Levan蔗糖酶及其在Levan果聚糖合成中的应用

Levan果聚糖是一类分子中含有大量β-(2,6)果糖苷键主链和少量β-(2,1)果糖苷键支链的聚糖。部分微生物来源的Levan果聚糖具有抗肿瘤、抗糖尿病、免疫增强、降血脂等重要的生物活性,在医药和功能食品方面具有巨大的应用潜能。由于微生物发酵液提取法产量相对较低,而化学法合成过程繁琐,Levan果聚糖的酶法合成备受关注。Levan蔗糖酶(Levansucrase,EC 2.4.1.10)属于糖苷酶家族GH68,是一类β-螺旋桨家族蛋白,其催化糖类合成遵循non-Leloir糖基转移酶机制,以蔗糖为底物转果糖基合成Levan果聚糖。部分微生物Levan蔗糖酶的分子结构及基因的表达调控已经得到阐明,Levan果聚糖的酶法合成得到广泛研究。本文综述了Levan蔗糖酶的催化机制、酶分子结构、酶基因表达调控以及酶在合成Levan果聚糖中的应用,以促进微生物Levan蔗糖酶及Levan果聚糖的研究和应用。     

青霉菊粉酶的产生和性质

由淀粉通过葡萄糖异构化生产高果糖糖浆,生产上受到原料的限制。果糖另一种来源是菊粉（Inulin）。菊粉是以β-2,1果糖苷键连接的一种多聚果糖,其末端含有一个蔗糖残基,它作为贮存性多糖大量存在于菊芋（Helianthus tuberosus）、菊巨(chicoryintybus)、大丽花（Dahlia pinnata）等多种植物中,至今尚未得到很好利用。菊粉可通过化学法或酶法水解生成果糖。利用菊粉酶     

外切菊粉酶的酶学研究进展

菊粉富含于菊芋、菊苣等多种菊科植物中,是一种来源丰富的可再生资源。菊粉是一种由D 呋喃果糖经β-2, 1-糖苷键连接,还原端经α-1, 2-糖苷键连接1个葡萄糖残基构成的果聚糖。菊粉能被菊粉酶水解,生产果糖、高果糖浆、菊粉寡糖,可通过微生物发酵生产燃料酒精等产品,在食品、生物能源、医疗保健等方面都有重要应用,受到广泛关注。介绍外切菊粉酶的分类、来源、结构和催化机理,重点总结近10年微生物来源外切菊粉酶的重组表达和酶学性质情况,简述外切菊粉酶在食品、能源等方面的应用,展望外切菊粉酶的研究热点及方向。     

高等植物果聚糖研究进展

果聚糖是高等植物重要的贮藏碳水化合物,因植物种类和发育阶段而异,主要存在5种类型的结构：线型菊糖型果聚糖、菊糖型果聚糖新生系列、线型梯牧草糖型果聚糖、混合型梯牧草糖型果聚糖和梯牧草糖型果聚糖新生系列。果聚糖的代谢模型随着代谢酶—蔗糖：蔗糖果糖基转移酶、蔗糖：果聚糖_6_果糖基转移酶、果聚糖：果聚糖果糖基转移酶、果聚糖：果聚糖_6_果糖基转移酶、果聚糖外水解酶等的发现、纯化和克隆日趋清晰。此外,果聚糖分子生物学研究也取得了一定的进展。     

高等植物果聚糖研究进展

果聚糖是高等植物重要的贮藏碳水化合物 ,因植物种类和发育阶段而异 ,主要存在 5种类型的结构 :线型菊糖型果聚糖、菊糖型果聚糖新生系列、线型梯牧草糖型果聚糖、混合型梯牧草糖型果聚糖和梯牧草糖型果聚糖新生系列。果聚糖的代谢模型随着代谢酶—蔗糖 :蔗糖果糖基转移酶、蔗糖 :果聚糖_6_果糖基转移酶、果聚糖 :果聚糖果糖基转移酶、果聚糖 :果聚糖_6_果糖基转移酶、果聚糖外水解酶等的发现、纯化和克隆日趋清晰。此外 ,果聚糖分子生物学研究也取得了一定的进展     

具有生物活性的川牛膝果聚糖的结构

从传统中药川牛膝(Cyathula offcinalis kuan)中分离提取到了一种具有生物活性的多糖RCP.核磁共振、甲基化分析、还原裂解和GC-MS分析揭示了RCP是一高度分支的果聚糖,它以(2→1)连接为骨架,其上有大量的(2→6)连接的分支,且属于新蔗果三糖系列.在93.17%果糖残基中,24.15%是末端果糖,26.24%是1-连接果糖,20.46%是6-连接果糖.在6.83%的葡萄糖残基中,2.14%是末端葡萄糖,4.69%是6-连接葡萄糖.RCP的平均聚合度是15.     

