Recent Advances in the use of Immobilized Lipases Directed Toward the Asymmetric Syntheses of Complex Molecules

Water-insoluble compounds can be substrates for enzymatic reactions when lipases are immobilized properly and suitable organic solvents are used. In this review, three type of lipase immobilization method and their application to the asymmetric syntheses of complex molecules are described. Lipases immobilized with Celite or synthetic prepolymers such as urethane prepolymer and photo-crosslinkable resin prepolymer have been applied for the kinetic resolution of many kinds of water-insoluble substrate.

Phospholipid-lipase aggregates with ether linkages are novel and have been found to function effectively as immobilized lipases in asymmetric hydrolysis or esterification reactions in water-saturated organic solvent. The phospholipid-lipase aggregates are considered to have a stacked bilayer based on X-ray diffraction analysis structure of the lipid in the crystalline phase.     

Hydrogen evolution by co-immobilized Chlorella vulgaris and Clostridium butyricum cells

Whole cells of Chlorella vulgaris and Clostridium butyricum were co-immobilized in 2% agar gel. NADP was suitable as an electron carrier. The rate of hydrogen evolution increased with increasing NADP concentration. The optimum conditions for hydrogen evolution were pH 7.0 and 37°C. The immobilized C. vulgaris-NADP-immobilized Cl. butyricum system continuously evolved hydrogen at a rate of 0.29–1.34 μmol/h per mg Chl for 6 days. On the other hand, the system without NADP evolved only a trace amount of hydrogen.     

Development of light-shielding hydrogel for nitrifying bacteria to prevent photoinhibition under strong light irradiation

Microalgae-nitrifying bacteria consortia have gained attention because photooxygenation of algae can supply oxygen to bacteria which eliminates the need for costly mechanical aeration. However, nitrifying bacteria are known to suffer from photoinhibition. In this study, we developed “Light-shielding hydrogel”, in which bacteria were immobilized in hydrogel and light-shielding particles (carbon black) were incorporated, and evaluated its effectiveness to mitigate photoinhibition for bacteria under strong light irradiation. For comparison, “Hydrogel”, in which bacteria were immobilized in hydrogel without carbon black, and “Dispersion” which was simply suspended bacteria were prepared. At 1600 μmol photons m⁻² s^–1, the nitrification performance markedly decreased to 15.1 and 48.0% compared to the dark condition in the Dispersion and the Hydrogel, respectively. Meanwhile, it was successfully maintained for the Light-shielding hydrogel. Our results showed that the effectiveness of light-shielding hydrogel to mitigate photoinhibition on nitrifying bacteria even under strong light irradiation.     

固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

三乙醇胺基强碱性聚苯乙烯树脂共价固定胰酶的研究

用多孔强碱型三乙醇胺基聚苯乙烯阴离子交换树脂做为载体,用CNBr与载体上的多羟基作用共价偶联了胰酶。红外光谱表明:其共价偶联反应机理与用CNBr活化多糖类载体并接酶的机理相类似。最适偶联条件研究表明:CNBr用量增多,酶蛋白载量增加。但比活下降。偶联pH为10时,固定化酶有适宜的载量和较高的比活。由于胰酶水解蛋白反应释放出H~+质子,这些质子在载体内积累,使微环境内H~+质子浓度增加,进而使得固定化胰酶的pH—活性曲线在pH9～11范围内未出现下降。在变温和60℃恒温下对固定化酶的热稳定性测试表明:固相酶的热稳定性比天然酶的热稳定性有所提高。     

Two-dimensional maps in very acidic immobilized pH gradients

Up to the present time it has been impossible to perform two-dimensional (2-D) separations in very acidic immobilized pH gradients (IPG), due to the lack of suitable buffering acrylamido derivatives to be incorporated into the polyacrylamide matrix. The advent of the pK 3.1 buffer (2-acrylamido glycolic acid; Righetti et al., J. Biochem. Biophys. Methods 16, 1988, 185–192) allowed the formulation of such acidic gradients. We report here separations in IPG pH 2.8–5.0 intervals of polypeptide chains from total lysates of rat intestinal and liver cells and 30S and 50S ribosomal proteins from Halobacterium marismortui. Conditions are given for highly reproducible first and second dimensions gels and for a proper silver staining of 2-D maps with practically no background deposition.     

固定化虫荧光素酶光纤传感器

固定化虫荧光素酶光纤传感器蔡谨,王顺光,杨歧生,吉鑫松（浙江大学化工系生化教研室,杭州３１００２７;中国科学院上海生物化学研究所,２０００３１）关键词虫荧光素酶,ＡＴＰ,固定化酶,光纤生物传感器ＡＴＰ是生物体内极为重要的能量物质。如何准确快速地定量Ａ...     

有机硅橡胶固定化细胞进行的生物转化

有机硅橡胶固定化细胞进行的生物转化潘冰峰,戴学倩,冯青,李祖义（中国科学院上海有机化学研究所,２０００３２）关键词硅橡胶;白地霉;固定化细胞;生物转化近年来,有机化学领域的一个重要进展是用酶或微生物进行生物转化反应。其中研究和应用较多的是碳基还原,特...