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.     

Preparation of nano-enzyme aggregates by crosslinking lipase with sodium tripolyphosphate

Cross-linked enzyme aggregates (CLEAs) are considered as an effective tool for the immobilization of enzyme. The ionic cross-linking agent-sodium tripolyphosphate (TPP) was first used in preparing CLEAs. Aspergillus niger lipase was precipitated with ammonium sulfate and further cross-linked by TPP. The factors including enzyme concentration, pH of cross-linking medium, TPP dosage and cross-linking time were optimized. Maximum recovery activity (99.5 ± 0.634 %) and cross-linking yield (88.4 ± 0.46 %) can be obtained under the optimal process conditions, which can illustrate TPP had little effect on enzyme activity. CLEAs showed improved activity over broad pH and temperature range compared to the free enzyme. The thermal stability was obviously improved compared to free enzyme under the optimal temperature (40℃) and the half-life was 7.5-fold higher than that of free enzyme. Moreover, scanning electron microscopy (SEM) revealed that CLEAs had a cavity with porous structure and the particle size was 249 ± 3.98 nm. X-ray diffraction (XRD) showed the crystallinity of the CLEAs decreased. The changes in secondary structures of CLEAs revealed the increment in conformational rigidity. Such results suggested that the CLEAs has ideal application prospects.     

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.     

Characteristic features and biotechnological applications of cross-linked enzyme aggregates (CLEAs)

Cross-linked enzyme aggregates (CLEAs) have many economic and environmental benefits in the context of industrial biocatalysis. They are easily prepared from crude enzyme extracts, and the costs of (often expensive) carriers are circumvented. They generally exhibit improved storage and operational stability towards denaturation by heat, organic solvents, and autoproteolysis and are stable towards leaching in aqueous media. Furthermore, they have high catalyst productivities (kilograms product per kilogram biocatalyst) and are easy to recover and recycle. Yet another advantage derives from the possibility to co-immobilize two or more enzymes to provide CLEAs that are capable of catalyzing multiple biotransformations, independently or in sequence as catalytic cascade processes.     

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.     

Cross-linked esterase aggregates (CLEAs) using nanoparticles as immobilization matrix

Abstract

The present study focusses on the enhancement of the catalytic activity and stability of an acetylesterase enzyme isolated from Staphylococcus spp. as Cross-Linked Enzyme Aggregates (CLEAs). The various parameters governing the activity of CLEAs were optimized. The magnetite and graphene oxide nanoparticles were successfully prepared via the chemical co-precipitation and Hummer's method, respectively. These nanoparticles supported the preparation as magnetite nanoparticle-supported cross-Linked Enzyme Aggregates (MGNP-CLEAs) and graphene oxide-supported Cross-Linked Enzyme Aggregates (GO-CLEAs). The activity and stability of these immobilized CLEAs were compared with the free enzyme at various temperature, pH, and organic solvents along with its storage stability and reusability. The immobilized preparations were analyzed by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR) techniques. Acetylesterase precipitated with 60% saturated ammonium sulfate salt (SAS) solution and cross-linked with 100?mM glutaraldehyde for 4?h at 30?°C was found to be optimal to produce CLEAs with highest activity recovery of 99.8%. The optimal pH at 8.0 and temperature at 30?°C remained the same for both the free and immobilized enzyme, respectively. Storage stability significantly improved for the immobilized enzyme as compared to free enzyme. SEM showed type-I aggregate and FT-IR revealed the successful immobilization of the enzyme. MGNP-CLEAs were found to have better activity and stability in comparison to other immobilized preparations.     

Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.     