微生物酶法合成低聚半乳糖的新进展

低聚半乳糖(GOS)是目前国际上已开发的功能性低聚糖之一,其商业化产品是应用微生物β-半乳糖苷酶以乳糖为原料进行转糖基反应获得,不同来源的酶合成GOS的结构不同,转糖基效率也存在差异.天然酶合成GOS的产量一般为20%～45%,分子改造获得的人工酶能将90%的乳糖底物转化为GOS;采用两相体系或反相胶束可以在一定程度上提高GOS产量.应用填充床反应器、活塞流反应器、膜反应器可规模化合成GOS;采用色谱柱法、酶法、纳滤膜法和微生物发酵法可纯化GOS产品,去除单糖及乳糖组分,扩大其应用范围.     

蔗糖磷酸化酶的分离纯化及其催化合成α-熊果苷的研究

考察了肠膜明串珠菌(Leuconostoc mesenteroides)G123厌氧发酵产蔗糖磷酸化酶下游的分离纯化工艺.收集的菌体经超声破碎得到粗酶液,通过硫酸铵沉淀、透析、阴离子交换层析分离后获得了电泳纯的蔗糖磷酸化酶,酶活回收率为31.7%,酶的分子量约为55.7 kD,纯化后的蔗糖磷酸化酶比活为115.3 U/mg.该酶在中性及偏酸性(pH5.5-8.0)情况下,酶稳定性较好,较报道的肠膜明串珠菌(Leuconostoc mesenteroides)B-1149的pH稳定范围宽.同时该酶在37℃保存2 h,酶活几乎没有下降.利用获得的纯酶以氢醌和蔗糖为底物催化合成α-熊果苷,在23 U/mL的酶反应体系中,60%蔗糖、5%氢醌、pH7.5,37℃,反应12 h,氢醌转化率达到16.3%,α-熊果苷的产量为20g/L.     

小麦植株中可溶性糖纸层析定量方法

小麦、大麦、黑麦等作物以及某些单子叶和菊科植物的营养器官或貯藏器官中的可溶性糖类,除一般单糖(葡萄糖、果糖)双糖(蔗糖)外,还有大量果聚糖类存在,而且有时作为貯藏形式。它们的分子结构一端是蔗糖,蔗糖的果糖基通过2,1或2,6糖甙链连接着若干果糖基,形成一系列结构相似的寡糖或多糖,不具还原性。一般的分析方法不能分类测定。我们     

蔗糖:蔗糖-1-果糖基转移酶的表面展示及酶学性质分析

【目的】蔗糖:蔗糖-1-果糖基转移酶催化1分子蔗糖上的果糖基转移到另一个蔗糖分子上,形成1-蔗果三糖和葡萄糖。在低聚果糖中,1-蔗果三糖益生素活性最高。本研究将该酶展示在酵母菌细胞表面上,并用于1-蔗果三糖的制备。【方法】将来自莴苣的蔗糖:蔗糖-1-果糖基转移酶基因克隆到用于酵母细胞表面展示的表达载体上,并在解脂亚罗酵母菌中进行异源表达,表达的酶展示在该细胞表面上,然后以蔗糖为底物,研究表面展示的蔗糖:蔗糖-1-果糖基转移酶的性质。【结果】免疫荧光实验结果表明蔗糖:蔗糖-1-果糖基转移酶已展示在酵母菌的细胞表面上,高效液相色谱结果表明酵母表面展示的该酶具有转移酶的催化活性。该酶的最适作用温度、最适作用p H分别为45°C和7.5;该酶的催化活性受Zn2+和Cu2+的抑制,受Ca2+激活;该酶重复使用7次后,酶活下降50%。表面展示的蔗糖:蔗糖-1-果糖基转移酶和3%蔗糖混合后在40°C条件下孵育30 min后,所产1-蔗果三糖含量最高为20.8 mmol/L。【结论】蔗糖:蔗糖-1-果糖基转移酶在解脂亚罗酵母菌中得到成功表达,并展示在其细胞表面上,生化研究表明该重组蛋白具有果糖基转移酶活性,且催化蔗果三糖的生成。表面展示的蔗糖:蔗糖-1-果糖基转移酶作为一种全细胞催化剂能够用于1-蔗果三糖的制备。     

Influence of nutritional factors on the nature, yield, and composition of exopolysaccharides produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.     

Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.     

Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis

Levan, fructo-oligosaccharides and fructosyl derivatives were formed from sucrose using recombinant levansucrase from Rahnella aquatilis. Levan formation was optimal at 30 °C resulting 57 % of the theoretical yield. The more suitable substrate concentration for levan formation was 200 g sucrose/L. Oligosaccharides was accumulated selectively at high substrate concentration. The increase of levan and oligosaccharides formation was not achieved by adding water-miscible organic solvents. Alkyl fructosides were synthesized from various alcohols as fructosyl acceptors by R. aquatilis levansucrase. © Rapid Science Ltd. 1998     

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.     

Fructan Biosynthesis by Intra‐ and Extracellular Zymomonas mobilis Levansucrase after Simultaneous Production of Ethanol and Levan

The chemical composition of the Zymomonas mobilis biomass and the culture liquid after ethanol and levan synthesis were studied. The activities of intra‐ and extracellular levansucrase produced by the Z. mobilis strain 113 “S” under optimum conditions both for levan and fructooligosaccharide (FOS) synthesis were also determined. It was shown that levan production relates to the reduction of the carbohydrate and lipid content in the biomass by increasing the nucleic acid and protein content. The levan producing activity of cellular levansucrase after ethanol and levan synthesis was approximately 30–40% of the total activity in the second fermentation stage. It was established that the cell free culture liquid, containing ethanol, levan, gluconic acid and sucrose (15%) at 25 °C, did not show any additional levan synthesising activity. At optimum FOS synthesis conditions (45 °C and 70% sucrose), the cell‐free culture liquid exhibited a high FOS synthesising activity (31% from total carbohydrates), with slightly reduced biomass activity. It was concluded that as a result of the simultaneous ethanol and levan production, the remaining biomass as well as the cell‐free culture liquid could be used for FOS production.     

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight? and compared with the activity of Fructozyme?. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme?, which is important for its potential application in the tequila industry.     

Biochemical and Economical Aspects of Levan Synthesis by Zymomonas Mobilis

With flocculent cells of Zymomonas mobilis levan was produced in a continuous upflow-tower fermentation with a productivity of up to 16 gl^-1h^-1. A large-scale process basing on whole Zymomonas cells is thought to be economical, if levan and ethanol production can be carried out simultaneously. Levan sucrase as the enzyme responsible for levan biosynthesis was purified and partially characterized.     

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.     

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.     

In vitro digestibility and fermentability of levan and its hypocholesterolemic effects in rats

This study describes the in vitro digestibility and fermentability of high molecular weight (ca. 2,000,000) levan and its effect on the metabolism of lipids in growing rats fed cholesterol-free diets. Levan was synthesized from sucrose using bacterial levansucrase immobilized on a honeycomb-shaped ceramic support. Although body weight gain, weight of visceral organs, morphologic changes in the digestive tract, and the serum triacylglycerol and glucose concentrations were not affected by feeding levan diets for 4 weeks, a significant hypocholesterolemic effect was observed. Serum cholesterol level was decreased to 83% or 59% by feeding a 1% or 5% levan diet, respectively. The hypocholesterolemic effect was accompanied by a significant increase in fecal excretion of sterols and lipids. High molecular weight levan, though not hydrolyzed by the salivary amylases, was hydrolyzed by artificial gastric juice and was changed to a low molecular weight (ca. 4,000) levan with a small amount of fructose, but did not produce any fructooligosaccharides. Low molecular weight (ca. 6,000) levan was not hydrolyzed by either pancreatic juice or small intestinal enzymes. This suggests that, in vivo, low molecular weight levan derived from the high molecular weight material is not further digested and reaches the colon intact. The fermentation of low molecular weight levan (ca. 6,000) by several strains of bifidobacteria was not observed. These results showed that the hypocholesterolemic effect of levan may result from the prevention of intestinal sterol absorption, and not from the action of the fermentation products of levan.