Influence of ionic liquids as additives on sol–gel immobilized lipase

The immobilization of lipase from Candida rugosa, using ionic liquids as additives to protect the inactivation of lipase by released alcohol and shrinking of gel during sol–gel process, was investigated. The influence of various factors, such as structure of ionic liquids, content of ionic liquids and types of precursor in the sol–gel process on the activity and stability of immobilized lipase was also studied. The highest hydrolytic activity of immobilized lipase was obtained when the hydrophilic ionic liquid, [C₂mim][BF₄], was used as an additive, while the highest stability of immobilized lipase was obtained by using hydrophobic ionic liquid, [C₁₆mim][Tf₂N]. Therefore, the binary mixtures of these ionic liquids as additives were used to obtain the optimal immobilized lipase, which shows both high activity and stability. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1:1 at molar ratio of [C₂mim][BF₄] and [C₁₆mim][Tf₂N] were 10-fold and 14-fold greater than in silica gel without ionic liquids (ILs), respectively. After 5 days incubation of this immobilized lipase in n-hexane at 50 °C, 84% of initial activity was remained, while the residual activity of the lipase immobilized without ILs was 28%.     

Comparing soluble and co-immobilized catalysts for 2-ketoaldose production by pyranose 2-oxidase and auxiliary enzymes

The tri-enzyme system pyranose 2-oxidase (P2O), laccase, and catalase was used to study major parameters in the homogeneous and heterogeneous application of a multi-component enzymatic machinery. P2O oxidizes aldoses to 2-ketosugars, which are interesting intermediates in carbohydrate chemistry, and concomitantly reduces oxygen or alternative electron acceptors. The enzyme was immobilized on eleven agarose or acrylic resins using various coupling methods. The binding capacity was determined and an acrylic carrier with the most suitable properties selected for detailed studies. As P2O shows higher turnover numbers with the electron acceptor 1,4-benzoquinone than with oxygen, the use of this alternative electron acceptor was enabled by employing laccase for the continuous reoxidation of hydroquinone. The laccase regeneration system was found to increase the specific productivity up to 3-fold. Catalase was used to disproportionate the formed hydrogen peroxide in close proximity to the oxygen consuming enzymes and applied in different amounts to adjust the hydrogen peroxide concentration, which was found to be the main reason for enzyme deactivation under turnover conditions. In contrast to homogeneous catalysis, the specific productivity of heterogeneous catalysts under the applied experimental conditions was limited primarily by oxygen transfer, an effect significantly reduced by the laccase regeneration system.     

Enhanced bio-decolorization of azo dyes by co-immobilized quinone-reducing consortium and anthraquinone

In the present study, the accelerating effect of co-immobilized anthraquinone and quinone-reducing consortium was investigated in the bio-decolorization process. The anthraquinone and quinone-reducing consortium were co-immobilized by entrapment in calcium alginate. The co-immobilized beads exhibited good catalytic activity and increased the decolorization rate for many kinds of azo dyes. The reusability of the co-immobilized beads was evaluated with repeated-batch decolorization experiments. After ten repeated experiments, the decolorization rate of co-immobilized beads retained over 92.8% of their original value. Furthermore, acclimatized quinone-reducing consortium was analyzed by denaturing gradient gel electrophoresis (DGGE) and 16S rDNA gene sequencing to get the complete picture of its diversity. This study explored a great improvement of conventional treatment system and the new bio-treatment concept.     

Immobilization of lignin peroxidase on nanoporous gold: Enzymatic properties and in situ release of H2O2 by co-immobilized glucose oxidase

Immobilization of enzymes on porous inorganic materials is very important for biocatalysis and biotransformation. In this paper, nanoporous gold (NPG) was used as a support for lignin peroxidase (LiP) immobilization. NPG with a pore size of 40–50 nm was prepared by dealloying Au/Ag alloy (50:50 wt%) for 17 h. By incubation with LiP aqueous solution, LiP was successfully immobilized on NPG. The optimal temperature of the immobilized LiP was ca. 40, 10 °C higher than that of free LiP. After 2 h incubation at 45 °C, 55% of the initial activity of the immobilized LiP was still retained while the free LiP was completely deactivated. In addition, a high and sustainable LiP activity was achieved via in situ release of H₂O₂ by a co-immobilized glucose oxidase. The present co-immobilization system was demonstrated to be very effective for LiP-mediated dye decolourization.     

