右旋糖酐-α-1,6键水解酶的家族、结构及其应用

右旋糖酐（dextran）水解酶种类繁多,其中右旋糖酐-α-1,6键水解酶（D-α-1,6 H）是主要的水解酶类.该类酶包括右旋糖酐酶（EC 3.2.1.11）、葡萄糖右旋糖酐酶（EC 3.2.1.70）、异麦芽糖右旋糖酐酶（EC 3.2.1.94）等,分属不同糖苷水解酶家族.D-α-1,6 H的结构和催化方式多样,分类和进化关系复杂,是糖苷水解酶催化机制研究和酶蛋白分子进化研究的好材料.D-α-1,6H及该类酶的催化产物在工业和医学中均有重要而广泛的应用.近年来对D-α-1,6H的理论和应用研究逐渐增加,但仍缺乏深入的系统性研究.本文对D-α-1,6H的家族、结构和功能进行分析,并对其在工业和医学中的最新应用研究作以总结.     

右旋糖酐酶开发及应用研究进展

右旋糖酐酶是一类能特异性催化右旋糖酐中α-1,6-糖苷键水解的酶,其作用底物——右旋糖酐则是蔗糖经某些微生物发酵生成的高分子葡萄糖聚合物。右旋糖酐被广泛应用于医药、食品、材料等领域,但也给口腔健康、制糖生产等带来不良影响。随着人们对右旋糖酐、右旋糖酐水解物及其他多糖研究的深入,右旋糖酐酶发挥着越来越重要的作用,但右旋糖酐酶制剂的整体开发水平仍不高,应用深度仍有限。综述了右旋糖酐酶菌种构建、发酵、纯化、固定化、酶学性质表征、酶活性增强等多个方面的开发研究,以及右旋糖酐酶在制糖、食品、医药、材料等领域的应用研究进展,其中包括本团队在开发和应用方面的研究成果,讨论了在开发和应用过程中存在的问题,并对未来的开发和应用研究方向进行了展望。     

一株产耐热右旋糖酐酶真菌的筛选、鉴定及其酶学性质

【目的】从土壤中筛选得到1株产耐热右旋糖酐酶的真菌。【方法】采用营养缺陷型培养基,结合稀释涂布法和平板透明圈法分离筛选出产耐热右旋糖酐酶的菌株。通过观察菌落形态、菌体形态和培养特征,结合ITS r DNA序列分析对菌株进行鉴定。研究菌株所产右旋糖酐酶的酶学性质。【结果】通过筛选得到1株产耐热右旋糖酐酶的菌株DG001,经鉴定为淡紫拟青霉(Paecilomyces lilacinus)。菌株DG001所产右旋糖酐酶的最佳催化条件为55°C,p H 5.0;最适底物为5%Dextran T70。酶在60°C以下和p H 4.0–7.0之间稳定。urea、Mn~(2+)和Mg~(2+)对酶活均有促进作用,低浓度的Mn~(2+)和urea可使酶活分别提高到116.91%和110.14%,而Cu~(2+)则对其有强烈抑制作用。该酶水解右旋糖酐T2000的产物主要是异麦芽糖和异麦芽三糖,被确定为内切右旋糖酐酶。酶对底物的亲和性随底物分子量的增加而增强。【结论】成功筛选获得1株产耐热右旋糖酐酶的菌株DG001,所产酶在较宽温度范围内具有较高活力,热稳定性好。该酶在制糖工业及不同分子量右旋糖酐的制备中具有很好的应用前景。     

