Effect of Ethanol on Formation of Cyclodextrin from Soluble Starch by Bacillus macerans Cyclodextrin Glucanotransferase

Formation of cyclodextrin (CD) from soluble starch by Bacillus macerans CGTase (cyclodextrin glucanotransferase) was studied experimentally in both systems with and without the addition of ethanol. The yields of α-, β- and γ-CD after long time reaction were almost independent of initial concentration of starch (5–50 g/l) in both the systems. The α-CD yield in the presence of ethanol was increased by 1.8–1.9 times of that in the absence of ethanol, while the β-CD yield was decreased by the amount almost comparable to the increase in α-CD yield. Initial rates of CD formation were decreased with increasing ethanol concentration, which was remarkable especially for α-CD.     

Alpha‐Cyclodextrin Production using Cyclodextrin Glycosyltransferase from Klebsiella pneumoniae AS‐22

Alpha‐cyclodextrin (α‐CD) production, using cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS‐22, was investigated in the presence of various complexing agents using raw wheat starch and dextrin as substrates. The addition of many alcohols resulted in increased conversion of both raw wheat starch and dextrin to α‐CD. With 125 g/L raw wheat starch, 42.5% (w/w) conversion to CDs was obtained in the presence of 2% (v/v) butan‐1‐ol. The ratio of α:β‐CD formed was 97:3, with negligible formation of γ‐CD and malto‐oligosaccharides. The production of α‐CD was optimized using two‐level factorial designs in three variables: dextrin, hexan‐1‐ol and enzyme concentrations. Under optimum conditions, 12.1% (w/w) conversion of 500 g/L dextrin to total‐CDs was achieved. The ratio of α:β:γ‐CD formed was 91:3:6, with negligible production of malto‐oligosaccharides. This CGTase was strongly inhibited by α:‐CD; 50% inhibition of reaction rate was observed at an initial concentration of 4 g/L α:‐CD. The effectiveness of an ultrafiltration membrane bioreactor for continuous removal of product was also tested.     

Chain Length Specifity of Cyclodextrin Glycosyltransferase

Bacillus macerans CGTase recognizes the terminal 6 glucose units of the non-reducing end of α-(1→4)-glucans. Analysis of the chain-length distributions produced by the action of CGTase on well-defined maltodextrins gave information on the enzyme's specifity in regard of the size of the transferred glucanosyl-moiety. It was found that in the presence of α-cyclodextrin, maltohexaosyl-groups could be highly specifically coupled to an acceptor like maltose. The product, maltooctaose, appeared in a yield of 45 % molar amount. The affinity for hexaosylresidues was explored by using homodisperse substrates from glucose to maltotriadecaose. HPTLC-analysis of the product patterns in early states of CGTase catalyzed reactions revealed that the yield of the primary coupling product decreased with increasing DP. This was due to the effect, that disproportion was more rapid on higher oligomers. The intermolecular transfer of glucanosylgroups was also found to be size-dependent, inasmuch as the relative CGTase transfer activity decreased gradually from maltohpxaosyl- to glucosylresidues. In an investigation of the enzymic degradation of maltodextrins maltononaose gave the highest yield of α-CD.     

环糊精糖基转移酶定点突变及其产物特异性分析

环糊精葡萄糖基转移酶（cyclodextrin glycosyltransferase,CGTase）是一种多功能酶,能够催化淀粉生成环糊精（cyclodextrin,CD）,本研究考察了Bacillus cereus的CGTase活性区域43位氨基酸与CGTase酶活力及其催化玉米淀粉形成γ-CD能力的联系。实验中共构建17 株突变株,并在大肠杆菌BL21中实现异源表达。结果表明：相比于原始酶,H43E、H43F、H43W、H43Y突变酶的活力均有所提高;产物特异性方面,除H43W突变酶外,其余突变酶的催化产物中γ-CD比例均有所升高,将43位组氨酸突变成脯氨酸、异亮氨酸和苏氨酸后得到的突变酶作用于玉米淀粉,酶的催化产物中β-CD含量均下降,γ-CD含量由20%分别升高到30.2%、28.4%、29.2%,实际产量由4.76 g/L升高到7.64、6.05、6.29 g/L,由此可见Bacillus cereus的CGTase活性区域中43位氨基酸对于CGTase的活力和产物特异性具有重要影响。     