多酶共固定化反应体系的研究进展

近年来,生物催化为化学、生物学和生物工程学等领域提供了一种绿色研究工具,其中多酶体系在这些领域中的应用越来越受到关注,其克服了以往单个酶不能满足催化需求的局限性,同时多酶共固定化在级联反应过程中,可增加酶周围的反应物浓度,并将不同酶的催化特性结合起来,能排除干扰因素,从而提高酶的整体催化效率。对多酶共固定化反应体系的研究进展进行了综述,包括多酶反应体系的类别、共固定化技术的特点以及相关应用,并对共固定化多酶反应体系进行了展望。     

Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase

We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40 °C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10–700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4 °C.     

Production,characterization and (co-)immobilization of dextranase from <Emphasis Type="Italic">Penicillium aculeatum</Emphasis>

Fermentation kinetics of Penicillium aculeatum ATCC 10409 demonstrated that fungal growth and dextranase release are decoupled. Inoculation by conidia or mycelia resulted in identical kinetics. Two new isoenzymes of the dextranase were characterized regarding their kinetic constants, pI, MW, activation energy and stabilities. The larger enzyme was 3-fold more active (turnover number: 2,230 ± 97 s⁻¹). Pre-treatment of bentonite with H₂O₂ did not affect adsorption characteristics of dextranase. Enzyme to bentonite ratios above 0.5:1 (w/w) resulted in a high conservation of activity upon adsorption. Furthermore, dextranase could be used in co-immobilizates for the direct conversion of sucrose into isomalto-oligosaccharides (e.g. isomaltose). Yields of co-immobilizates were 2–20 times that of basic immobilizates, which consist of dextransucrase without dextranase.     

亚栖热菌透性化细胞的耦合固定化研究

将海藻酸盐凝胶包埋法与交联法和聚电解质静电自组装覆膜法相耦合,对含有海藻糖合酶活性的亚栖热菌的透性化细胞进行了固定化研究。结果表明,利用重氮树脂和聚苯乙烯磺酸钠对海藻酸凝胶微球交替覆膜,可以显著提高凝胶微球在磷酸盐缓冲液中的稳定性,以碳二亚胺对固定化细胞进行交联处理则可以提高固定化细胞中海藻糖合酶的热稳定性。透性化细胞经包埋－交联－覆膜耦合固定化后,酶活回收率为32％,最适酶反应pH值由6.5左右升至7.0左右,最适反应温度未变,仍为60℃。所得固定化细胞间歇反应时,催化麦芽糖转化为海藻糖的转化率可达60％,重复使用4次（每次50℃、反应24h）,酶活损失小于20％,转化率可保持在50％以上。     

蔗糖酶和GOD的共固定化研究

本文研究了用吸附交联技术共固定化蔗糖酶和葡萄糖氧化酶（ＧＯＤ）的方法,考查了共固定化酶的动力学性质。试验结果表明：与溶液酶相比较,固定化蔗糖酶和ＧＯＤ的响应滞迟期分别为３分钟和２分钟,４态响应时间增加６分钟和４分钟,Ｋｍ值增大,ｐＨ─活力曲线变宽,最适ｐＨ值分别增大０．７和０．６４,最适温度则降低７．３℃和１６℃。以活性氧化铝作载体,戊二醛作交联剂制备的共固定化蔗糖酶和ＧＯＤ,其蛋白质固定化率为９２．９％,分解葡萄糖的总速度为４４１．６ＩＵ,当蔗糖浓度在０．２％以内时其反应速度与蔗糖浓度呈正相关（ｒ＝０．９９６）,使用半衰期１６２３次,在４℃下保存１２０天活力残存为８３．７％。     

Continuous cider fermentation with co-immobilized yeast and Leuconostoc oenos cells

Ca-alginate matrix was used to co-immobilize Saccharomyces bayanus and Leuconostoc oenos in one integrated biocatalytic system in order to perform simultaneously alcoholic and malo-lactic fermentation of apple juice to produce cider, in a continuous packed bed bioreactor. The continuous process permitted much faster fermentation compared with the traditional batch process. The flavor formation was also better controlled. By adjusting the flow rate of feeding substrate through the bioreactor, i.e. its residence time, it was possible to obtain either “soft” or “dry” cider. However, the profile of volatile compounds in the final product was modified comparatively to the batch process, especially for higher alcohols, isoamylacetate, and diacetyl. This modification is due to different physiology states of yeast in two processes. Nevertheless, the taste of cider was quite acceptable.