核苷类药物酶法合成研究进展

由于核苷类似物具有很高的抗病毒活性,因为而已成为医药工作者研究的重点。运用酶法合成核苷类似物．已经显示了巨大的优势。本文综述了酶法合成核苷类似物的产生菌种和酶系,以及它们的催化机理,并罗列了已经用于生产或较有使用价值的菌种。     

Enzymatic Synthesis of Cinnamic Acid Derivatives

Using Novozym 435 as catalyst, the syntheses of ethyl ferulate (EF) from ferulic acid (4-hydroxy 3-methoxy cinnamic acid) and ethanol, and octyl methoxycinnamate (OMC) from p-methoxycinnamic acid and 2-ethyl hexanol were successfully carried out in this study. A conversion of 87% was obtained within 2 days at 75 °C for the synthesis of EF. For the synthesis of OMC at 80 °C, 90% conversion can be obtained within 1 day. The use of solvent and high reaction temperature resulted in better conversion for the synthesis of cinnamic acid derivatives. Some cinnamic acid esters could also be obtained with higher conversion and shorter reaction times in comparison to other methods reported in the literature. The enzyme can be reused several times before significant activity loss was observed. Revisions requested 10 January 2006; Revisions received 17 January 2006     

Enzymatic Synthesis of Trehalose from Maltose

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.

Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.     

Studies on Enzymatic Synthesis of Oligosaccharides

The overproduction of tumor necrosis factor-α (TNF-α) was suppressed by orally administering a perilla leaf extract (PLE). When mice were successively injected with OK-432, severe TNF-α was induced in the serum, but this elevated TNF-α level was reduced after an oral administration of PLE (400 μl/mouse). Oral administration of PLE also inhibited TNF-α production that was induced by muramyl dipeptide (500 μg/mouse) and OK-432 (3 KE/mouse). These characteristics were obtained from all strains of perilla. The inhibitory activity against TNF-α production was heat-stable, and the existence of several active molecules was suggested. When PLE was passed through an ultrafilter, the inhibitory activity against TNF-α production was collected in those fractions with a mass of 0.5 to 1 kDa and more than 10 kDa. When PLE was solvent-extracted, the strongest activity was recognized with aqueous preparation, although significant activity was also detected in preparations extracted with n-hexane and ethyl acetate. These findings suggest that the daily use of certain functional foods may be useful for controlling the host defense system.     

酶法合成抗病毒药物阿糖腺苷

目的:为了开发一种生产阿糖腺苷的有效方法。方法:研究了以产气肠杆菌完整细胞为催化剂酶法合成阿糖腺苷,优化了菌体培养条件以及酶反应条件。结果:在培养基中添加0.5%葡萄糖,33℃下培养16h,既能得到较多菌体,又能使菌体的催化活性保持较高。酶反应在pH7.0、25mmol/L的磷酸钾缓冲液中进行,底物浓度为阿糖尿苷30mmol/L,腺嘌呤10mmol/L,加入10%湿菌体,在60℃下振荡反应48h,腺嘌呤转化率可达90%。结论:酶法合成阿糖腺苷可应用于大规模工业化生产。     

海藻糖酶法合成途径及其酶基因的重组表达研究

在生物抗逆研究中,海藻糖合酶基因是继甘露醇、脯氨酸、甜菜碱合成酶基因之后又一个与抗逆相关的基因。海藻糖具有独特的生物学功能,能提高生物体对干旱、高温、冷冻和渗透压的抗性,发现以来就受到人们的普遍关注。随着对海藻糖化学性质、生理功能、作用机理及代谢途径等方面研究的深入,其在生物制品、食品、医药、作物育种及精细化工等领域广阔的应用前景日益显现。就海藻糖在生物体中的合成途径,以及海藻糖合成酶的基因工程研究进展进行了综述。     

生物酶法生产生物柴油的研究进展

生物柴油是一种对环境友好的绿色可再生能源,是石油燃料的理想替代品。目前用于制造生物柴油的方法主要有化学法、生物酶法、超临界法。将生物酶法与化学法及超临界法进行了比较,指出了生物酶法具有其他两种方法无法比拟的优势,但酶法也存在着反应效率低、生产成本高的缺点;针对如何提高酶法生产效率及降低生产成本的问题进行了综述,并对生物柴油酶法技术的发展提出了建议。     

Enzymatic Synthesis of Acarviosyl-maltooligosaccharides Using Disproportionating Enzyme 1

The soluble and insoluble fractions obtained after sonication and centrifugation of Bifidobacterium adolescentis M101–4 cells were examined, and both of these fractions exhibited mitogenic activity in art assay of murine splenocytes and Peyer’s patch cells in vitro. The soluble fraction was further treated by a 6-step procedure involving proteinase K-treatment, ultrafiltration with a 50-kDa cut-off molecular-sieving membrane, anion-exchange chromatography, dialysis, ultrafiltration through a 6-kDa cut-off membrane filter, and gel-filtration to yield a soluble high molecular weight fraction (SHF) which was effective for stimulating the proliferation of murine splenocytes. Almost three quarters of this fraction by weight was found to consist of carbohydrates containing glucose and galactose as major constituents, and the average molecular weight was estimated to be between 60,000 and 2,460,000, with the main peak at 1,550,000 Da, by the retention time of gel permeation chromatography. A structural analysis by ¹H- and ¹³C-nuclear magnetic resonance and methylation indicated that SHF contained polysaccharides consisting of -4Galp1-, -4Glcp1-, and -6Glcp1- as the major residues, and Galf1- and -6Galf1- as the minor residues. Immunopotentiating SHF was found to contain galactofuranosyl residues as characteristic constituents which had not been previously detected in other soluble fractions from Gram-positive bacteria.     

