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.     

Preparation and characterization of cross-linked laccase aggregates and their application to the elimination of endocrine disrupting chemicals

Laccase from the white rot fungus Coriolopsis polyzona was immobilized for the first time through the formation of cross-linked enzyme aggregates (CLEAs). Laccase CLEAs were produced by using 1000g of polyethylene glycol per liter of enzyme solution as precipitant and 200muM of glutaraldehyde as a cross-linking agent. These CLEAs had a laccase activity of 148Ug(-1) and an activity recovery of 60.2% when using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as substrate. CLEAs formed by co-aggregation with bovine serum albumin (BSA) as a stabilizer showed lower laccase activity and affinity for ABTS than those without BSA. The CLEAs co-aggregated with BSA showed higher residual activity against a protease, NaN(3), EDTA, methanol and acetone. The thermoresistance was higher for CLEAs than for free laccase and also higher for CLEAs co-aggregated with BSA than for simple CLEAs when tested at a pH of 3 and a temperature of 40 degrees C. Finally, laccase CLEAs were tested for their capacity to eliminate the known or suspected endocrine disrupting chemicals (EDCs) nonylphenol, bisphenol A and triclosan in a fluidized bed reactor. A 100-ml reactor with 0.5mg of laccase CLEAs operated continuously at a hydraulic retention time of 150min at room temperature and pH 5 could remove all three EDCs from a 5mgl(-1) solution.     

Cross-linked enzyme aggregates of β-glucosidase from Prunus domestica seeds

Cross-linked enzyme aggregates (CLEAs) of β-glucosidase were prepared and characterized. Under the optimum conditions, the activity recovery of CLEAs reached 84?%. The reduction by NaBH(4) resulted in slightly lower activities of CLEAs, while their thermostability was enhanced. CLEAs were more thermally stable than free enzyme (half lives, 973 vs. 518?min at 50?°C), while less stable than seed meal (half life, 1,090?min). In 90?% (v/v) t-butanol, the half lives of CLEAs and free enzyme were 53 and 6.7?h, respectively. Besides, the catalytic efficiency (V (max)/K (m)) of CLEAs was comparable to free enzyme (0.42 vs. 0.47?min(-1) mg(-1)). This carrier-free immobilized enzyme had a network structure with multiple layers. The productivity of salidroside using CLEAs reached 150?g/l?g catalyst, while being 6.3?g/l?g with seed meal.     

Enhanced esterification activity through interfacial activation and cross‐linked immobilization mechanism of Rhizopus oryzae lipase in a nonaqueous medium

Interfacial activation via surfactant (Tween 80, Triton X‐100) treatment was conducted to improve the esterification activity of Rhizopus oryzae lipase that had undergone immobilization through cross‐linked enzyme aggregates (CLEA®) technique. Surfactant pretreated immobilized enzymes exhibited better esterification activity compared to free and non‐pretreated immobilized enzyme (Control CLEAs) since higher conversion rates were obtained within shorter times. The superiority of surfactant pretreated CLEAs, especially Tween 80 pretreated CLEAs (T 80 PT CLEAs), were clearly pronounced when longer alcohols were used as substrates. Conversion values exceeded 90% for octyl octanoate, oleyl octanoate and oleyl oleate synthesis with T 80 PT CLEAs whereas Control CLEAs and free enzyme showed no activity. Maximum conversions were achieved in the case equal molars of the substrates or in the case excess of the alcohol to acid in cyclohexane. In solvent free medium containing equal molars of substrates the conversion rates were 85% and 87% with T 80 PT CLEAs respectively for octyl octanoate and oleyl oleate within 2 hours. T 80 PT CLEAs showed 59% of its original activity after 7 consecutive usage for oleyl oleate synthesis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:899–904, 2016     

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.     

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.     

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.     

Kinetic resolution of racemic naproxen methyl ester by magnetic and non-magnetic cross-linked lipase aggregates

Abstract

In this study, the non-magnetic and the magnetic cross-linked enzyme aggregates (CLEAs) from Candida rugosa lipase were synthesized to catalyze the kinetic resolution reaction of naproxen methyl ester (NME). Magnetic iron oxide nanoparticles (MIONPs) were produced through co-precipitation method and their surfaces were modified by silanization reaction. The MIONPs were used as a platform to synthesize the magnetic CLEAs (M-CLEAs). The biocatalysts and MIONPs synthesized were characterized by FTIR spectroscopy and SEM analysis. The kinetic resolution of racemic NME was studied in aqueous buffer solution/isooctane biphasic system to compare the performance of M-CLEAs and CLEAs. The effects of reaction parameters such as temperature, pH, stirring rate on the enantiomeric excess of the substrate (ee_s%) were investigated in a batch reactor system. The activity recovery of CRL enzyme in CLEAs was higher than M-CLEAs. Compared with M-CLEAs, CLEAs biocatalysts had previously reached ee_s% values. Although both biocatalysts showed similar cavity structure from SEM analysis, the lower performance of M-CLEAs may be due to the different microenvironments of M-CLEAs from CLEAs. However, the reusability performance of M-CLEAs was higher than that of CLEAs. The optimal reaction conditions for M-CLEAs and CLEAs were found to be 37?°C, pH 7.5, and 300?rpm.     

