Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

海藻酸-SiO2杂化凝胶固定化洋葱伯克霍尔德茵脂肪酶的研究

利用四乙氧基硅烷（TEOS）原位水解法将SiO2掺杂于海藻酸（ALG）凝胶中,通过双交联制备出新型ALG—SiO2杂化凝胶以固定化洋葱伯克霍尔德菌脂肪酶。结果表明,固定化酶的最优条件：质量分数为2．0％的ALG、0．2mol／LCaCl2、V（ALG）／V（TEOS）为5、加酶量为1gALG加100mg酶粉、固定化60min、采用直径为0．8mm的针头滴定、真空冷冻干燥。在此条件下,酶蛋白的包埋率可达100％,酶活回收率可达91％。固定化酶的最适pH为8．0,最适作用温度为50℃,重复使用8次后,酶活性仍能保持80％以上。ALG—Si02杂化凝胶的场扫描电镜（FESEM）观察发现凝胶的整体构造仍然是海藻酸凝胶骨架;与ALG凝胶平滑的内部相比较,杂化凝胶仍具有完整的网络结构,但内部更为粗糙,结构更为致密。     

Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles

Mucor javanicus lipase was effectively immobilized on silica nanoparticles which were prepared by Stöber method. Glycidyl methacrylate (GMA), which bears a reactive epoxide group, was incorporated onto the surface of the nanoparticles and the epoxide groups were directly used for multipoint coupling of the enzyme. We also introduced amine residues by coupling ethylene diamine (EDA) to the epoxide group of GMA. M. javanicus lipase was covalently immobilized onto the amine-activated silica nanoparticles by using glutaraldehyde (GA) or 1,4 phenylene diisothiocyanate (NCS) as a coupling agent. The lipase loading capacities of the EDA-GA and EDA-NCS nanoparticles (81.3 and 60.9 mg g⁻¹, respectively) were much higher than that of the unmodified GMA nanoparticles (18.9 mg g⁻¹). The relative hydrolytic activities in an aqueous medium of the lipases immobilized on EDA-GA and EDA-NCS attached silica nanoparticles (115% and 107%, respectively) were significantly high and almost in the same range with the free enzyme. This may be due to the improvement of the enzyme–substrate interaction by avoiding the potential aggregation of free lipase molecules. The immobilized lipases were also more resistant to temperature inactivation than the free form. This work demonstrates that the size-controlled silica nanoparticles can be efficiently employed as host materials for enzyme immobilization leading to high activity and stability of the immobilized enzymes.     

Immobilization of lipase into mesoporous silica particles by physical adsorption

Mesoporous silica particles for immobilization of lipase from Candida rugosa were prepared by precipitation and aggregation of primary particles from highly basic sodium silicate solution but without addition of templates. The average pore size of the material was 15.8 nm, which allowed enzyme adsorption inside the pores and high enzyme loading. Specific surface area of the material was found to be 359 m²g^?1. A loading of 100 mglipasegdrysilica^?1 was obtained at initial enzyme concentration of 1.8 mgmL^?1 by physical adsorption. The FTIR spectrum showed the structural conformation of lipase to be retained after adsorption into the mesoporous silica support. Although the efficiency of the mesoporous biocatalyst was shown to be lower than that of the free enzyme, the immobilized enzyme showed enhanced thermal stability and could be desorbed with Triton X-100, indicating the hydrophobic nature of the adsorption.     

包埋法固定化真菌漆酶及其应用研究

采用海藻酸钠包埋法固定真菌漆酶,海藻酸钠和CaCl2的最佳浓度分别为3%和4%,最佳给酶量为30U,最大回收率为48.0%.与游离漆酶相比,固定化漆酶的热稳定性有明显改善,最适反应pH向酸性方向漂移0.5,最适反应温度提高了5℃.使用固定化酶处理低浓度造纸废水,运行8批次后残留酶活为64%.     

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.     