Bacillus sp. Y112环糊精葡萄糖基转移酶位点R81定点突变提高产物特异性

通过对来源于海洋芽孢杆菌Y112的环糊精葡糖基转移酶进行同源建模及氨基酸序列比对,发现N末端81位精氨酸可能影响环糊精产物特异性。本研究通过定点突变将第81位点处的精氨酸突变为苏氨酸,降低了α-环糊精的生成量,将β-环糊精的比例从64%上升至71%,提高了产物的专一性。分析原因可能与取代氨基酸残基的大小及氢键作用力的变化有关。同时,突变酶的酶学性质方面保持了原始酶的嗜热性、热稳定性及耐碱性。     

环糊精系列综述之一 环糊精葡萄糖基转移酶的筛选及其定向改造

环糊精因其特殊性质得到了食品、化妆品、医药等行业的青睐,全球消费量逐年稳步增加。环糊精的巨大需求也使其生产中所必需的环糊精葡萄糖基转移酶(CGT酶,EC 2.4.1.19)成为研究热点。目前,对该酶的研究多集中在作用机制、产物专一性机理等,鲜见从菌株筛选出发的系统性报道。因此,作者从野生CGT酶菌株的筛选出发,立足未来环糊精工业的商业诉求,综述了优质CGT酶获取的多种途径。同时结合国内外对CGT酶的大量研究工作以及我国的资源优势,对该领域未来的研究提出更高的期望。     

Electron Microscopic Study of Cyclodextrin Derivatives

The importance of cyclodextrin (CD) derivatives and inclusion complexes in drug research is increasing. Numerous modern methods are applied for their investigation, but electron microscopic studies do not appear to have been reported in the literature. Electron microscopic investigations of α-, β- and γ-CD, dimethyl-β-CD, cross-linked CD and nitroglycerine-β-CD inclusion complexes are now described.     

Purification of Cyclodextrin Glycosyltransferase by Immunochromatography

Cyclodextrin glycosyltransferase (CGTase) was produced in recombinant Escherichia coli which contains the cgt gene of Bacillus macerans in the pTCGT1 plasmid. Antiserum against CGTase was prepared in rabbits. Anti-CGTase IgG in Serum was purified using an immobilized Protein A affinity column and an immobilized E. coli cell lysate affinity column. The purified anti-CGTase IgG was coupled to Sepharose 4B (5.3mg IgG/mL gel), which was activated with cyanogen bromide. Crude CGTase was purified with this immunoadsorbent with CGTase binding capacity of 0.27 mg/mL and eluted with 0.1M glycine buffer, pH 10.6. Approximately 65% of enzyme activity was recovered from the crude CGTase preparation by this method. The purified CGTase was electrophoretically and antigenically homogeneous, appearing as a single band. After 10 cycles, the recovery yield was not reduced significantly. There was no cross reaction between anti-CGTase IgG and other tested amylolytic enzymes. This immunoaffinity column is, therefore, effective, efficient, and convenient to use, and can be used widely to purify CGTase from various sources.     

环糊精制备过程的催化机制及其影响因素

环糊精主要是由环糊精葡萄糖基转移酶(CGT酶,EC 2.4.1.19)作用淀粉制备而得。除受CGT酶的来源及种类的影响之外,影响环糊精产率及产物特异性的因素还有很多,诸如酶作用时间、作用温度、pH、加酶量、添加物、底物种类、底物浓度、原料预处理程度等;此外,这些因素还将影响环糊精的制备工艺过程。然而,目前尚未有环糊精制备过程影响因素的系统报道。作者从环糊精糖基转移酶的催化机理分析入手,阐述影响环糊精产率及产物特异性的影响因素,同时结合环糊精的制备工艺,对环糊精制备相关研究进展进行综述,以期为在环糊精制备相关领域开展研究提供参考。     

环状糊精葡萄糖基转移酶的研究进展

环状糊精葡萄糖基转移酶是催化淀粉等合成环状糊精的酶,环状糊精由于具有特殊的理化性质,能将多种化学物质包接在其环形空隙中,因此在食品等领域有着广泛的应用前景。本文对环状糊精葡萄糖基转移酶的来源、酶学性质、作用机理、筛选及分子生物学方面进行了系统的综述。     

Isolation and Partial Properties of Cyclomaltodextrin Glucanotransferase-producing Alkaliphilic Bacillus spp. from a Deep-sea Mud Sample

Cyclomaltodextrin glucanotranferase (CGTase) producing alkaliphilic bacteria were isolated from deep-sea bottom mud samples. Isolates no. 3–22 and no. 1–7 were identified as an alkaliphilic Bacillus sp. and an alkaliphilic Bacillus subtilis strain from their taxonomic characteristics, respectively. Isolate no. 3–22 grew at 4°C whereas isolate no. 1–7 did not. The crude enzymes of both strains showed activity in both broad temperature and broad pH (5–9) ranges, Isolate no. 3–22 produced the main product. β-cyclodextrin (β-CD), and minor products, α- and γ-CDs from starch. On the other hand, isolate no. 1–7 produced the main product, β-CD, and the minor product, γ-CD from starch; no α-CD was detected.     