基于右旋糖酐蔗糖酶转糖基作用的槲皮素葡萄糖苷的合成研究

右旋糖酐蔗糖酶是一种以蔗糖为唯一底物,将蔗糖分子中D-葡萄糖基催化转移到受体分子上的葡萄糖基转移酶。利用右旋糖酐蔗糖酶的转糖基作用,以蔗糖为葡萄糖糖基供体,槲皮素为糖基受体,对槲皮素糖苷的酶法合成进行了探索。通过对该酶催化反应体系、催化反应条件及产物分析的研究,结果表明：在25℃下,右旋糖酐蔗糖酶能够在30％DMSO-70％乙酸-乙酸钙（0.02 mol/L,pH值5.4）的反应体系中催化合成一种槲皮素葡萄糖苷,在这个反应体系下,以10％的蔗糖作为糖基供体,槲皮素为糖基受体,右旋糖酐蔗糖酶活力为40 U/mL,转速为150 r/min,槲皮素糖苷的转化率最高,可达39.5％。通过质谱分析确定是一种槲皮素单糖苷,分子量为464。该研究结果为黄酮类物质的糖基化修饰奠定了基础。     

长野芽孢杆菌普鲁兰酶的同源建模及三维结构分析

普鲁兰酶(Pullulanase)是脱支酶,因其能水解葡聚糖的α-1,6-糖苷键而有不同的工业应用潜力。本研究通过同源建模和分子对接的方法对长野芽孢杆菌(Bacillus naganoensis)普鲁兰酶进行建模及其三维结构分析,表明该酶由CBM41-X45aX25-X45b-CBM48-GH13_14多结构域组成,酶蛋白中心形成其催化区,催化区的Asp619、Glu648和Asp733三个残基构成酶的催化三联体。同时,通过柔性对接研究了酶与底物分子相互作用的关系,并预测构成酶的活性中心相关氨基酸残基,为进一步改良酶的特性提供重要的理论依据。     

嗜极菌的极端酶

近年来,嗜极菌极端酶的分离鉴定取得了很大进展。本文简述了极端酶的分离纯化及其某些生化特性、极端酶的稳定因素和应用。极端酶的发现与研究,拓宽了传统的生物催化及应用的范围。但是,关于极端酶的稳定机制及其工业应用,仍有许多难题需要解决。     

工业蛋白质构效关系的计算生物学解析

工业催化用酶已经成为现代生物制造技术的核心"芯片"。不断设计和研发新型高效的酶催化剂是发展工业生物技术的关键。工业催化剂创新设计的科学基础是对酶与底物的相互作用、结构与功能关系及其调控机制的深入剖析。随着生物信息学和智能计算技术的发展,可以通过计算的方法解析酶的催化反应机理,进而对其结构的特定区域进行理性重构,实现酶催化性能的定向设计与改造,促进其工业应用。聚焦工业酶结构-功能关系解析的计算模拟和理性设计,已成为工业酶高效创制改造不可或缺的关键技术。本文就各种计算方法和设计策略以及未来发展趋势进行简要介绍和讨论。     

嗜极菌的极端酶

近年来,嗜极菌极端酶的分离鉴定以得了很大进展,本文简述了极端酶的分离纯化及其某些生化特性,极端酶的稳定因素和应用。极端酶的发现与研究,拓宽了传统的生物催化及应用的范围,但是,关于极端酶的稳定机制及其工业应用,仍有许多难题需要解决。     

人参皂苷单体定向转化的生物催化及应用进展

人参是我国传统中药,药效显著、应用广泛。通过定向修饰与转化人参皂苷糖基可产生高抗癌活性稀有人参皂苷。传统化学法由于制备工艺极其复杂、成本过高,不能应用于临床,微生物及其酶系转化成为解决该瓶颈问题的最可行手段。有关全细胞催化、糖苷酶重组表达、固定化及其催化分子识别机制和溶剂工程的生物转化已有大量综述报道,但尚无在人参皂苷转化应用中的系统研究。文中通过对人参皂苷单体生物转化理论和应用研究最新进展的回顾,结合目前广泛采用的生物催化方法的讨论,系统梳理归纳了能够改善产物专一性、提高催化效率,且具有工业应用前景的人参皂苷单体定向转化方法。基于酶分子设计以及离子液体溶剂工程,对人参皂苷单体抗癌药物和食品、保健品市场的开发、规模化制备进行了展望。     

