菊糖果糖转移酶基因在毕赤酵母中的分泌表达

将PCR获得的不包含信号肽序列的菊糖果糖转移酶基因(ift)与毕赤酵母分泌表达载体pPIC9K进行连接,构建重组载体pPIC9 K-IFTase.重组载体经线性化后电转入毕赤酵母GS115感受态细胞,利用G418抗性梯度筛选及PCR鉴定,获得一株高拷贝菊糖果糖转移酶重组毕赤酵母菌株.该重组菌株经甲醇诱导,能够分泌具有活性的菊糖果糖转移酶,且60h时酶活为10.3U/mL.结果表明,菊糖果糖转移酶可以实现在毕赤酵母的分泌表达.     

超滤法浓缩菊糖果糖转移酶的初步研究

超滤浓缩的菊糖果糖转移酶粗酶根据用途可直接使用或进一步纯化,采用截留分子量为10kDa的Pellicon-2盒式超滤膜包,对菊糖果糖转移酶超滤浓缩进行了初步研究。结果表明,酶解菊糖产物为DFAIII;酶收率85%以上,膜通量减少至3.2L/m2·h;单位体积酶活(U/mL)增加8.5倍,比酶活达到6.7U/mg;再生后,膜通量可以恢复95%以上。     

超高压加工对菊糖果糖转移酶活力和构象的影响

运用高效液相、荧光光谱和圆二色谱等方法研究了超高压加工对菊糖果糖转移酶活力和构象的影响,并分析了压力处理后的酶构象变化与酶活力大小之间的联系。结果表明：在80℃的条件下,与未处理组相比,经压力处理后的菊糖果糖转移酶活力随处理压力的升高及保压时间的延长先上升后下降,200MPa处理30rain后相对酶活力达最高,为124．08％。菊糖果糖转移酶的内源荧光主要来自Trp残基,压力处理后酶的相对荧光强度和相对酶活力之间呈正线性相关,表明酶活力变化与压力处理后的Trp微环境的变化有关。圆二色谱的结果显示该酶仅在219nm有负峰,表明β-折叠是菊糖果糖转移酶的主要二级结构;219nm负峰的峰值大小与相对酶活力呈负线性相关,表明β-折叠是该酶发挥催化活力的结构基础。压力引起酶构象变化的研究为超高压加工提高酶活力的机理研究提供了一定的理论依据．     

填充床反应器固定化菊糖果糖转移酶催化合成双果糖酐Ⅲ的研究

本文建立了填充床反应器固定化菊糖果糖转移酶水解菊糖的工艺。以菊糖为底物,探究该工艺水解菊糖的条件,以单因素实验为基础,依据正交实验优化,考察菊糖浓度、反应时间、反应温度及底物流速等因素对双果糖酐Ⅲ含量的影响,获得最佳的工艺条件。该工艺采用填充床反应器的容积为20m L,固定化酶的装载量为15m L。结果表明:菊糖浓度为100g/L,反应时间为1h,温度为60℃,底物流速为20m L/h,在该工艺条件下制取双果糖酐Ⅲ的浓度为67.38g/L。该工艺能持续操作48d以上,半衰期为48d,为该工艺大规模生产提供理论与操作的基础。     

果聚糖果糖转移酶的研究现状

果聚糖果糖转移酶(levan fructotransferase,即LFTase)[EC 4.2.2.16]是将果聚糖(levan)催化水解为双果糖酐IV(DFA IV)的酶。文中综述了果聚糖果糖转移酶的制备菌种、发酵工艺、分离纯化、酶学性质、酶解产物(DFA IV)及其生理功能等。     

菊糖低聚果糖与功能性糖果

本文简要叙述菊糖、低聚果糖的性状及其保健功能,同时介绍菊糖、低聚果糖在功能性糖果中的应用等。     

微生物菊糖蔗糖酶及在食品中的应用研究进展

菊糖作为一种可溶性膳食纤维,具有良好的理化性质和重要的生理功能,因此,在食品中应用广泛。菊糖蔗糖酶能够以蔗糖为唯一底物,一步合成高分子质量的微生物菊糖。目前鉴定的菊糖蔗糖酶有15 种微生物来源,其中一种的晶体结构已经被解析。国内关于菊糖蔗糖酶的研究鲜有报道。本文对菊糖蔗糖酶的晶体结构和反应机理进行了综合分析。着重讨论了菊糖的链长调控以及菊糖蔗糖酶在生产高分子质量菊糖、低聚果糖、新型低聚糖和菊糖纳米材料方面的应用。最后,结合菊糖蔗糖酶的研究,探讨其发展趋势。     

