亚位点?7处突变对碱性芽胞杆菌CGT酶产物特异性的影响

为了研究来源于碱性芽胞杆菌的γ-环糊精葡萄糖基转移酶(CGT酶)具有较高产物特异性的作用机理,对其氨基酸序列和模拟结构进行了分析,确定其亚位点7处氨基酸的缺失可能影响其产物特异性。运用重叠PCR的方法,在其亚位点7处添加缺失的6个氨基酸,造成插入突变。将突变基因与pET-20b(+)连接并在大肠杆菌BL21(DE3)中表达。以可溶性淀粉为底物进行酶转化,HPLC分析转化产物中的环糊精含量。结果表明,相对于野生型γ-CGT酶,突变酶转化生成的3种环糊精中,γ-环糊精所占的比例从76.0%降至12.5%,α-、β-环糊精分别从8.7%和15.2%提高至37.5%和50%。分析其可能机理为:与α-、β-CGT酶相比,野生型γ-CGT酶的亚位点7处缺失6个氨基酸,该构象为葡萄糖的结合提供了更大的空间,从而更适合γ-环糊精的生成;而在其亚位点7处插入6个氨基酸,造成插入突变后,葡萄糖链结合的空间变小,这种构象不利于γ-环糊精的生成。     

利用高效阴离子色谱快速检测筛选环糊精糖基转移酶产生菌>

利用高效阴离子色谱快速直接地检测微生物发酵液中的环糊精成分,尤其是大环环糊精的组成,进而创造了一种能快速准确地从土壤中筛选产环糊精糖基转移酶菌种的方法。共分离了149个产胞外淀粉水解酶的微生物菌株,利用高效阴离子交换色谱共检测了其中11株菌,其中6株主要产CD6 ,5株主要产CD7,主要产CD8的没有。在直接鉴定产生环糊精糖基转移酶菌株的过程中,也可以定量检测各种环糊精包括大环糊精（CD大于8）的含量。     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

基于分子动力学模拟的高γ-环糊精专一性环糊精糖基转移酶理性改造

【背景】环糊精糖基转移酶的分子动力学模拟较传统基因改造而言能有效提高改造效率,减少盲目性。【目的】探究环糊精糖基转移酶的催化专一性机理,为获得产γ-环糊精专一性更高的环糊精糖基转移酶提供高效突变菌株方法。【方法】通过分子对接和分子动力学模拟,获得3种产物类型CGTase与底物的对接模拟结构,并通过定点突变实验进行验证。【结果】分子动力学模拟结果显示α-和β-CGTase与十糖链在酶蛋白S1区域呈现闭合的形态,而γ-CGTase和十糖链在S1区域呈现更易于生成γ-环糊精的张开形态;3种CGTase与十糖链在相同位置存在氢键的氨基酸共有17个相对应位点,其中14个位点的氨基酸种类一致,不一致的3个氨基酸对应α-CGTase位点分别为Y89、D234和Y262。本研究对Y262位点进行定点突变和产物专一性实验,结果显示经过分子动力学预测的Y262L有助于提高产γ-CD专一性,从野生酶的13.7%提高到39.9%,γ-环糊精产物比例提高了3倍。【结论】分子动力学模拟结果对于指导环糊精糖基转移酶的专一性内在机理具有一定的正向指导意义。     

作为非病毒基因载体的环糊精及其衍生物

环糊精由于自身的生物相容性和结构易裁剪性,通过结构修饰、聚合或超分子组合等设计被逐渐应用于非病毒基因载体系统。本文将分别从环糊精、其小分子衍生物、含环糊精聚合物以及超分子结构综述国内外近几年的设计思路和研究进展,并探讨含环糊精及其衍生物的非病毒基因载体的"结构-安全性-基因转染效率"关系。     

α-环状糊精葡萄糖基转移酶的固定化及其性质研究

研究了一种α-环糊精葡萄糖基转移酶的固定化和固定化酶的性质。通过对戊二醛浓度、酶量和交联时间各单因素的考察,确定了最佳的固定化条件。与游离酶相比,以DEAE纤维素为载体的固定化酶最适pH向酸性偏移,最适温度不变,pH稳定性和热稳定性都有所提高。在40℃、150r/min下反应3h,转化率可以达到32%。固定化酶可以连续使用4次以上。固定化酶在4℃、5mmol/L CaCl2溶液里保存18d,还剩余80%以上的活力。     

环糊精葡萄糖基转移酶的基因改造与高效表达

环糊精葡萄糖基转移酶(CGTase,EC 2.4.1.19)是一种多功能酶,主要用于生产环糊精(CD)、糖基化碳水化合物,同时在食品行业也有重要作用。为改善CGTase在这些方面的应用性能,筛选出优势突变酶,异源表达、定点突变、固定化等技术被研究和应用,取得了实质性的进展。综述了CGTase基因高效异源表达策略,概述了基因改造CGTase的研究进展,并且还总结了用于改造CGTase的其他手段,例如固定化酶、嵌合酶、化学添加剂等,以期为在相关CGTase研究领域开展研究提供参考。     

