亲水性离子液体[EMIM]BF4对固定化β-葡萄糖醛酸苷酶催化活性的影响

以壳聚糖为固定化载体,采用吸附交联法制备固定化β-葡萄糖醛酸苷酶;以1-乙基-3-甲基咪唑四氟硼酸盐([EMIM]BF4)/缓冲溶液均相体系为介质,研究了亲水性离子液体[EMIM]BF4对固定化酶生物催化甘草酸(GL)合成单葡萄糖醛酸基甘草次酸(GAMG)的影响.实验结果表明,当均相体系中[EMIM]BF4的体积分数为16%,pH为5.4,反应温度为50℃及摇床转速为200 r/min时,酶活力达到最高,并且明显优于纯缓冲溶液体系中的最高酶活.重复利用性实验结果表明,与纯缓冲液介质体系相比,固定化酶在含亲水性离子液体[EMIM]BF4的均相介质中表现出较好的操作稳定性.表观动力学参数和活化能数据表明,亲水性离子液体[EMIM]BF4在催化体系中能够增强酶和底物GL的亲和力,有效稳定酶-底物的过渡态,并降低反应活化能,而使固定化β-葡萄糖醛酸苷酶表现出较高的催化活性.     

同位素标记及未标记的1-脱氧-D-木酮糖-5-磷酸和2-甲基-D-赤藓糖醇-4-磷酸的合成方法

萜类化合物构成了最大的一个天然产物家族,结构复杂多变,且有许多重要的生理活性.2-甲基-D-赤藓糖醇4-磷酸(MEP)途径是近年来发现并建立的一条萜类化合物的生物合成途径,其中所涉及到的酶均可作为靶标来进行新抗菌素的筛选.综述了以化学合成及酶催化合成方法制备MEP途径中关键中间体1-脱氧-D-木酮糖5-磷酸和2-甲基-D-赤藓糖醇4-磷酸的进展,并着重介绍了同位素标记的这两个化合物的制备方法.     

双层固定化酵母催化还原4-氯-乙酰乙酸乙酯的研究

以海藻酸钠为内层载体,利用机械强度良好的聚乙烯醇(PVA)改性海藻酸钠混合物作为外层载体,双层固定化高活性酵母,得到固载的生物催化剂,用于由磷酸缓冲溶液和环己烷组成的双相体系,对4-氯-乙酰乙酸乙酯(COBE,ethyl 4-cloro-3-oxobutyrate)进行不对称催化加氢合成(S)-4-氯-3-羟基丁酸乙酯[(S)-CHBE],ethyl[(S)-4-chloro-3-hydro butanoate].在最适条件下,双层固定化酵母的不对称催化还原反应优于游离细胞和单层固定化细胞.在底物COBE的最佳浓度和进料速率分别为170mmol/L,125mL/h的连续操作条件下,最高(S)-CHBE转化率为94.2%,最高对映体过量值为98%.与游离细胞催化COBE相比,反应转化率提高3%,最佳底物浓度比游离细胞增加1倍;与单层固定化细胞催化还原相比,对映体过量值提高8%.连续性实验表明,双层固定化酵母细胞在连续使用5d,其转化率和对映选择性没有明显下降.     

糖苷合成研究(XXI)2-N-(β-D-吡喃葡萄糖-1-基)-6-(4-卤代苯基)-3-哒嗪酮的合成

哒嗪酮葡萄糖苷;相转移催化;糖苷合成研究(XXI)2-N-(β-D-吡喃葡萄糖-1-基)-6-(4-卤代苯基)-3-哒嗪酮的合成     

新型海参硫酸软骨素葡萄糖受体及葡萄糖醛酸受体的高效合成

以D-葡萄糖为起始原料,经9步反应合成了2-O-苄基-3-O-烯丙基-1-O-对甲氧基苯基α-D-葡萄糖(9);将9的6-位伯羟基经叔丁基二苯基硅烷基(TBDPS)保护,首次合成了正交保护的新型葡萄糖受体2-O-苄基-3-O-烯丙基6-O-叔丁基二苯基硅烷基1-O-对甲氧基苯基α-D-葡萄糖(Ⅱ),总收率28.2%;将9的6-位伯羟基氧化糖醛酸化后,再经甲酯化,以25.0%的总收率首次合成了新型葡萄糖醛酸受体2-O-苄基-3-O-烯丙基-1-O-对甲氧基苯基α-D-葡萄糖醛酸甲酯(Ⅲ),化合物结构经¹H NMR,¹³C NMR, IR和HR-MS(ESI)表征。     