双酶水解提取牛蒡菊糖的研究

采用双酶水解法提取牛蒡菊糖。首先从8 种酶中选取3 种对牛蒡菊糖提取率最高的酶,分别为木瓜蛋白酶、植物蛋白酶和酸性蛋白酶,对牛蒡菊糖的提取率分别为8.83%、8.67% 和8.21%。然后对每一种酶采用单因素试验方法研究pH 值、固液比、加酶量、温度以及时间对牛蒡菊糖提取率的影响;再通过3 种酶之间的相互组合试验,选出一组最佳组合为：木瓜蛋白酶+ 植物蛋白酶,其提取率为11.43%。最后采用单因素和正交试验设计方法,研究加酶量、固液比、温度、时间以及pH 值对牛蒡菊糖提取率的影响,得到双酶水解提取牛蒡菊糖的最佳条件组合为：在木瓜蛋白酶加酶量10%、温度50℃、pH7、时间4h、固液比1:15(m/V)的条件下进行酶解,4h 后加入植物蛋白酶,加酶量20%、温度45℃、pH8,时间4h、固液比1:15(m/V),提取液经乙醇沉淀、真空浓缩,得到粗菊糖,菊糖提取率为13.41%,产品中菊糖含量为67.86%,蛋白质含量为1.32%。     

双果糖酐水解酶分子改造提升酶活性研究

双果糖酐水解酶(difructose anhydrideⅢhydrolase, DFA-Ⅲase)能将新型功能甜味剂双果糖酐转化为益生元菊二糖，然而DFA-Ⅲase酶活性较低，为了提高DFA-Ⅲase酶法合成菊二糖的能力，该研究对来源于Arthrobacter chlorophenolicus A6的双果糖酐水解酶AcDFA-Ⅲase进行分子改造。基于已有的AcDFA-Ⅲase的晶体结构，选择酶活性口袋上方loop区和活性口袋中的14个关键位点进行定点突变，得到17种DFA-Ⅲase突变体。利用96孔板作为高通量培养容器，结合DNS法快速测定酶活性，初筛得到一个酶活性提高的突变体D380A。用HPLC法测定分离纯化出的酶活性，验证结果表明其酶活性有显著提高，酶比活性是AcDFA-Ⅲase的162.3%。同源建模分析，380位天冬氨酸突变成丙氨酸后，loop环区域疏水性增强，保证了催化中心的疏水微环境，提高了酶的催化活性。该研究建立的高通量检测方法为DFA-Ⅲase的筛选以及菊二糖的大量生产奠定了基础。     

牛蒡菊糖酶法提取

采用固定化酶法提取牛蒡菊糖。结果表明酶水解提取牛蒡菊糖的最佳工艺为：13.5g/100mL 中性蛋白酶、pH 7、固液比1:15、50℃、酶水解6h,菊糖提取率为14.57%;固定化酶制备最佳工艺为：以甲醛(40%):NaOH(2mol/L)=2:3 为凝结液、pH7.5、壳聚糖2.5g/100mL、60℃、加酶量7.5mg/mL,固定8h,酶活力回收率可达到39.13%;固定化酶提取牛蒡菊糖最适条件为：pH7、固液比1:15、60℃、固定化酶加入量13. 5 g/100mL、酶解5h,在此条件下菊糖提取率达到12.89%。固定化酶的稳定性与游离酶相比有显著的提高,连续反应10 次后,固定化酶仍然具有良好的使用性能,此时牛蒡菊糖的提取率为9.42%。     

DFA III production from inulin with inulin fructotransferase in ultrafiltration membrane bioreactor

An ultrafiltration membrane bioreactor was used for the production of DFA III from enzymatic conversion of inulin. Compared with the traditional batch reactor, the productivity and purity of DFA III could be markedly enhanced and product inhibition was removed and IFTase could be continuously used for six runs in the UF membrane bioreactor. When the substrate concentration was 100 g/L, the concentration of DFA III was about 78.4 g/L, while the productivity and purity of DFA III could attain about 2385 and 92%, respectively.     

