Preparation and characterization of immobilized lipase on magnetic hydrophobic microspheres

A novel magnetic poly(vinyl acetate (VAc)–divinyl benzene (DVB)) material (8–34 μm) was synthesized by copolymerization of vinyl acetate and divinyl benzene using oleic acid-stabilized magnetic colloids as magnetic cores. The magnetic colloids and the copolymer microspheres were characterized with transmission and scanning electron microscopes, respectively. Magnetization of the microspheres could be described by the Langevin function. All the observations indicated that the microspheres were superparamagnetic. Magnetic sedimentation of the microspheres was achieved within 3 min, over 300 times faster than the gravitational sedimentation. Candida cylindracea lipase (CCL) was immobilized to the porous carrier at up to 6750 IU/g carrier, remarkably higher than the previous studies. The pH and temperature dependencies of the immobilized CCL were investigated and the optimum temperature and pH for the immobilized CCL were determined. Activity amelioration of the immobilized CCL for the hydrolysis of olive oil was observed, indicating an interfacial activation of the enzyme after immobilization. Moreover, the immobilized CCL showed enhanced thermal stability and good durability in the repeated use after recovered by magnetic separations.     

Regioselective Enzymatic Hydrolysis of 3',5'-DI-O-Acetylthymidine and Observation of a Surface Effect Due to Polystyrene

The selective enzymatic hydrolysis of 3',5'-di-O-acetylthyidine (1) was studied. The lipases from porcine pancreas and Aspergillus niger, and pig liver esterase, all catalysed selective hydrolysis of the 5'O-acetyl group, but the lipase from Candida cylindracea catalysed selective hydrolysis of the 3'-O-acetyl group. Highest selectivity, leading to essentially pure 3'-O-acetylthymidine, was achieved using porcine pancreatic lipase in dilute solution at pH 7.5. Provision of an artificial interface in the form of polystyrene beads led to a significant increase in the rate of hydrolysis, accompanied by a marked fall in selectivity. Other changes in the hydrolysis conditions, such as raising the concentration of substrate or adding cosolvent, also led to a fall in selectivity.     

Alcohol Induced Reversal of Enantioselectivity in a Lipase Catalyzed Resolution of 2-Chloropropionic Acid

Alcohol induced reversal of enantioselectivity in the esterification of 2-chloropropionic acid using lipase from Candida cylindracea has been investigated. It was found that an alcohol having substituents both at the α- and the β-carbon preferentially esterified the S-acid, while a straight chain alcohol preferentially esterified the R-acid. Intermediate enantioselectivities were obtained with alcohols having substituents either at the α- or the β-carbon, but still in favor of the R-acid. An acyl binding domain composed of three subsites is proposed for this lipase; one for the hydrocarbon chain, a second for a methyl substituent and a third for an electronegative substituent.     

Purification and carbohydrate-binding specificity of Agrocybe cylindracea lectin

A lectin was isolated from fruiting bodies of Agrocybe cylindracea by two ion-exchange chromatographies and gel filtration on Toyopearl HW55F. The lectin was homogeneous on polyacrylamide gel electrophoresis and its molecular mass was determined to be 30 000 by gel filtration, and 15 000 by sodium dodecylsulfate polyacrylamide gel electrophoresis, signifying a dimeric protein. Its carbohydrate-binding specificity was investigated both by sugar-hapten inhibition of hemagglutination and by enzyme-linked immunosorbent assay. The inhibition tests showed the affinity of the lectin to be weakly directed toward sialic acid and lactose, and the enhanced affinity toward trisaccharides containing the NeuAcα2,3Galβ-structure. Importantly, the lectin strongly interacted with glycoconjugates containing NeuAcα2,3Galβ1,3GlcNAc-/GalNAc sequences. This revised version was published online in November 2006 with corrections to the Cover Date.     