The Influence of Oxygen Volumetric Mass Transfer Rates on Cyclodextrin Glycosyltransferase Production by Alkaliphilic Bacillus circulans in Batch and Fed-Batch Cultivations

In this work, the influence of oxygen mass transfer rates on the production of cyclodextrin glycosyltransferase (CGTase) by the alkaliphilic bacterium Bacillus circulans ATCC 21783 was investigated. Experimental design and response surface methodology were applied to optimize agitation speed and air flow rate in batch cultivations, in order to identify their significant effects and interactions with the synthesis of CGTase. Results were expressed as the volumetric mass transfer rates of oxygen (k_la, [per hour]). The maximal CGTase productivity of 155 U mL⁻¹ h⁻¹ was achieved with k_la of 48 h⁻¹. CGTase production was also studied in fed-batch cultures using the optimized parameters obtained in the batch experiments. The maximal CGTase productivity on fed-batch cultivations was 137 U mL⁻¹ h⁻¹ with feeding rates of starch at 0.17 g L⁻¹ h⁻¹.     

环糊精及肽配体介导的毒死蜱非竞争检测模式

以毒死蜱作为对象,构建了一种由环糊精和肽配体介导的非竞争检测模式。首先利用分子模拟和光谱学方法对β-环糊精包合毒死蜱的效应进行了证实;然后将β-环糊精与牛血清蛋白进行偶联,并证实偶联状态的环糊精分子仍能包合毒死蜱;以β-环糊精-牛血清蛋白偶联产物为核心材料,从商品化的噬菌体展示线性十二肽库中筛选获得了5个可以特异识别环糊精-毒死蜱包合物的肽配体;最后,利用亲和力最强的肽配体(8#克隆)构建毒死蜱的非竞争检测模式,该检测模式能够特异性地识别毒死蜱,而对丙溴磷、除线磷和溴硫磷等结构类似农药分子无识别。本研究所构建的检测模式无需对小分子物质进行改造和固定,可为小分子化学污染物的快速检测提供新的参考。     

高产环糊精葡萄糖基转移酶菌株发酵工艺条件优化

以筛选、诱变所获得的一株高产环糊精葡萄糖基转移酶CGTase 的芽孢杆菌为出发菌株,对其进行发酵工艺条件的优化。结果表明：该菌株产CGTase 的最佳条件为碳源1% 可溶性淀粉、氮源0.1% 酵母膏、碳酸盐1% Na2CO3、pH10、培养温度33℃、接种量3%、摇床转速180r/min,此条件下CGTase 酶活为2842U/ml。     

环麦芽糊精葡聚糖转移酶高活性的土壤微生物菌种筛选

根据环麦芽糊精葡聚糖转移酶(CGTase)催化淀粉和糊精生成环状糊精(CD),环状糊精可以吸附筛选培养基中的刚果红和甲基橙使菌落周围形成透明圈,且透明圈越大,透明度越好,CGTase的转糖基活性越高的酶催化特性,设计筛选模版,进行产酶菌株分离筛选。经初筛、复筛得到了YS-19和YS-40的2株转糖基活性较高CGTase菌种,酶活分别为2.90U/mL和3.15U/mL。     

Characterization of a Maltogenic Amylase of Thermononospora viridis and Application in Branched Cyclodextrin Production

Some kinetic characteristics and actions on various cyclodextrins (CDs) of a maltogenic α-amylase of T. viridis (TVA) were examined to apply in the branched CD production. Michaelis constants (Km) for hydrolysis of α-, β- and γ-CDs by the enzyme were 6.5, 7.6 and 10.8 mM, and apparent molecular activities (k_θ) for these saccharides were 11.0, 67.2 and 432.8 s⁻¹, respectively. The (k_θ) values for various branched CDs were remarkably smaller than those for unbranched CDs. The TVA cleaved first the ring of maltosyl-β-CD, and the branched nonasaccharide formed was immediately degraded to give branched hexa- and penta-saccharides with simultaneous formation of smaller unbranched saccharides. As a minor action, the enzyme released a glucose molecule from the side chain of maltosyl-β-CD to give glucosyl-β-CD which was readily degraded to smaller saccarides. The enzyme had an ability to hydrolyze unbranched CDs preferentially in the presence of significant amounts of branched CDs. These characteristics of the enzyme are consistent with its potential industrial use in the branched CD production.     