Characterization of <Emphasis Type="Italic">Mucor miehei</Emphasis> lipase immobilized on polysiloxane-polyvinyl alcohol magnetic particles

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding. The resulting immobilized biocatalyst was recycled by seven assays, with a retained activity around 10% of its initial activity. K_m and V_max were respectively 228.3 M and 36.1 U mg of protein^–1 for immobilized enzyme. Whereas the optimum temperature remained the same for both soluble and immobilized lipase (45 °C), there was a shift in pH profiles after immobilization. Optimum pH for the immobilized lipase was 8.0. Immobilized enzyme showed to be more resistant than soluble lipase when assays were performed out of the optimum temperature or pH.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Immobilization of lipase by salts and the transesterification activity in hexane

Lipases from six different sources were immobilized on Celite and five types of salt. The transesterification activities in hexane for lipases immobilized on EDTA-Na₂ increased by 463% for the lipase from Candida rugosa (CRL), 2700% for the lipase from Candida sp. (CSL) and 1215% for the lipase from Pseudomonas sp. (PSL), compared to the salt-free enzyme. With 0.5% sucrose for CRL or 1% sorbitol for PSL as the lyoprotectant during lyophilization process, transesterification activity increased by 100% and 13%, respectively, compared to the immobilized enzyme on EDTA-Na₂ without lyoprotectant.     

Sugar ester synthesis by a mycelium-bound Mucor circinelloides lipase in a micro-reactor equipped with water activity sensor

The mycelium-bound Mucor circinelloides lipase was used for the synthesis of esters of saccharides and fatty acids in 37 ml reactor equipped with magnetic stirrer and water activity sensor. Either di-n-pentyl ether or the mixture of di-n-pentyl and petroleum ethers were applied as reaction media. Water activity sensor provided on-line monitoring of this parameter and control of continuous processes of ester synthesis. It was found that two natural antioxidants, i.e. carotene and astaxanthin activated this lipase in organic solvents that could be beneficial for the synthesis of esters of compounds sensitive to oxidation, e.g. polyunsaturated fatty acids.     

Synthesis of alginate‐silica hybrid hydrogel for biocatalytic conversion by β‐glucosidase in microreactor

The organic–inorganic hybrid materials have been used in different fields to immobilize biomolecules since they offer many advantages. The aim of this study was to optimize and characterize the alginate‐silica hybrid hydrogel as a stable and injectable form for microfluidic systems using internal gelation method and increase the stability and activity of immobilized enzyme for biocatalytic conversions as well. Characterization was carried out by scanning electron microscopy, energy dispersive spectroscopy/mapping, Brunauer–Emmett–Teller, Barrett–Joyner–Halenda, and Fourier‐transform infrared spectroscopy analyses, and the shrinkages of monoliths were evaluated. Subsequent to optimizing the enzyme concentration (40 μg), hydrolytic conversion of 4‐nitrophenyl β‐d ‐glucopyranoside (pNPG) was performed to understand the behavior of the bioconversion in the microfluidic system. The yield was 94% which reached the equilibrium at 24 h indicating that the alginate‐silica gel derived microsystem overcome some drawbacks of monolithic systems. Additionally, bioconversion of Ruscus aculeatus saponins was carried out at the same setup in order to obtain aglycon part, which has pharmaceutical significance. Although pure aglycon could not be achieved, an intermediate compound was obtained based on the HPLC analysis. The developed formulation can be utilized for various life science applications.     

Effects of water and silica gel on enzyme agglomeration in organic solvents

It has been observed that water, which is absolutely essential for enzyme activity, can induce the agglomeration of enzyme particles in organic media. Although enzyme agglomeration is significant in that it usually reduces enzyme activity and stability, little attention has been paid to the quantitative analysis of enzyme agglomeration behavior in nonaqueous bioactalytic systems. In this study, the effects of water and silica gel on enzyme agglomeration were investigated usingCandida rugosa lipase and cyclohexane as a model enzyme and an organic medium. The extent of enzyme agglomeration was quantified by sieve analysis of freeze-dried agglomerates. Increasing the water content of the medium increased the size of the enzyme agglomerates, and it was found that water produced during the esterification reaction could also promote the agglomeration of enzyme particles suspended in organic media. On the other hand, the size of the enzyme agglomerates was remarkably reduced in the presence of silica gel at the same water content. We also show that this increase in the size of enzyme agglomerates results in lower reaction rates in organic solvents.     