13-顺-4-羰基维甲酸葡萄糖酯的合成与表征

13-顺维甲酸与溴代-2,3,4,6-四-O-乙酰基-α-D-吡喃葡萄糖在4-二甲氨基吡啶催化下合成了13-顺维甲酸葡萄糖酯(5),以二氧化锰为氧化剂,5经氧化成功地合成了新化合物13-顺-4-羰基维甲酸葡萄糖酯(6)。5和6的结构经1H NMR,13C NMR,IR和MS表征,结果表明5和6均为β-构型糖酯。     

羰基还原酶CR2重组酶体系不对称合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺

构建了羰基还原酶CR2重组酶体系,并优化了相关的酶促催化反应条件.通过在催化体系中添加辅酶NADP+(0.1 mmol/L)和辅底物葡萄糖(120 g/L),在30℃及p H=8.0的条件下反应4 h,CR2重组酶体系不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP,10 g/L),合成了高光学纯度(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺[(S)-DHTP,e.e.值99.9%],产率为62%.在酶促催化过程中,由于辅酶循环生成葡萄糖酸导致反应体系p H值下降而影响催化效率.通过调控反应体系p H值,(S)-DHTP的产率提高到68%.不同浓度底物的反应过程表明底物对CR2酶促反应具有抑制作用,且在10 g/L底物浓度下反应的时空产率可达1.3 g·L-1·h-1.     

N-丁基-1-脱氧野尻霉素的合成新方法

以2,3,4,6-四-O-苄基-α-D-葡萄糖为原料,经4步反应制得中间体2,3,4,6-四-O-苄基-D-葡萄糖酸-δ-内酰胺(6);在碱性条件下6与正溴丁烷进行氮原子上的烃基化反应得N-丁基-2,3,4,6-四-O-苄基-D-葡萄糖酸-δ-内酰胺(7);用氢化铝锂将7的羰基还原为亚甲基得N-丁基-2,3,4,6-四-O-苄基-1-脱氧野尻霉素(8);8经催化氢解脱去苄基合成了N-丁基-1-脱氧野尻霉素,其结构经1H NMR,13C NMR和MS确证。     

多糖激活的酪氨酸酶生物电极测定酚类的研究

研制了以麦芽糊精作为酪氨酸酶的激活剂 ,采用Nafion117膜抗阴离子干扰 ,聚乙烯亚胺 (PEI)作包埋剂以抗阳离子干扰的酪氨酸酶碳糊电极。工作电位为 - 10 0mV [vs.Hg/Hg2 Cl2(s) ],pH为 5 .4 0 ,测量时间为 1.0min。在此测量条件下 ,电极对苯酚的检出限为 5 .0× 10 - 8mol·L- 1,线性范围为 2 .0× 10 - 7～ 5 .0× 10 - 5mol·L- 1,RSD为 1.2 %。用所研制电极测定炼油废水中酚的含量 ,加标回收率为 10 0 .7%。     

1-脱氧-1-氨甲基-4,6-O-亚苄基-β-D-吡喃葡萄糖的合成

1-脱氧-1-氨甲基-4,6-O-亚苄基-β-D-吡喃葡萄糖是合成糖碳苷类糖肽的重要潜在中间体.本文以无水D-葡萄糖为原料经过两步反应合成1-脱氧-1-硝基甲基-4,6-O-亚苄基葡萄糖,然后通过催化转移氢化反应选择性地对硝基进行还原而不影响4,6位的亚苄基.     

Enzymatic Synthesis of α-Glucose-1-phosphate: A Study Employing a New α-1,4 Glucan Phosphorylase from Corynebacterium callunae 1

Abstract

In synthetic pathways to complex carbohydrates such as oligosaccharides or nucleotide sugars the activated sugar 1-phosphates serve as important starting molecules. In this study the enzymatic synthesis of α-glucose-1-phosphate (Glc-1-P) has been investigated using a new bacterial α-glucan phosphorylase from Corynebacterium callunae. The major factors governing the rate of reaction and the attainable degree of substrate conversion have been identified and, accordingly, for optimizing the yield and limiting reaction time for the enzymatic process several points must be considered: (i) the pH-dependent equilibrium of reaction, (ii) product inhibition of the phosphorylase and (iii) enzymatic cleavage of α-1,6 glycosidic linkages present in α-1,4-glucans such as starch or maltodextrins by pullulanases to improve their phosphorolytic conversion. Results obtained in continuous experiments with the phosphorylase retained in an ultrafiltration membrane reactor confirmed the complete operational stability of the enzyme for several days at 30 °C. Since no more than approximately 18 % of the inorganic phosphate can be converted into Glc-1-P an efficient procedure for phosphate and product recovery will be particularly important.     