重组β-环糊精葡萄糖基转移酶生产β-环糊精的工艺条件优化

将来自于Bacillus circulans 251的β-CGTase编码基因克隆到表达载体pET-20b(+),转化Escherichia coli BL21(DE3)。经酶活检测培养基上清中的β-CGTase酶活为20 U/mL。对酶转化淀粉生成β-环糊精的反应条件进行了优化,结果表明,当底物马铃薯淀粉浓度15%,反应初始pH5.5,温度30℃,加酶量10 U/g干淀粉,环己烷浓度2.5%-5%(V/V),转化周期24 h,β-环糊精转化率达到最高值75.3%,是国内外报道的酶法生产β-环糊精的最高水平。     

Role of capillary electrophoresis methods in the drug development process

In the past several years, capillary electrophoresis (CE) has generated considerable interest from pharmaceutical companies for control of both the chiral and achiral purity of bulk drugs and drug products. This paper evaluates the use of CE as: (1) a technique complementary to HPLC for the determination of peak homogeneity of a drug, (2) for determination of chiral purity, and (3) for determination of achiral purity. It would be greatly advantageous if CE could be used to determine both the chiral and achiral purity in a single assay. This investigation compares the results obtained for the separation of the enantiomers of duloxetine using several neutral cyclodextrins to those obtained using anionic cyclodextrins (sulfobutyl ether derivatives) as chiral selectors added to the separation buffer. In addition, it reports chiral separations obtained by using neutral cyclodextrins in a sulfonic acid-coated capillary column, which give a negatively charged capillary surface and electro-osmotic flow even in low pH buffers. The possible mechanism of separation is discussed. © 1996 Wiley-Liss, Inc.     

Sulfated cyclodextrins for the chiral separations of catecholamines and related compounds in the reversed electrophoretic polarity mode

The utility of negatively charged sulfated cyclodextrins (SCD) as chiral additives (CA) in capillary electrophoresis (CE) was studied in the chiral resolution of several compounds of pharmaceutical interest, including catecholamines such as norepinephrine, epinephrine, DOPA and their precursors, phenylalanine, and tyrosine. Experiments were conducted using 10 mM sodium phosphate monobasic solution and 2% SCD adjusted to pH 3.2 with phosphoric acid. Chiral recognition mechanisms were explored using structurally related analytes including basic, acidic, and neutral compounds as well as 3,5-dinitrobenzoyl phenylglycine, phenylalanine, and homophenylalanine. The advantage of the reversed electrophoretic polarity mode for the enantioresolution of these compounds is also discussed. © 1996 Wiley-Liss, Inc.     

Epididymal sperm profiles in young adult,middle-aged,and testosterone-supplemented old rats

Both ejaculated semen and epididymal contents from an individual male contain sperm that differ in various physicochemical characteristics. An experiment is reported in which epididymides from rats 5–24 months old were subjected to density gradient centrifugation to separate gametes of different stages of maturity. The research was designed to examine typical changes in “profiles” of sperm maturity during the reproductive lifetime of rats. Also, testosterone complexed with cyclodextrin that mimics the episodic release of the endogenous hormone was used to supplement the decreased circulating titers of some of the old males. Results revealed clear ontogenetic patterns of gradually decreasing reproductive competence as measured by absolute numbers of sperm, circulating levels of testosterone, and various other physiological markers of fertility. Sperm profiles also revealed age-specific changes with a shift toward progressively more mature, perhaps senile, gametes that begins at middle age. Testosterone supplementation (400 μg/kg b.w./day for 30 days) failed to restore sperm numbers or other measures of physiology in the old males, but the steroid modified sperm profiles to approximate more closely the profiles characteristic of young adult males than either untreated middle-aged or old males. The data were interpreted as suggesting that epididymal sperm profiles clearly identify males of different ages, and that the aging epididymis retains its capacity to respond to manipulations that modify the endocrine milieu.     

