sorbierite, then cross-linking with 1.5% (v/v) glutaraldehyde at 0 °C for 4 h. As a result, the immobilized Lipozyme had high specific bioactivity (34.64 U/g) of transesterification in non-aqueous media. With these immobilized enzymes, the optimum transesterification conditions were investigated systematically, including CLEA loading, the mole ratio of vinyl acetate versus sucrose, temperature and reaction time, etc. The results showed that the highest concentration and yield of S-6-a was 49.8 g/L and 87.46%, respectively. Further experiments showed that the resulting CLEAs also had much higher operational stability than the commercial Lipozyme TLIM. The present work has paved a new path for the large-scale bioproduction of S-6-a with immobilized lipase in the future.     

Kinetic resolution of (+/-)-1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases

Lipases from two different sources Candida rugosa (CRL) and Burkholderia cepacia (BCL) were formulated as enzyme precipitated and rinsed with organic solvents, organic solvent rinsed enzyme preparation, cross-linked enzyme aggregates (CLEAs) and protein coated micro-crystals (PCMCs). These various enzyme formulates were evaluated for the kinetic resolution of (+/-)-1-phenylethanol in ionic liquid [Bmim][PF(6)] by transesterification with vinyl acetate. Of all the enzyme forms evaluated EPRP and PCMC in the case of CRL showed the best results with 26 % (E value=153) and 53% (E value=79) conversion, respectively, at 35 degrees C in 24h. Carrying out this conversion with PCMC at lower temperature of 25 degrees C further improved the E value to 453 (with 44% conversion in 12h). For BCL the acetone-rinsed enzyme preparation (AREP), CLEA and PCMC performed equally well with % conversion of 50 and 99 ee(p) (%) (E value >1000) in just 2h, whereas, the free lipase gave only 8% conversion.     

聚丙烯腈纤维固定化青霉素酰化酶合成头孢氨苄的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键结合到聚丙烯腈纤维的衍生物上。制成的丝状固定化青霉素酰化酶表现活力达 1 5 3U g(湿重 )。固定化酶合成头孢氨苄的最适pH为 6 5 ,最适温度为 40℃。 7 ADCA的投料浓度以 4%为好 ,7 ADCA与PGME的投料量比率为1∶2 ,最佳用酶量为 1 70U g 7 ADCA。在pH6 5、温度 3 0℃时 ,固定化酶对 7 ADCA的表观米氏常数K7 ADCA为 0 1 6 2mol L ,对PGME的表观米氏常数KPGME为 0 3 6 4mol L ,最大反应速度Vmax为0 0 4 6 2mol·L- 1·min- 1,用固定化酶合成头孢氨苄 ,使用 5 0次保留酶活力 83 9%     

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.     

Preparation of cross-linked tyrosinase aggregates

Tyrosinase from mushroom was immobilized as a cross-linked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. The effects of precipitation and cross-linking on CLEA activity were investigated and the immobilized tyrosinase was characterized. Sixty percent ammonium sulfate saturation and 2% glutaraldehyde were used; a 3-h cross-linking reaction at room temperature, at pH 7.0 was performed; particle sizes of the aggregates were reduced; consequently, 100% activity recovery was achieved in CLEAs with enhanced thermal and storage stabilities. Slight changes in optimum pH and temperature values of the enzyme were recorded after immobilization. Although immobilization did not affect V_max, substrate affinity of the enzyme increased. Highly stable CLEAs were also prepared from crude mushroom tyrosinase with 100% activity recovery.     

Cross-linked aggregates of penicillin acylase: robust catalysts for the synthesis of β-lactam antibiotics

A novel method for the immobilization of penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11) is reported. It involves the physical aggregation of the enzyme, followed by chemical cross-linking to form insoluble cross-linked enzyme aggregates (CLEAs). Compared with conventionally immobilized penicillin G acylases, these CLEAs possess a high specific activity as well as a high productivity and synthesis/hydrolysis (S/H) ratio in the synthesis of semi-synthetic antibiotics in aqueous media. Moreover, they are active in a broad range of polar and apolar organic solvents.     

Quantitative characteristic of the catalytic properties and microstructure of cross-linked enzyme aggregates of penicillin acylase

The microstructure and the catalytic properties of cross-linked enzyme aggregates (CLEA) of penicillin acylase (PA) obtained under different conditions were investigated. The period of time left between the enzyme precipitation and the cross-linking step was found to influence the structural organization of the resulting enzyme preparation. Confocal fluorescent microscopy of the so-called “fresh” and “mature” CLEAs PA allowed to estimate the “characteristic” diameter of CLEA PA particles, which appeared to be about 1.6 μm, and revealed that the “mature” type was composed of relatively big particles as compared to the “fresh” type. Complementary kinetic studies showed that the “mature” CLEA PA were more effective in both hydrolytic and synthetic reactions. It was suggested that the aggregate size might regulate the extent of covalent modification of PA and thereby influence the catalytic properties of CLEA.