Maltodextrin as stabilizer for emulsion polymerization: Adsorption and grafting behavior

Emulsion polymerization of the three-monomer system butyl acrylate–styrene–methacrylic acid was performed in batch using a commercial maltodextrin derived from starch degradation as stabilizer. Stable latexes with narrow particle size distributions were obtained in all examined cases. A method was developed to analyze and quantify the partitioning of the maltodextrin between the continuous phase (supernatant) and the particle phase. Significant differences between the polysaccharides adsorbed onto particles with or without emulsion polymerization reaction were observed. The possible reactions of maltodextrin in presence of a radical initiator were studied in aqueous phase, thus confirming maltodextrin degradation. The formation of copolymers involving the original monomers and the stabilizer according to two different reactive pathways was also confirmed. In terms of adsorbed maltodextrin, two different contributions were observed: maltodextrin physically adsorbed and maltodextrin chemically grafted and/or physically incorporated into the polymer.     

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with β-cyclodextrin (β-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 μmol of β-CD/(min·g_IF) at 50°C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distin ctranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24h of reaction, namely, 10.4 mM β-CD and 2.3 mM γ-CD.     

Innovative method for determining glycogen content in the Pacific oyster (Crassostrea gigas) by ion chromatography

A precise analytical assay was developed and validated for the determination of Pacific oyster glycogen using ion chromatography. The Pacific oyster glycogen was quantified by the determination of glucose-6-phosphate (Glc-6-P), which was derived from glucose that was hydrolyzed from glycogen. Glc-6-P, adenosine triphosphate, and adenosine diphosphate were separated by ion chromatography. The method was validated over the curve range 0.5-100mg/L for the abovementioned analytes. The recoveries were between 95% and 102%. The relative standard deviations (RSDs, c=10mg/L, n=9) were less than 4.37%. Unlike a traditional method, this validated method was inexpensive and stable.     

大肠杆菌O152中wfgD基因编码的β-1,3-葡萄糖基转移酶产物结构的质谱表征

大肠杆菌O152抗原的寡糖重复单位中含葡萄糖-β-1,3-N-乙酰葡萄糖胺(Glc-β-1,3-GlсNAc)连接键。本研究采用电喷雾离子化多级串联质谱技术对以人工合成的天然受体底物的结构类似物苯氧基十一烷二磷酸-N-乙酰葡萄糖胺(GlcNAc-β-PO3-PO3-(CH2)11-O-phenyl(GlcNAc-PP-PhU))为受体底物,尿苷二磷酸葡萄糖(UDP-Glc)为给予体底物的酶促反应产物进行了详细的结构表征。电喷雾离子化多级串联质谱图中观察到的主要碎片源于磷酸二酯键部分和糖苷键的裂解。此外,由观察到的二糖产物非还原端碎片获得了序列信息;跨环断裂碎片及源于吡喃环取代基消除的‘内在’碎裂离子可提供组成产物的单糖残基连接方式信息。广泛的碎裂信息表明wfgD基因编码UDP-Glc:GlcNAc-pyrophosphate-lipid中的β-1-3葡萄糖基转移酶。     

Research activities in the field of enzyme engineering in the framework of the russian state scientific program “novel methods in bioengineering”

The article describes the research activities in the field of enzyme engineering in Russia. The discussion is focused on fundamental studies of biocatalytic processes that expand utilization of enzymes, biocatalytic synthesis of organic products from renewable raw mate rials, enzymes for hydrolysis of cellulose and lignocellulose materials, immobilized cells, new enzyme-based drugs, enzymes in fine organic synthesis, bioanalytic devices, biosensors, and biofuels.     

Enantioanalysis of S-Ketoprofen Using Enantioselective,Potentiometric Membrane Electrodes

Abstract

Maltodextrins with different dextrose equivalent (DE) values (maltodextrin I: DE 4.0–7.0; maltodextrin II: DE 13.0–17.0; maltodextrin III: DE 16.5–19.5) were used for the design of three enantioselective, potentiometric membrane electrodes (EPMEs) for the assay of S-ketoprofen. The linear concentration ranges for the proposed electrodes were 10^?10 to 10^?8, 10^?9 to 10^?5, and 10^?10 to 10^?7 mol/L, with slopes of 58.0, 58.67, and 58.93 mV/decades of concentration and limits of detection of 1.49 × 10^?8, 2.43 × 10^?8, and 4.19 × 10^?11 mol/L for EPMEs based on maltodextrin I, II, and III, respectively. The EPMEs showed high reliability and effectiveness for the enantioanalysis of S-ketoprofen raw material and its pharmaceutical formulations.     