Bacillussp.HA-1产环糊精葡萄糖基转移酶发酵工艺研究

在10 L 发酵罐中,研究了环糊精葡萄糖基转移酶产生菌 Bacillus sp．HA-1的发酵规律,结果表明,在菌体对数增长期,发酵液溶氧处于较低状态,发酵液 pH 在对数增长前期持续下降,中期后回升,菌体产酶呈增长趋势；当菌体增长进入稳定期,发酵液溶氧迅速回升至初始状态,pH 也回升至起始值,菌体产酶接近峰值。根据发酵规律优化发酵工艺,通过有效增加菌体对数增长期发酵液溶氧,使菌株产酶到达高峰的时间缩短了 50%以上。应用该菌株在5 m~3生产罐中发酵,24 h 产酶水平达到6 800 IU/mL。     

Modification of pasting properties of wheat starch by cyclodextrin glycosyltransferase

The effect of cyclodextrin glycosyltransferase (CGTase) on wheat starch functionality was assessed by determining the pasting properties and the dynamic rheology. The addition of CGTase lowered the peak and final viscosity, increased breakdown and markedly decreased the total setback. The influence of CGTase on the ability of starch to form complex with lipids was explored in the presence of fatty acids (stearic and lauric) and an emulsifier (diacetyl tartaric acid ester of monoglyceride, DATEM). Further information about the effect of CGTase on the pasting properties of wheat starch was obtained by analysing the first derivative of the viscograph curves. The cyclodextrins formed in the starch as a result of the cyclization activity of the CGTase were quantified and it was observed that α‐cyclodextrin occurred in the highest concentration followed by β‐ and γ‐cyclodextrins. The starch obtained with CGTase after pasting exhibited better emulsifying properties as it contained the cyclodextrins. The CGTase treatment also modified the dynamic rheology of the starch slurry leading to a decrease in the elastic and viscous modulus. Copyright © 2004 Society of Chemical Industry     

Towards Understanding Formation and Stability of Cyclodextrin Inclusion Complexes: Computation and Visualization of their Molecular Lipophilicity Patterns[1]

Five cyclodextrin inclusion complexes - typical examples for their solid state as well as exemplary „frozen molecular images”︁ of their status in solution - were subjected to a molecular modelling study comprising the computer-assisted generation of the molecular lipophilicity patterns (MLP's), i. e. the p-iodoaniline inclusion into α-CD, the complexes of β-CD with 1,4-butanediol, adamantane-1-carboxylic acid, and adamantane-1-methanol, and the unique 12-crown-4 ether inclusion compound of γ-CD. Based on their solid state structures, the „solvent-accessible”︁ contact surfaces were generated by the MOLCAD program. Calculation and color-coded projection of the MLP's onto these surfaces easily allows to locate the hydrophobic and hydrophilic surface regions of CD-host and guest alike. Their detailed analysis revealed a far-reaching, mostly complete conformity between the hydrophobic surface areas of the guest and the hydrophobic domains in the CD cavity. This striking tendency to optimize the reciprocal concurrence of lipophilic as well as hydrophilic domains at the guest-host-interface may accordingly be concluded to be an important, if not the decisive element in orienting the guest into the cavity and in determining the stability of the complex, particularly in cases where the guest is devoid of polar groups; if these are present, dipole-dipole alignments and the need for their solvation may diminish the importance of hydrophobic attractions for orienting and stabilizing the guest in the CD cavity.     

Kinetic Characterization of Chimeric Cyclomaltodextrin Glucanotransferase from Genes of Two Alkalophilic Bacillus

The chimeric CGTase, which was constructed genetically with two β-CD forming type enzyme genes of different species of alkalophilic Bacillus, was characterized by comparing with the actions of parent enzymes. The final composition of CDs in the reaction digest from starch by the chimeric enzyme, especially in the yield of α-CD, was different from those by parent enzymes, and total yields of CDs by these enzymes were almost the same. The chimeric enzyme catalyzed the formation of β-CD as a major action from starch, the rapid synthesis of α-CD from other CDs and maltooligosaccharides and the slow degradation and interconversion of α-CD to others. From these results, the role of second and third segments in the enzyme molecules was discussed.