Hybrid Magnetic Cross-Linked Enzyme Aggregates of Phenylalanine Ammonia Lyase from Rhodotorula glutinis

Novel hybrid magnetic cross-linked enzyme aggregates of phenylalanine ammonia lyase (HM-PAL-CLEAs) were developed by co-aggregation of enzyme aggregates with magnetite nanoparticles and subsequent crosslinking with glutaraldehyde. The HM-PAL-CLEAs can be easily separated from the reaction mixture by using an external magnetic field. Analysis by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) indicated that PAL-CLEAs were inlayed in nanoparticle aggregates. The HM-PAL-CLEAs revealed a broader limit in optimal pH compared to free enzyme and PAL-CLEAs. Although there is no big difference in K_m of enzyme in CLEAs and HM-PAL-CLEAs, V_max of HM-PAL-CLEAs is about 1.75 times higher than that of CLEAs. Compared with free enzyme and PAL-CLEAs, the HM-PAL-CLEAs also exhibited the highest thermal stability, denaturant stability and storage stability. The HM-PAL-CLEAs retained 30% initial activity even after 11 cycles of reuse, whereas PAL-CLEAs retained 35% of its initial activity only after 7 cycles. These results indicated that hybrid magnetic CLEAs technology might be used as a feasible and efficient solution for improving properties of immobilized enzyme in industrial application.     

Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases

Cross-linked enzyme aggregates (CLEA^®) were prepared from laccases from three different sources: Trametes versicolor, Trametes villosa and Agaricus bisporus. The effect of the various parameters – nature of the precipitant, pH, temperature, glutaraldehyde concentration and cross-linking time – on the activity recovery and storage and operational stability of the resulting CLEAs was different. The laccase CLEAs exhibited the expected increased stability compared to the free enzyme but there was no direct correlation with the number of surface lysine residues in the latter. It is clearly not the only parameter influencing the properties of the CLEA. Co-aggregation with albumin did not improve the stability. The laccase CLEAs, in combination with the stable N-oxy radical, TEMPO, were shown to be active and stable catalysts for the aerobic oxidation of linear C₅–C₁₀ aliphatic alcohols, to the corresponding aldehydes, in aqueous buffer (pH 4). Rates were an order of magnitude higher than those observed with the corresponding free enzyme and the CLEAs could be recycled several times without appreciable loss of activity. The addition of water immiscible or water miscible solvents showed no further improvement in rate compared with reactions in aqueous buffer alone.     

Utilization of cross‐linked laccase aggregates in a perfusion basket reactor for the continuous elimination of endocrine‐disrupting chemicals

A perfusion basket reactor (BR) was developed for the continuous utilization of insolubilized laccase as cross‐linked enzyme aggregates (CLEAs). The BR consisted of an unbaffled basket made of a metallic filtration module filled with CLEAs and continuously agitated by a 3‐blade marine propeller. The agitation conditions influenced both the apparent laccase activity in the reactor and the stability of the biocatalyst. Optimal laccase activity was obtained at a rotational speed of 12.5 rps and the highest stability was reached at speeds of 1.7 rps or lower. The activity and stability of the biocatalyst were affected drastically upon the appearance of vortices in the reaction medium. This reactor was used for the continuous elimination of the endocrine disrupting chemicals (EDCs) nonylphenol (NP), bisphenol A (BPA), and triclosan (TCS). Optimization of EDC elimination by laccase CLEAs as a function of temperature and pH was achieved by response surface methodology using a central composite factorial design. The optimal conditions of pH and temperature were, respectively, 4.8 and 40.3°C for the elimination of p353NP (a branched isomer of NP), 4.7 and 48.0°C for BPA, and 4.9 and 41.2°C for TCS. Finally, the BR was used for the continuous elimination of these EDCs from a 5 mg L^?1 aqueous solution using 1 mg of CLEAs at pH 5 and room temperature. Our results showed that at least 85% of these EDCs could be eliminated with a hydraulic retention time of 325 min. The performances of the BR were quite stable over a 7‐day period of continuous treatment. Furthermore, this system could eliminate the same EDCs from a 100 mg L^?1 solution. Finally, a mathematical model combining the Michaelis–Menten kinetics of the laccase CLEAs and the continuous stirred tank reactor behavior of the BR was developed to predict the elimination of these xenobiotics. Biotechnol. Bioeng. 2009;102: 1582–1592. © 2008 Wiley Periodicals, Inc.     