生物过程酶反应及产品技术研究

生物过程酶反应技术具有催化效率高、环境友好等优点,发展势头迅猛,得到了越来越广泛的应用。综述了近年来单酶催化、多酶催化、化学酶法耦合催化,以及酶的混沌催化等方面的研究现状、重大突破及其在重要医药化工产品合成方面的应用,并对其发展趋势进行了展望。     

Dextranase production from Penicillium funiculosum

Twenty-eight Penicillium cultures were screened for dextranase activity. Dextranase yield of about 2000 DU/ml was obtained with Penicillium funiculosum SH-5. Maximum dextranase concentration was attained only when cell mass decreased. The kinetics of the dextranase production was correlated with the cell mass by a two-parameter model. The optimum pH and temperature for dextranase were 5.0-5.5 and 55°C, respectively. Crude dextranase preparation was inhibitory to insoluble glucan formation by streptococcus mutans 6715 in vitro.     

Isolation of a dextranase constitutive mutant of Lipomyces starkeyi and its use for the production of clinical size dextran

A derepressed and partially constitutive mutant for dextranase of Lipomyces starkeyi was selected after ethyl methane sulphonate mutagenesis by zone clearance on blue dextran agar plates. The mutant produced dextranase when grown on glucose, fructose and sucrose as well as on dextran, and more enzyme was produced by the mutant than by the parental strain when grown on 1% dextran. The pH and temperature optima for the mutant dextranase were 5.5 and 55°C, respectively. Dextranase produced on sucrose produced more isomaltose and less glucose after dextran hydrolysis than the equivalent enzyme produced on dextran. The clinical size dextran (average mol. wt of 75000 ± 25000) yield of mixed culture fermentation with the mutant and Leuconostoc mesenteroides was 94% of the total dextran produced.     

Action patterns of immobilized dextranase

Dextranase, isolated from Penicillium funiculosum and P. lilacinum, was immobilized on porous, silanized-silica beads and a phenol-formaldehyde resin. A commercial dextran of relatively low molecular weight (～2 × 10⁶) was degraded by immobilized dextranase, with the formation of reducing sugars, but with little decrease in viscosity. In contrast, soluble dextranase caused rapid loss of viscosity, but only a slight increase in reducing sugar. Native dextran of high molecular weight, from Leuconostoc mesenteroides NRRL B-512 (F), was attacked very slowly by immobilized dextranase, with the release of oligosaccharides of low molecular weight.     

The purification and characterization of a dextranase from Lipomyces starkeyi

Dextranase produced by Lipomyces starkeyi was purified 43-fold, by carboxymethyl-Sepharose chromatography followed by agarose gel-filtration chromatography. The purified enzyme showed four bands by SDS/polyacrylamide gel electrophoresis with estimated mass 74 kDa, 71 kDa, 68 kDa and 65 kDa. This preparation exhibited multiple isoelectric points between 5.6 and 6.1. All the isoelectric forms were active and catalytically similar. The dextranase contained a carbohydrate moiety (8%). The physical properties of the enzyme were pH and temperature optima of 5.0 and 55 degrees C, respectively. This dextranase was stable between pH 2.5 and 7.0 at temperatures below 40 degrees C. Lipomyces dextranase was a typical endodextranase with the final product of dextran hydrolysis being isomalto-oligosaccharides from glucose to isomaltotetrose.     

Purification and characterization of Paecilomyces lilacinus dextranase

Two dextranase isoenzymes [endo-(1,6)-α-d-glucan-6-glucanohydrolase, EC 3.2.1.11] have been isolated from a crude enzyme powder prepared from the culture supernatant of Paecilomyces lilacinus. Purification was achieved by means of a two-stage ion-exchange chromatography on DEAE-cellulose. Dextranase I was recovered with a 35.3-fold increase in specific activity and a yield of 16%; dextranase II was purified 19-fold with a yield of 4%. The characteristics of the isoenzymes were very similar; both exhibited maximum hydrolytic activity at pH 4.5 and 55°C. Activation energies for thermal inactivation were 402 and 330 kJ mol^?1 for dextranase I and II, respectively. The dextranases were not inhibited by EDTA or N-ethylmaleimide.     