Activation of immobilized lipase in non-aqueous systems by hydrophobic poly-DL-tryptophan tethers

Many industrially important reactions use immobilized enzymes in non-aqueous, organic systems, particularly for the production of chiral compounds such as pharmaceutical precursors. The addition of a spacer molecule ("tether") between a supporting surface and enzyme often substantially improves the activity and stability of enzymes in aqueous solution. Most "long" linkers (e.g., polyethylene oxide derivatives) are relatively hydrophilic, improving the solubility of the linker-enzyme conjugate in polar environments, but this provides little benefit in non-polar environments such as organic solvents. We present a novel method for the covalent immobilization of enzymes on solid surfaces using a long, hydrophobic polytryptophan tether. Candida antarctica lipase B (CALB) was covalently immobilized on non-porous, functionalized 1-microm silica microspheres, with and without an intervening hydrophobic poly-DL-tryptophan tether (n approximately 78). The polytryptophan-tethered enzyme exhibited 35 times greater esterification of n-propanol with lauric acid in the organic phase and five times the hydrolytic activity against p-nitrophenol palmitate, compared to the activity of the same enzyme immobilized without tethers. In addition, the hydrophobic tethers caused the silica microspheres to disperse more readily in the organic phase, while the surface-immobilized control treatment was less lipophilic and quickly settled out of the organic phase when the suspensions were not vigorously mixed.     

有机相中固定化脂肪酶促有机硅烷醇的转酯

探讨了有机相中固定化脂肪酶（Lipozyme）催化非天然的有机硅院醇与脂肪酸酯转酯反应的可能性;系统地研究了有机溶剂特性、水活度、有机硅烷醇结构、脂肪酸酯碳链长等因素对转酯反应的影响。     

Enhancement of Candida rugosa lipase activity in non-aqueous media by co-lyophilization with amphiphiles from aqueous solution

Modified Candida rugosa lipase was co-lyophilized with two gemini-type amphiphiles, l- and d-2-(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionylamino)-pentanedioic acid didodecyl ester or dodecanoic acid 2-[(3-bis-[3-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl -propionyl)-(2-dodecanoyloxy-ethyl)-amino]-ethyl ester. Enzymatic activities of the modified lipases in the transesterification between racemic 2,2-dimethyl-1,3-dioxolane-4-methanol and vinyl butyrate in cyclohexane were enhanced as much as by 37-78, 1.5–5- and 41–83-fold of magnitude relative to that of native enzyme, respectively. The lack of significant enhancement of the enzymatic activity, only in the case of the d-isomeric amphiphile-modified lipase, was considered from the topological view of the amphiphile.     

Kinetics of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl Acetate Hydrolysis Catalyzed by <Emphasis Type="Italic">Mucor javanicus</Emphasis> Lipase in AOT Reverse Micellar Solutions Formulated in Different Organic Solvents

The rate of hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by Mucor javanicus lipase has been measured in AOT reverse micellar solutions formulated in aliphatic hydrocarbons, aromatic hydrocarbons and a chlorinated compound. The study has been performed at a single value of W = ([water]/[AOT]) = 6.0. Fluorescence decay measurements of intrinsic enzyme fluorescence, mainly due to tryptophan residues, in the different reverse micellar systems were also carried out, in an attempt to obtain some insight on the effect of the organic solvent on the protein conformation. Differences observed in the kinetics of the fluorescence decays of tryptophan residues of the lipase incorporated to the micelles with the different external organic solvents were also found in the catalytic behaviour of the enzyme. In particular, it is observed that the contribution of the long lived component of the fluorescence decay is considerably higher (ca. 40%) in aliphatic than in aromatic solvents (ca. 15%), indicating significant differences in the protein conformation. This effect of the organic solvent on the protein conformation is also observed in the enzymatic activity, which is higher in the aromatic than in the aliphatic solvents.     

