D-阿洛酮糖酶法生产的研究进展

稀有单糖D-阿洛酮糖是D-果糖的C-3差向异构体,主要通过D-塔格糖3-差向异构酶或D-阿洛酮糖-3-差向异构酶对D-果糖进行异构化获得。D-阿洛酮糖不仅可以作为食品添加剂和膳食补充剂,而且具有改善胰岛素抵抗、增强抗氧化和降血糖控制等多种生理功能。因此,D-阿洛酮糖作为传统高能量糖如蔗糖、果糖等的健康替代品,具有重要的研究价值。作者综述了D-阿洛酮糖的理化性质、来源、体内代谢、生理功能、应用和最新生产技术,讨论了D-阿洛酮糖生产中存在的问题及解决方案。针对低废物生成、低能耗、高糖产量等特点,提出了一种绿色、可循环利用的D-阿洛酮糖生产工艺技术。     

Biosynthetic Gene Cluster of Cetoniacytone A,an Unusual Aminocyclitol from the Endosymbiotic Bacterium Actinomyces sp. Lu 9419

A gene cluster responsible for the biosynthesis of the antitumor agent cetoniacytone A was identified in Actinomyces sp. strain Lu 9419, an endosymbiotic bacterium isolated from the intestines of the rose chafer beetle (Cetonia aurata). The nucleotide sequence analysis of the 46 kb DNA region revealed the presence of 31 complete ORFs, including genes predicted to encode a 2‐epi‐5‐epi‐valiolone synthase (CetA), a glyoxalase/bleomycin resistance protein (CetB), an acyltransferase (CetD), an FAD‐dependent dehydrogenase (CetF2), two oxidoreductases (CetF1 and CetG), two aminotransferases (CetH and CetM), and a pyranose oxidase (CetL). CetA has previously been demonstrated to catalyze the cyclization of sedoheptulose 7‐phosphate to the cyclic intermediate, 2‐epi‐5‐epi‐valiolone. In this report, the glyoxalase/bleomycin resistance protein homolog CetB was identified as a 2‐epi‐5‐epi‐valiolone epimerase (EVE), a new member of the vicinal oxygen chelate (VOC) superfamily. The 24 kDa recombinant histidine‐tagged CetB was found to form a homodimer; each monomer contains two βαβββ scaffolds that form a metal binding site with two histidine and two glutamic acid residues. A BLAST search using the newly isolated cet biosynthetic genes revealed an analogous suite of genes in the genome of Frankia alni ACN14a, suggesting that this plant symbiotic nitrogen‐fixing bacterium is capable of producing a secondary metabolite related to the cetoniacytones.     

变形假单胞菌2-酮基葡萄糖酸差向异构酶基因的原核表达

采用降落聚合酶链式反应（touchdown polymerase chain reaction,TD-PCR）技术,从2-酮基葡萄糖酸工业生产菌株变形假单胞菌JUIM01中克隆了编码2-酮基葡萄糖酸差向异构酶的基因kguE。将目的基因与质粒pET-28a（＋）重组后转入宿主表达菌中,获得了重组菌株大肠杆菌BL21（DE3）/pET-28a（＋）-kguE。在异丙基-β-D-硫代半乳糖苷的诱导下,该重组菌株表达了一个分子质量约为30.5?ku的融合蛋白,且该蛋白的Western-blot鉴定结果显示为阳性。生物信息学分析结果表明,该蛋白是一种由256?个氨基酸残基组成的分子质量为28.5?ku的亲水性蛋白,与恶臭假单胞菌的2-酮基葡萄糖酸差向异构酶在氨基酸序列上的一致性达78%,定位于细胞质中,其二级结构中α-螺旋、延伸链和无规则卷曲所占的比例分别为40.62%、17.19%和42.19%。本研究结果为变形假单胞菌2-酮基葡萄糖酸差向异构酶的功能研究提供了一定理论支持。     

Lactulose production from lactose by recombinant cellobiose 2‐epimerase in permeabilised Escherichia coli cells

Caldicellulosiruptor saccharolyticus cellobiose 2‐epimerase (CsCE) catalyses the single substrate lactose into lactulose and is the most efficient enzyme ever found for the enzymatic synthesis of lactulose. Ethanol‐permeabilised Escherichia coli cells containing CsCE were used as biocatalysts for lactulose production. The reaction conditions for maximum lactulose production were optimised to be 600 g L^?1 lactose, pH 7.5, 80 °C and 12.5 U mL^?1 of whole‐cell biocatalyst. After incubated at Na₂HPO₄–NaH₂PO₄ buffer for 2 h, approximately 390.59 g L^?1 lactulose was obtained with a conversion yield of 65.1%. The lowest production and conversion yield of epilactose were also found in Na₂HPO₄–NaH₂PO₄ buffer with a final concentration of 11.7 g L^?1 and a conversion yield <2%. The results represent a promising technology to attain high production and conversion yield of lactulose with a high purity on industrial scale.     

D-塔格糖-3-差向异构酶及其应用

D-塔格糖-3-差向异构酶（D-tagatose-3-epimerase,DTE）家族,是催化酮糖类单糖C-3位置的差向异构化反应的一类酶,也是稀有糖生产中的核心酶.随着稀有糖在食品、保健品、医药中间体等领域的广泛应用,利用DTE酶家族制备稀有糖备受关注.本文对DTE酶家族的DTEase和DPEase的来源、酶学特性、蛋白结构以及DTE酶家族在稀有糖生产中的应用现状等方面进行了综述,最后对DTE酶家族的研究趋势进行了展望.