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC₅₀ and MBRC₅₀) of dextranase were 2 U ml^?1 and 5 U ml^?1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.     

Screening,production, and characterization of dextranase from Catenovulum sp.

A psychrotolerant dextranase-producing bacterium was isolated from the Gaogong island seacoast near Jiangsu, China. The bacterium, denoted as DP03, was identified as Catenovulum sp. based on its phenotype, biochemical characteristics, and 16S rRNA gene comparison. The optimal enzyme production time, initial pH, temperature, and aeration conditions of strain DP03 were found to be 28 h, 8.0, 30 °C, and 25 % volume of liquid in 100-ml Erlenmeyer flasks, respectively. The ability of 1 % dextran T20 to induce dextranase was investigated. Dextranase from strain DP03 displayed its maximum activity at pH 8.0 and 40 °C and was found to be stable at 30 °C and over a broad range of pH values (pH 6–11). Scanning electron microscopy showed that dextranase from the isolate DP03 could at least partially prevent Streptococcus mutans from forming biofilms on glass coverslips.     

Hydrolytic and chromatographic studies on the PEGylation of dextranase from Penicillium sp.

Dextranases catalyze the hydrolysis of the α-l,6-glucosidic bond of the polysaccharide dextran. Dextranases have been isolated from bacteria, yeast and fungi. Purified dextranase enzyme from Penicillium sp. was PEGylated (polyethylene glycol modification) with mPEG (5000 Da) and showed an increase in the dextranase protein molecular weight as estimated by Superose 12 (23 ml) column and this increment in the molecular weight is directly proportional to mPEG (5000 Da) concentration until a complete dextranase enzyme PEGylation (disappearance of dextranase peak). The residual activity of partially PEGylated dextranase (mPEG 5000 of 5.8 mg/ml) was 33.8% and for the completely PEGylated dextranase (mPEG 5000 of 29 mg/ml) it was 25.75%. Dextranase PEGylated with mPEG (30,000 Da) showed a little PEGylation at mPEG concentration of 5.8 mg/ml but at a concentration of 29 mg/ml several PEGylated peaks were produced with a difference in dextranase activity toward dextran T500, retardation in the activity with the increasing in the molecular weight was clearly appeared with Sephadex G75 but for Sephadex G200 a little retardation than Sephadex G75 has been appeared.     

Modification of alternan by dextranase

Alternan is a unique glucan with a backbone structure of alternating α-(1 → 6) and α-(1 → 3) linkages. Previously, we isolated strains of Penicillium sp. that modify native, high molecular weight alternan in a novel bioconversion process to a lower molecular weight form with solution viscosity properties similar to those of commercial gum arabic. The mechanism of this modification was unknown. Here, we report that these Penicillium sp. strains secrete dextranase during germination on alternan. Furthermore, alternan is modified in vitro by commercial dextranases, and dextranase-modified alternan appears to be identical to bioconversion-modified alternan. This is surprising, since alternan has long been considered to be resistant to dextranase. Results suggest that native alternan may have localized regions of consecutive α-(1 → 6) linkages that serve as substrates for dextranase. Dextranase treatment of native alternan, particularly with GRAS enzymes, may have practical advantages for the production of modified alternan as a gum arabic substitute. U.S. Department of Agriculture—Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Dextranase from Penicillium minioluteum: reaction course,crystal structure,and product complex

Dextranase catalyzes the hydrolysis of the alpha-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme and product-bound forms. The main domain of the enzyme is a right-handed parallel beta helix, which is connected to a beta sandwich domain at the N terminus. In the structure of the product complex, isomaltose was found to bind in a crevice on the surface of the enzyme. The glycosidic oxygen of the glucose unit in subsite +1 forms a hydrogen bond to the suggested catalytic acid, Asp395. By NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon, implying a single displacement mechanism. Both Asp376 and Asp396 are suitably positioned to activate the water molecule that performs the nucleophilic attack. A new clan that links glycoside hydrolase families 28 and 49 is suggested.