Stereochemistry of a diastereoisomeric amphiphile and the species of the lipase influence enzyme activity in the transesterification catalyzed by a lipase-co-lyophilizate with the amphiphile in organic media

Modified Candida rugosa and Pseudomonas cepacia lipase (CRL and PCL) were co-lyophilized with two pairs of synthetic diastereoisomeric amphiphiles, d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-propyl]-carbamoyl-propionylamino)-pentanedioic acid didodecyl ester (d- and l-BIG2C₁₂CA); d- and l-2-(2,3,4,5,6-pentahydroxy-hexanoylamino)-pentanedioic acid didodecyl ester (d- and l-2C₁₂GE). Enzyme activities of the modified lipase in the transesterification in organic solvent were evaluated. Both pairs of the diastereoisomeric amphiphiles showed enhanced enzyme activity in the transacetylation between racemic sulcatol and isopropenyl acetate in diisopropyl ether, catalyzed by the PCL-co-lyophilizate, by 19–48 fold when compared to the native lipase lyophilized from buffer alone independent of the stereochemistry of the amphiphiles, while in the case of the CRL-co-lyophilizate only the l-BIG2C₁₂CA showed enhanced enzyme activity in the transbutyrylation between racemic solketal and vinyl butyrate in cyclohexane as high as 68–78 fold.     

Spatial distribution of major bacterial species and different volatile fatty acids in a two-phase anaerobic biofilm reactor with PVA gel beads as bio-carrier

Abstract

Conventional completely mixed anaerobic treatment systems limit the chances of the different species of bacteria to spatially group together according to their mutual cooperation and as a result, show a lower efficiency and vulnerability towards shock situations. It is interesting to know about the stratification of the different bacterial species participating in the degradation process and the intermediates that they produce. In this study, we established and optimized a two-phase anaerobic packed bed biofilm reactor system (AnPBR) with porous PVA gel beads used as bio-carriers and ran the reactor system in a steady state to observe the VFAs produced along with the microbial diversity of the predominant species at different stages of the reactor system. We observed that acetate and butyrate were the predominant intermediate VFAs while concentrations of other VFAs such that propionic acid were low. Acetobacterium and Clostridium were found to be the most abundant bacterial species in acidogenic reactor while methanogenic reactor was highly enriched with Methanobacterium and Methanosarcina. Apart from the above, syntrophic populations such as Syntrophobactor wolinii were also observed to be dominant in both the reactors – especially towards the end of acidogenic reactor and the initial part of the methanogenic reactor.     

Study of DNA integrity in somatic cells and spermatozoa by single-cell gel electrophoresis with silver-nitrate staining

In the present work, genome instability in human and bovine peripheral blood lymphocytes and spermatozoa was studied by the method of DNA microelectrophoresis with subsequent staining of single cells with silver nitrate. A comparative analysis of the types of damage to human and bovine lymphocytes and spermatozoa genomes was performed. In the group of healthy donors, the spontaneous frequency of DNA damage revealed by single cell DNA microelectrophoresis did not exceed 9% and amounted, on average, to 4.8 ± 1.2%. In studying the effect of the duration of cryoconservation on bovine spermatozoa, no significant changes were revealed between the group of bulls whose spermatozoa were stored for less than one year (3.1 ± 0.9%) and the group of animals whose spermatozoa were under conditions of cryoconservation for more than 20 years (4.3 ± 0.5%). From the obtained single-cell DNA microelectropheretic data on the types and frequencies of DNA damage, a conclusion was made regarding the possibility of using a light variant based on cell staining with silver nitrate for the detection of genome instability, not only in somatic, but also in reproductive, cells.