Efficient induction of inulin fructotransferase by inulin and by difructose anhydride III in Arthrobacter aurescens SK 8.001

Inulin fructotransferase (IFTase; EC 4.2.2.18) has received great attention mainly due to its application in producing difructose anhydride III (DFA III), which is a novel functional sweetener. The object of this study was to investigate the induction of IFTase in Arthrobacter aurescens SK 8.001 with various carbon sources, especially inulin and DFA III. IFTase production could be significantly promoted by the supplement of inulin (5–50 g/L) and DFA III (5–20 g/L). Inulin at high initial concentrations gave no indication of catabolite repression, whereas 30 and 40 g/L DFA III intensely inhibited cell growth and IFTase activity. No fructose was detected in broth throughout the cultivation with inulin, and inulin was converted into DFA III and minor fructooligosaccharides. And when DFA III was the carbon source, DFA III was the only sugar detected in the broth. In conclusion, both DFA III and inulin are effective for IFTase induction, and inulin with higher IFTase activity proved to be a more potent inducer.     

Molecular cloning of the gene encoding the di-D-Fructofuranose 1,2':2,3' dianhydride hydrolysis enzyme (DFA IIIase) from Arthrobacter sp. H65-7

The gene encoding an intracellular enzyme hydrolyzing di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) (DFA IIIase) was cloned from the genomic DNA of Arthrobacter sp. H65-7 for the first time. The single open reading frame (ORF) of the DFA IIIase gene consisted of 1368-bp encoding 455 amino acids. DFA IIIase showed a phylogenetically distinct position from other inulin-degrading enzymes and showed similarity only with inulin fructotransferases (depolymerizing) (inulase II, EC 2.4.1.93) from Arthrobacter globiformis C11-1, Arthrobacter sp. A-6, and Arthrobacter sp. H65-7 (48.7-50.3%), and inulin fructotransferase (DFA I-producing) (EC 2.4.1.200) from A. globiformis S14-3 (44.4%). An Escherichia coli transformant harboring a recombinant plasmid, pINB2, in which the DFA IIIase gene was fused with the beta-galactosidase of pUC19 and under the control of the lac promoter, expressed DFA IIIase and the cloned enzyme produced inulobiose from DFA III similarly to the DFA IIIase of the wild-type strain, Arthrobacter sp. H65-7.     

Actinomycete Nonomuraea sp. isolated from Indonesian soil is a new producer of inulin fructotransferase

An actinomycete that excretes inulin fructotransferase to the culture supernatant was able to produce di-d-fructofuranose 1,2':2,3' dianhydride (DFA III) from inulin, with the greatest rate of enzyme activity at 65°C and at a pH of 5.5. Through chemotaxonomic and 16S rRNA gene analysis, this strain was identified as genus Nonomuraea in the Streptosporangiaceae family. This is the first report of an inulin fructotransferase producer in this family.     

Cloning and extracellular expression of inulin fructotransferase from Arthrobacter aurescens SK 8.001 in E. coli

BACKGROUND: Difructose anhydride (DFA) III is a natural and low‐calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III. RESULTS: IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over‐expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL^?1. This value was 4.1‐fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h. CONCLUSION: IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over‐expressed in E. coli. IFTase was reported for the first time to be over‐expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production. Copyright © 2011 Society of Chemical Industry     

Isolation and identification of Di-D-fructose dianhydrides resulting from heat-induced degradation of inulin

Dry heating of inulin up to 200 °C for 30 min resulted in a wide range of degradation products, from which five quantitatively dominating compounds were isolated using flash chromatography and semi-preparative HPLC–RI. It was possible to identify four different DFDAs (di-d-fructose dianhydrides) by means of one- and two-dimensional NMR, namely DFA III (α-D-Fruf-1,2′:2,3′-β-D-Fruf), DFA I (α-D-Fruf-1,2′;2,1′-β-D-Fruf), DFA VII (α-D-Fruf-1,2′;2,1′-α-D-Fruf), and β-D-Fruf-1,2′;2,1′-β-D-Fruf. For the fifth compound, the structure of the difructan α-D-Fruf-1,2′-β-D-Fruf was proposed. Using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), it was possible to quantify those isolated DFDAs in inulin caramels to a total amount of up to 25%, indicating that dry heating of inulin may be a suitable tool to enrich caramels with DFDAs.     