Complexation of Tocotrienol with γ-Cyclodextrin Enhances Intestinal Absorption of Tocotrienol in Rats

To determine the bioavailability of tocotrienol complex with γ-cyclodextrin, the effects of tocotrienol/γ-cyclodextrin complex on tocotrienol concentration in rat plasma and tissues were studied. Rats were administered by oral gavage an emulsion containing tocotrienol, tocotrienol with γ-cyclodextrin, or tocotrienol/γ-cyclodextrin complex. At 3 h after administration, the plasma γ-tocotrienol concentration of the rats administered tocotrienol/γ-cyclodextrin complex was higher than that of the rats administered tocotrienol and γ-cyclodextrin. In order to determine the effect of complexation on tocotrienol absorption, rats were injected with Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoprotein by lipoprotein lipase, and then administered by oral gavage an emulsion containing tocotrienol, tocotrienol with γ-cyclodextrin, or tocotrienol/γ-cyclodextrin complex. The plasma γ-tocotrienol concentration of the Triton-treated rats administered tocotrienol/γ-cyclodextrin complex was higher than that of the other Triton-treated rats. These results suggest that complexation of tocotrienol with γ-cyclodextrin elevates plasma and tissue tocotrienol concentrations by enhancing intestinal absorption.     

Reevaluation of the role of the multidrug-resistant P-glycoprotein in cellular cholesterol homeostasis

The multidrug resistance P-glycoprotein (P-gp) was recently proposed to redistribute cholesterol in the plasma membrane, suggesting that P-gp could modulate cholesterol efflux to cholesterol acceptors. To address this hypothesis and to reevaluate the role of P-gp in cholesterol homeostasis, we first analyzed the role of P-gp expression on cholesterol efflux in P-gp stably transfected drug-selected LLC-MDR1 cells. Cholesterol efflux to methyl-beta-cyclodextrin (CD) was 4-fold higher in LLC-MDR1 cells compared with control LLC-PK1 cells, indicating that the accessible pool of plasma membrane cholesterol was increased by P-gp expression. However, using the P-gp-inducible cells lines HeLa MDR-Tet and 77.1 MDR-Tet, cholesterol efflux to CD, apolipoprotein A-I, or HDL was not associated with P-gp expression. In addition, we did not observe any effect of P-gp expression on cellular free and esterified cholesterol content, cholesteryl ester uptake from LDL and HDL particles, or acyl-CoA:cholesterol acyltransferase activity. Therefore, we conclude that P-gp expression does not play a major role in cholesterol homeostasis in P-gp-inducible cells and that the effects of P-gp on cholesterol homeostasis previously described in drug-selected cells might result from non-P-gp pathways that were also induced by selection for drug resistance.     

Anchorage of cyclodextrin glucanotransferase on the outer membrane of Escherichia coli

The gene encoding cyclodextrin glucanotransferase (CGTase) was successfully cloned from B. macerans by PCR. A recombinant plasmid pCS005 with a gene encoding the Lpp-OmpA-CGTase trifusion protein was constructed and transformed into E. coli for the surface display of CGTase. Results of immunoblotting analysis and protease accessibility on the fractionated cell membranes confirmed that the Lpp-OmpA-CGTase trifusion protein was successfully anchored on the outer membrane of E. coli. However, only 50% of the membrane-anchored trifusion proteins were displayed on the outer surface of E. coli with the remaining 50% un-translocated. The low efficiency of surface display is attributed to the large size of CGTase. Only a trace amount of CGTase activity was detected for both the whole cells and the cell debris fractions. Because the results of the protease accessibility study suggested that the trypsin-resistant conformation of CGTase was preserved in the membrane-anchored CGTase, we believe that the lack of enzyme activity is mainly due to the inaccessibility of the CGTase active site, near the N-terminus, for substrate molecules. It can be estimated that the critical size for surface display of protein in E. coli is approximately 70 kDa.     

Evaluation of enantiomeric purity of selected amino acids in honey

Highly sensitive and accurate HPLC methods were used for the determination of total amounts of proline, leucine and phenylalanine and their enantiomeric ratios in a variety of different honey samples. Significant amounts of D -leucine and D -phenylalanine and relatively low concentrations of D -proline have been found in honeys of different botanical and geographical origins. It is suggested that the enantiomeric ratios of amino acids could be used to test for storage effects, age, and the quality of the processing of the honey. © 1994 Wiley-Liss, Inc.     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

Formation of Inclusion Complexes of Cycldextrin with Ethanol under Anhydrous Conditions

Complex formation of poorly water soluble organic compounds with cyclodextrin (CD) is quite difficult in an aqueous cyclodextrin system. Formation of the inclusion complex of d-limonene, phenyl ethanol, acetophenone, or menthol was investigated in a slurry form of α-, β-, or γ-CD in organic solvents or alcohol under anhydrous conditions. Ethanol and methanol were found to be good solvents for this method. The use of ethanol as the solvent was investigated in greater detail. There existed an optimal amount of ethanol for the maximum inclusion of d-limonene as the guest compound. However, an excess of ethanol inhibited the inclusion. An adsorption model of alcohol on CD, analogous to the substrate inhibition model of enzyme kinetics, could correlate the inclusion ratio with the amount of alcohol added to CD.