Reactivity of Sphingosine Base in Enzymatic Synthesis of Ceramide

The reactivity of sphingosine bases as substrates in the enzymatic synthesis of ceramide was in decreasing order of erythro-4-sphingenine, erythro-sphinganine, threo-4-sphingenine and thero-sphinganine in the microsomal fraction obtained from chicken liver. Unsaturated base-containing ceramide was enzymatically formed from 4-sphingenine and saturated base-containing ceramide from sphinganine. The formation of unsaturated base-containing ceramide proceeded similar to that of saturated base-containing ceramide. The data explain in part, the fact that 4-sphingenine is generally superior to sphinganine as the constituent sphingosine base in sphingolipids, the biochemical derivatives of ceramide.     

Enzymatic Synthesis of Rhamnose Containing Chemicals by Reverse Hydrolysis

Rhamnose containing chemicals (RCCs) are widely occurred in plants and bacteria and are known to possess important bioactivities. However, few of them were available using the enzymatic synthesis method because of the scarcity of the α-L-rhamnosidases with wide acceptor specificity. In this work, an α-L-rhamnosidase from Alternaria sp. L1 was expressed in Pichia pastroris strain GS115. The recombinant enzyme was purified and used to synthesize novel RCCs through reverse hydrolysis in the presence of rhamnose as donor and mannitol, fructose or esculin as acceptors. The effects of initial substrate concentrations, reaction time, and temperature on RCC yields were investigated in detail when using mannitol as the acceptor. The mannitol derivative achieved a maximal yield of 36.1% by incubation of the enzyme with 0.4 M L-rhamnose and 0.2 M mannitol in pH 6.5 buffers at 55°C for 48 h. In identical conditions except for the initial acceptor concentrations, the maximal yields of fructose and esculin derivatives reached 11.9% and 17.9% respectively. The structures of the three derivatives were identified to be α-L-rhamnopyranosyl-(1→6'')-D-mannitol, α-L-rhamnopyranosyl-(1→1'')-β-D-fructopyranose, and 6,7-dihydroxycoumarin α-L-rhamnopyranosyl-(1→6'')-β-D-glucopyranoside by ESI-MS and NMR spectroscopy. The high glycosylation efficiency as well as the broad acceptor specificity of this enzyme makes it a powerful tool for the synthesis of novel rhamnosyl glycosides.     

Enzymatic Routes for the Synthesis of Rhododendrin and Epi-Rhododendrin

The synthesis of (R)- and (S)-3-(4-hydroxyphenyO-1-methylpropyl-β-D-glucopyranosides has been achieved by two enzymatic steps, namely an oxido-reduction step involving alcohol dehydrogenases from different origin for the preparation of both aglycones in enantiomeric pure form, and a transglycosidation step involving a thermophilic β-glucosidase from the archaeon Sulfolobus solfataricus.     

Papain Immobilization Study in Enzymatic Synthesis of Dipeptide Gly-Phe

Papain catalysed peptide synthesis was followed to determine the optimum conditions for adsorption and covalent binding immobilization methods. The synthesis of the dipeptide Gly-Phe was studied in two different reaction systems: a) For adsorbed papain, in an organic medium containing low water concentration, b) For covalently bound enzyme, in a two-liquid phase system, using trichloroethylene as organic phase. The amount of enzyme offered to the immobilization support and the p^H of the immobilization procedure were the main parameters studied.     

An Enzymatic Platform for the Synthesis of Isoprenoid Precursors

The isoprenoid family of compounds is estimated to contain ∼65,000 unique structures including medicines, fragrances, and biofuels. Due to their structural complexity, many isoprenoids can only be obtained by extraction from natural sources, an inherently risky and costly process. Consequently, the biotechnology industry is attempting to genetically engineer microorganisms that can produce isoprenoid-based drugs and fuels on a commercial scale. Isoprenoid backbones are constructed from two, five-carbon building blocks, isopentenyl 5-pyrophosphate and dimethylallyl 5-pyrophosphate, which are end-products of either the mevalonate or non-mevalonate pathways. By linking the HMG-CoA reductase pathway (which produces mevalonate) to the mevalonate pathway, these building block can be synthesized enzymatically from acetate, ATP, NAD(P)H and CoA. Here, the enzymes in these pathways are used to produce pathway intermediates and end-products in single-pot reactions and in remarkably high yield, ∼85%. A strategy for the regio-specific incorporation of isotopes into isoprenoid backbones is developed and used to synthesize a series of isotopomers of diphosphomevalonate, the immediate end-product of the mevalonate pathway. The enzymatic system is shown to be robust and capable of producing quantities of product in aqueous solutions that meet or exceed the highest levels achieved using genetically engineered organisms in high-density fermentation.