Enhancement of activity of cross-linked enzyme aggregates by a sugar-assisted precipitation strategy: technical development and molecular mechanism

The precipitation of enzyme causes the major activity loss in the conventional protocol for CLEAs preparation. Herein, a sugar-assisted strategy was developed to minimize the activity loss in the step of enzyme precipitation by adding sugar as the stabilizer, which contributed to improve the activity yield of resulting CLEAs. Penicillin G acylase (PGA) was employed as a model enzyme. The effects of glucose, sucrose and trehalose on the activity yields of CLEAs were investigated. The highest activity was obtained in the case of adding trehalose. Confocal laser scanning microscopy and Fourier transform infrared spectroscopy showed that the polar microenvironment and the secondary structure of native enzyme were preserved to some extent when PGA was prepared as sugar-assisted CLEAs, resulting in PGA's higher activity than sugar-free CLEAs. Scanning electron microscope revealed the different inner morphologies, and the kinetic studies showed the higher affinity and resist-inhibition capacity of sugar-assisted CLEAs. Furthermore, stability experiments demonstrated that CLEAs prepared in sugar-assisted strategy remained higher thermal stability when it was incubated at high temperature.     

Purification and in situ immobilization of papain with aqueous two-phase system

Papain was purified from spray-dried Carica papaya latex using aqueous two-phase system (ATPS). Then it was recovered from PEG phase by in situ immobilization or preparing cross-linked enzyme aggregates (CLEAs). The Plackett-Burman design and the central composite design (CCD) together with the response surface methodology (RSM) were used to optimize the APTS processes. The highly purified papain (96-100%) was achieved under the optimized conditions: 40% (w/w) 15 mg/ml enzyme solution, 14.33-17.65% (w/w) PEG 6000, 14.27-14.42% (w/w) NaH2PO4/K2HPO4 and pH 5.77-6.30 at 20°C. An in situ enzyme immobilization approach, carried out by directly dispersing aminated supports and chitosan beads into the PEG phase, was investigated to recover papain, in which a high immobilization yield (>90%) and activity recovery (>40%) was obtained. Moreover, CLEAs were successfully used in recovering papain from PEG phase with a hydrolytic activity hundreds times higher than the carrier-bound immobilized papain.     

Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis

Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH₂ groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.     

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.     

枯草芽孢杆菌FM208849产果胶酶的固定化及酶学性质研究

为了提高游离果胶酶的稳定性,对罗布麻脱胶具有特异性的枯草芽孢杆菌(FM208849)进行产果胶酶发酵时,采用交联酶聚集体(CLEAs)技术制备固定化果胶酶,并对交联果胶酶聚集体的制备条件、酶学性质进行研究。结果表明,游离果胶酶经80%饱和硫酸铵沉淀后,在30℃,经4%的戊二醛溶液交联135 min,所形成的交联果胶酶聚集体的活回收率为61.5%,其最适反应温度45℃和最适pH10,在对交联果胶酶聚集体的热稳定性和有机溶剂稳定性分析中,均显示了比游离酶更高的稳定性。     

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.     

Immobilization of Papain on the Mesoporous Molecular Sieve MCM‐48

The immobilization of papain on the mesoporous molecular sieve MCM‐48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM‐48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half‐life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12‐day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM‐48 with the aid of glutaraldehyde, can be used as a high‐performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.     

Improved biochemical characteristics of crosslinked β-glucosidase on nanoporous silica foams

Large mesoporous cellular foam (LMCF) materials were synthesized using the microemulsion templating route. For the enzyme stabilization, β-glucosidase was immobilized onto mesocellular silica foams (MCFs) in a simple and effective way, a process achieved using enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of crosslinked enzyme aggregates (CLEAs) of nanometer scale. The structural and chemical properties of these prepared materials were characterized by TG, CPMAS NMR and nitrogen adsorption measurements. The crosslinked immobilizates retained activity over wider ranges of temperature and pH than those of the free enzyme. Kinetic parameter (K_m) of the immobilized β-glucosidase is lower than that of its free counterpart. The resulting CLEA was proved to be active and recyclable up to 10 cycles without much loss in activity. This demonstrates its prospects for commercial applications. The immobilizate exhibited enhanced storage stability characteristics than the native enzyme. In contrast to adsorbed GL and covalently bound glucosidase, the resulting crosslinked enzyme aggregates (CLEAs) showed an impressive stability with high enzyme loadings.     

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。