D-Ala-D-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies

BACKGROUND: The D-alanyl-D-lactate (D-Ala-D-Lac) ligase is required for synthesis of altered peptidoglycan (PG) termini in the VanA phenotype of vancomycin-resistant enterococci (VRE), and the D-alanyl-D-serine (D-Ala-D-Ser) ligase is required for the VanC phenotype of VRE. Here we have compared these with the Escherichia coli D-Ala-D-Ala ligase DdlB for formation of the enzyme-bound D-alanyl phosphate, D-Ala(1)-PO(3)(2-) (D-Ala(1)-P), intermediate. RESULTS: The VanC2 ligase catalyzes a molecular isotope exchange (MIX) partial reaction, incorporating radioactivity from (14)C-D-Ser into D-Ala-(14)C-D-Ser at a rate of 0.7 min(-1), which approaches kinetic competence for the reversible D-Ala(1)-P formation from the back direction. A positional isotope exchange (PIX) study with the VanC2 and VanA ligases displayed a D-Ala(1)-dependent bridge to nonbridge exchange of the oxygen-18 label of [gamma-(18)O(4)]-ATP at rates of up to 0.6 min(-1); this exchange was completely suppressed by the addition of the second substrate D-Ser or D-Lac, respectively, as the D-Ala(1)-P intermediate was swept in the forward direction. As a third criterion for formation of bound D-Ala(1)-P, we conducted rapid quench studies to detect bursts of ADP formation in the first turnover of DdlB and VanA. With E. coli DdlB, there was a burst amplitude of ADP corresponding to 26-30% of the DdlB active sites, followed by the expected steady-state rate of 620-650 min(-1). For D-Ala-D-Lac and D-Ala-D-Ala synthesis by VanA, we measured a burst of 25-30% or 51% of active enzyme, respectively. CONCLUSIONS: These three approaches support the rapid (more than 1000 min(-1)), reversible formation of the enzyme intermediate D-Ala(1)-P by members of the D-Ala-D-X (where X is Ala, Ser or Lac) ligase superfamily.     

Glucoamylase immobilization on a magnetic microparticle for the continuous hydrolysis of maltodextrin in a fluidized bed reactor

Glucoamylase (GA) has been successfully immobilized through its carbohydrates previously oxidized with periodate onto a low-cost magnetic microparticle made of polyethyleneimine-coated magnetite crosslinked with glutaraldehyde (M-GAD) and derivatized with adipic dihydrazide (ADH). A stabilization posttreatment consisting of cross-linking its carbohydrates with ADH, increased the remaining activity from 54 to 71%, calculated on theV _m values and measured at 50°C and pH 4.5 with maltodextrin (DE 11-14) as substrate. This treatment also improved the enzyme stability and lowered the deactivation rate constantk _d to a third of its value. A 30% maltodextrin solution has been continuously hydrolyzed at 50°C and pH 4.5 in a recycled, fluidized bed reactor (FBR) containing GA immobilized on these magnetic microparticles. They easily settled in this highly viscous medium because of their high density (5 g/mL), and washout of ultrafines was prevented by surrounding the top of the bed with an electromagnet. The small particle size (20 μm) allowed a high enzyme loading in the reactor and also a high bed voidage, which is recommended to avoid extensive pressure drop and consequent channeling problems. The kinetic of hydrolysis fitted with the plug-flow model; this is explained by the insignificant backmixing effects observed. After 2 wk of hydrolysis under process conditions leading to a conversion of 70%, which corresponds to a high-conversion syrup, the immobilized GA only lost 4% of its initial activity.     

Application of capillary electrophoresis-mass spectrometry to synthetic in vitro glycolysis studies

We propose an approach designed to reconstitute a metabolic pathway composed of multistep biochemical reactions, rather than to dissect the individual reactions that make up the pathway. A synthetic in vitro glycolysis was reconstructed from ten purified Escherichia coli (E. coli) enzymes to obtain a better understanding of the regulation of sequential enzymatic reactions. The key to the success of this approach is the ability to perform direct and simultaneous determination of the diverse metabolic intermediates in the pathway by capillary electrophoresis-mass spectrometry. We observed that the pathway is regulated by a delicate balance between the changing metabolite concentrations and behaves like a natural biological oscillating network that has hitherto not been reported for E. coli glycolysis. The end-product, pyruvate, was periodically synthesized from glucose at an overall efficiency of 30%, corresponding to an average of 90% conversion efficiency for each of the ten steps involved. This approach is likely useful for the synthesis of natural products requiring complex sequential biocatalytic reactions.