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.     

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.     

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.     

Improved esterification activity of Candida rugosa lipase in organic solvent by immobilization as Cross-linked enzyme aggregates (CLEAs)

Cross-linked enzyme aggregates (CLEA^®s) were prepared from Candida rugosa lipase (CrL) using glutaraldehyde as the cross-linker. The optimum conditions of the immobilization process were determined (precipitant: ethanol, crosslinker concentration: 25 mM, enzyme concentration: 50 mg/ml, crosslinking time: 45 min.). CLEAs were shown to have several advantages compared to the free enzyme. They were more stable at 50 °C and 60 °C and had good reusability; retaining 40% of their initial activity after 15 recycles in aqueous media and remaining constant at that level thereafter, suggesting some initial leaching in water. The CLEAs catalyzed esterification reactions in cyclohexane, affording higher conversions than with the free enzyme, especially when longer fatty acids and alcohols were used as substrates.     

Cross-linking enzyme aggregates in the macropores of silica gel: A practical and efficient method for enzyme stabilization

Cross-linked enzyme aggregates of papain were prepared in commercial macroporous silica gel (CLEAs-MSG) in order to improve the operability and mechanical stability of CLEAs. CLEAs-MSG was obtained from simple adsorption, precipitation and one-step-cross-linking. CLEAs-MSG was characterized by stable structure that did not leak out enzyme from the macropores because of covalent bonding between CLEAs and MSG. The optimal temperature of papain CLEAs in MSG was 40–90 °C and the optimal pH was 7.0, which were improved compared to free papain and CLEAs. The CLEAs-MSG also enhanced the storage stability and thermal stability. Moreover, the CLEAs-MSG exhibited good reusability due to its suitable size and active properties. By using CLEAs-MSG of papain as biocatalyst, the kinetically controlled z-Ala-Gln synthesis was achieved with the yield of 32.9%, which was almost equal to that by using free papain as biocatalyst.     

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.     

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.     

Optimization protocols and improved strategies of cross-linked enzyme aggregates technology: current development and future challenges

Cross-linked enzyme aggregate (CLEA) technology has been regarded as an effective carrier-free immobilization method. This method is very attractive due to its simplicity and robustness, as well as for the possibility of using the crude enzyme extract and the opportunity to co-immobilize multiple different enzymes. The resulting CLEAs generally exhibit high catalyst productivities, improved storage and operational stability and are easy to recycle. Nowadays, although the technology has been applied to various enzymes, some undesirable properties have limited its further application. To overcome these limitations, novel strategies have been developing in recent years. This mini-review focuses on process optimization, new improved strategies and the latest advances on CLEAs technology.     

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。     

Effect of different parameters on the hydrolytic activity of cross-linked enzyme aggregates (CLEAs) of lipase from Thermomyces lanuginosa

Cross-linked enzyme aggregates (CLEAs) of lipase from Thermomyces lanuginosa (TLL) were synthesized using (NH₄)₂SO₄ as precipitant and glutaraldehyde as cross-linking agent. CLEAs were assayed for their hydrolytic activity in a reaction performed in an emulsioned medium. The effects of the amount of precipitant, cross-linker, and different additives such as protein cofeeder, oleic acid, n-heptane, sodium dodecyl sulfate (SDS), polyethylenglicol (PEG) and ethylendiamine were studied at selected ratios with respect to TLL mass. Traditional non-layered CLEAs of TLL showed recovered activities between 3 and 31% when compared with native lipase. Novel TLL layered CLEAs consisting of a protein cofeeder core and successive layers of target lipase showed an important increase in their retained activity. The highest recovered activity was found for the one-layered non-additivated CLEAs of TLL which showed a recovered activity of 75%.     

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.     

Co-aggregation of enzymes and polyethyleneimine: a simple method to prepare stable and immobilized derivatives of glutaryl acylase

We have developed a novel methodology that allowed the preparation of cross-linked enzyme aggregates (CLEAs) of glutaryl acylase (GAC) by co-aggregation of the enzyme with an aminated polymer: polyethyleneimine (PEI). The preparation of CLEAs of GAC from Pseudomonas sp. is not possible when using poly(ethylene glycol) and glutaraldehyde directly as precipitating and cross-linking agent, respectively. This problem arises probably from the low content of surface Lys groups of GAC which prevents an efficient cross-linking of the enzyme molecules in the aggregate. This fact was proven by the release of enzyme molecules from the aggregate and the solubilization of the enzyme when eliminating the precipitating agent. Our new co-aggregation system favors the cross-linking between the very reactive and abundant primary amino groups of the PEI and the primary amino groups on the enzyme surface. The use of PEI prevents the release of enzyme molecules from the aggregate. By this methodology, we prepared a very stable immobilized derivative of GAC. After optimization of the glutaraldehyde treatment conditions, the stability of the enzyme was significantly improved. It kept more than 60% of its initial activity after 72 h of incubation at 45 degrees C, whereas the soluble enzyme was fully inactivated in 2.5 h of incubation in the same conditions. Therefore, we have a new protocol for carrying out the preparation of cross-linked aggregates of enzymes with a low number of lysines on its surface.