柱状田头菇EST-SSR的开发及应用研究

借鉴香菇EST序列设计的40对引物对柱状田头菇进行了EST-SSR引物通用性研究。结果显示,挑选的28对引物中多态性好且稳定的有13对,通用率达46.4%。PCR扩增产物获得具有多态性条带81条,每对引物扩增条带的数目范围为2–14,平均5.86条,扩增片段大小在100–400bp之间。多态信息含量（PIC）在0.3750–0.8032,平均0.7084。研究结果表明,EST-SSR分子标记在食用菌的遗传多样性和比较基因组学研究中起到重要的作用。     

高分子载体固定化酵母脂肪酶的研究

合成了一系列丙烯酸甲酯－双甲基丙烯酸乙二醇酯、甲基丙烯酸羟乙酯－双甲基丙烯酸乙二醇酯、苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物作为固定化酵母脂肪酶的载体,比较了这些载体固定化酵母脂肪酶催化橄榄油水解反应的效果,结果发现疏水性较强的苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物固定化酵母脂肪酶的效果最好．比较了酵母脂肪酶溶液和苯乙烯－双甲基丙烯酸乙二醇酯文联共聚物固定化酶催化橄榄油水解反应的最适ｐＨ值、最适离子强度、最适温度和Ｋ＿ｍ值．同时考察了苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物固定化酶在有机溶剂中催化十六酸戊酯合成反应的活力．     

Hydrolysis and Esterification with Lipase from Candida Cylindracea. Influence of the Reaction Conditions and Acid Moiety on the Enantiomeric Excess

The enantiomeric ratio for hydrolysis and synthesis of 1-phenyl ethanol esters of straight chain aliphatic carboxylic acids catalyzed by Candida cylindracea lipase was determined. A distinct maximum in enantiomeric ratio was observed for valeric and caproic acid in the hydrolytic direction. No significant maximum could be determined in the esterification reaction. Even though the enzyme provided larger enantiomeric ratios in the synthetic direction the enantiomeric excess of the alcohol was not higher. The enantiomeric excess was depressed by racemization reactions in the esterification as the reaction approached thermodynamic equilibrium at an insufficient conversion. While choosing the optimal chain length of the acyl donor is important in hydrolytic reactions it seems to be of greater value to raise the equilibrium conversion in the esterification reactions.     

Enhanced Resolution of a Secondary Alcohol by Hydrolysis of a Bichiral Ester Catalyzed by Lipase from Candida cylindracea

The effect of a chiral centre in the acyl group on the resolution of esters prepared from a racemic alcohol was investigated. R-2-chloropropionic acid afforded a higher enantiomeric ratio than S-2-chioropropionic acid in the hydrolysis of the corresponding esters of racemic 1-phenylethanol catalyzed by Candida cylindracea lipase. Even when a mixture of esters prepared from racemic acid and racemic alcohol was used for resolution of the alcohol, a noteworthy high enantioselectivity was observed. The hydrolysis of a bichiral ester offers an amplification in the resolution of enantiomers of alcohols by the combination of a chemical diastereoselectivity and an enzymatic enantio- and diastereoselectivity.     

云芝、灵芝和柱状田头菇胞外多糖对果蝇寿命的影响

研究了云芝、灵芝、柱状田头菇等3种食药用真菌胞外多糖及复合多糖对黑腹果蝇寿命的影响.分别将3种食药用真菌胞外多糖及复合多糖以1、5g/L的剂量加入果蝇培养基中,观察对黑腹果蝇寿命的影响.结果表明,3种食药用真菌的胞外多糖及复合多糖对果蝇寿命均有不同程度的延长,云芝5g/L、柱状田头菇5g/L、云芝2.5g/L＋柱状田头菇2.5g/L、灵芝2.5g/L＋柱状田头菇2.5g/L对果蝇平均寿命延长率分别达到了36.38%、46.60%、36.69%、47.08%.     

Lipase activity in vesicular systems: characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation. (c) 1993 John Wiley & Sons, Inc.