Effects of difructose anhydride III (DFA III) administration on bile acids and growth of DFA III-assimilating bacterium Ruminococcus productus on rat intestine

The growth of DFA III-assimilating bacteria in the intestines of rats fed 3% DFA III for 2 weeks was examined. Sixty-four percent of the DFA III intake had been assimilated on day 3 of ingestion, and almost all of the DFA III was assimilated at the end of the experiment. The DFA III-assimilating bacterium, Ruminococcus productus, in DFA III-fed rats was in the stationary state of 10(8)-10(9) cells/g dry feces within a week from 10(6) cells/g dry feces on day 1 of DFA III ingestion. The number of R. productus cells was associated with the amount of DFA III excreted in the feces. The acetic acid produced from DFA III by R. productus lowered the cecal pH to 5.8. In control-fed rats and DFA III-fed rats, 94% of secondary bile acids and 94% of primary bile acids, respectively, were accounted for in the total bile acids analyzed. DFA III ingestion increased the ratio of primary bile acids and changed the composition of fecal bile acids. In conclusion, R. productus assimilated DFA III, produced short chain fatty acids, and the cecal pH was lowered. The acidification of rat intestine perhaps inhibited secondary bile acid formation and decreased the ratio of secondary bile acids. Therefore, it is expected that DFA III may prevent colorectal cancer and be a new prebiotic candidate.     

Short communication: Effect of difructose anhydride III on serum immunoglobulin G concentration in newborn calves

Difructose anhydride (DFA) III is an indigestible disaccharide that promotes paracellular absorption of calcium, magnesium, and other minerals in the intestine by acting on epithelial tight junctions. This study aimed to elucidate the effect of DFA III on serum IgG concentration. One hundred and twenty Holstein and Holstein/Japanese Black crossbred calves were randomly divided into 4 groups of 30 to receive untreated colostrum (DFA0) or colostrum containing 3, 6, or 18 g of DFA III (DFA3, DFA6, or DFA18, respectively). At 24h after birth, both serum IgG (ranging from 16.4 to 21.2mg/mL) and apparent efficiency of absorption (26.0 to 37.2%) showed increases with the amount of DFA III intake. By multiple regression analysis, the standardized partial regression coefficient for DFA III was 0.25, the second highest following that for the colostrum IgG concentration (0.80), indicating a positive effect of DFA III on serum IgG. A positive linear regression was found between colostrum IgG and serum IgG concentrations at 24h of age. These results indicate that IgG absorption occurred as a nonsaturable process, which might be characteristic of gradient-dependent paracellular transport. Thus, it was concluded that DFA III improves not only minerals but IgG absorption in calves.     

Effects of difructose anhydride III (DFA III) administration on rat intestinal microbiota

The effects of difructose anhydride III (di-D-fructofuranose-1,2':2,3'-dianhydride; DFA III) administration (3% DFA III for 4 weeks) on rat intestinal microbiota were examined using denaturing gradient gel electrophoresis (DGGE). According to DGGE profiles, the number of bacteria related to Bacteroides acidofaciens and uncultured bacteria within the Clostridium lituseburense group decreased, while that of bacteria related to Bacteroides vulgatus, Bacteroides uniformis and Ruminococcus productus increased in DFA III-fed rat cecum. In the cecal contents of DFA III-fed rats, a lowering of pH and an increase in short chain fatty acids (SCFAs), especially acetic acid, were observed. The DFA III-assimilating bacterium, Ruminococcus sp. M-1, was isolated from the cecal contents of DFA III-fed rats. The strain had 98% similarity with R. productus ATCC 27340T (L76595), and mainly produced acetic acid. These results confirmed that the bacteria harmful to host health were not increased by DFA III administration. Moreover, DFA III stimulated the growth of Ruminococcus sp. M-1 producing acetic acid, which may alter the intestinal microbiota towards a healthier composition. It is expected that DFA III would be a new candidate as a prebiotic.