D-塔格糖与L-阿拉伯糖异构酶的研究进展

D-塔格糖是一种天然的低能量填充型甜味剂,并具有抑制高血糖、改善肠道菌群和不致龋齿等多种生理功效。D-塔格糖是一种稀有糖,因此通常利用化学转化或生物转化方法进行大量生产。L-阿拉伯糖异构酶是生物法工业化生产D-塔格糖的有效酶。文中对D-塔格糖的性质、功能及利用L-阿拉伯糖异构酶生产D-塔格糖的研究新进展进行了综述。     

L-阿拉伯糖异构酶粗酶性质及其生物合成塔格糖的研究

从筛选的一株乳酸菌中得到L-阿拉伯糖异构酶,该酶能把D-半乳糖异构成D-塔格糖。L-阿拉伯糖异构酶粗酶的最适反应温度和pH分别为60℃和7.0;在pH4.0～8.0的范围内和30～60℃下具有较好的稳定性。Mn2+、Li+、Zn2+、Cu2+等离子对该酶有激活作用,而Ba2+、Mg2+、Fe2+几乎完全抑制酶活。用L-阿拉伯糖异构酶的粗酶、10%和40%的湿重细胞转化生成塔格糖,最适半乳糖浓度0.83mol/L,40%的湿重细胞反应72h后,转化率达到38.9%。      

L-阿拉伯糖异构酶粗酶性质及其生物合成塔格糖的研究

从筛选的一株乳酸菌中得到L-阿拉伯糖异构酶,该酶能把D-半乳糖异构成D-塔格糖。L-阿拉伯糖异构酶粗酶的最适反应温度和pH分别为60℃和7.0;在pH4.0～8.0的范围内和30～60℃下具有较好的稳定性。Mn2 、Li 、Zn2 、Cu2 等离子对该酶有激活作用,而Ba2 、Mg2 、Fe2 几乎完全抑制酶活。用L-阿拉伯糖异构酶的粗酶、10%和40%的湿重细胞转化生成塔格糖,最适半乳糖浓度0.83mol/L,40%的湿重细胞反应72h后,转化率达到38.9%。     

D-塔格糖的生理活性及生物合成研究进展

D-塔格糖是一种重要的稀有己酮糖,其甜度是蔗糖的92%,热量只有蔗糖的三分之一;D-塔格糖分别于2001年和2014年被美国食品药品监督管理局和我国卫生和计划生育委员会批准为食品新原料。D-塔格糖具有降低血糖、减少肥胖、改善肠道菌群、抗龋齿、抗氧化等功能。D-塔格糖作为一种理想功能性甜味剂,具有广泛的应用价值,然而塔格糖的生产成本以及产量一直是限制塔格糖发展和应用的重要原因。D-塔格糖的合成方法主要有化学合成法和生物转化法。化学合成法生产D-塔格糖虽然是一种经济有效的方法,但是在生产过程中需要高温高压的条件,并且容易生成山梨糖、甘露糖等杂糖,不利于分离纯化。为了克服这些缺点,生物转化法生产D-塔格糖得到了广泛的关注和研究,因此,系统阐述了以不同底物,如己糖(半乳糖醇、D-半乳糖和D-果糖)、乳糖、多糖(麦芽糊精和淀粉)转化成D-塔格糖的研究进展和优缺点,并对其未来研究趋势进行了展望,以期为D-塔格糖的研发与生产提供借鉴。     

交联海藻酸钠-明胶固定化L-阿拉伯糖异构酶的研究

以海藻酸钠和明胶为载体,对L-阿拉伯糖异构酶进行固定化。为增强固定化酶的稳定性,又用戊二醛对其进一步交联。研究了海藻酸钠及明胶浓度、CaCl2浓度、硬化时间以及戊二醛浓度等因素对固定化效果的影响,并对固定化酶的酶学性质进行了研究。结果表明最佳固定化条件为:海藻酸钠浓度2.0%、明胶浓度2.0%、硬化时间6h、CaCl2浓度4.0%、戊二醛浓度0.02%,该条件下所得酶活回收率最高为82%,且具有较好的操作稳定性,重复操作7次后酶活损失不到50%。与游离酶相比,固定化酶的最适反应pH及反应温度没有变化,但pH稳定性和耐热性都有所提高。      

交联海藻酸钠-明胶固定化L-阿拉伯糖异构酶的研究

以海藻酸钠和明胶为载体,对L-阿拉伯糖异构酶进行固定化。为增强固定化酶的稳定性,又用戊二醛对其进一步交联。研究了海藻酸钠及明胶浓度、CaCl2浓度、硬化时间以及戊二醛浓度等因素对固定化效果的影响,并对固定化酶的酶学性质进行了研究。结果表明最佳固定化条件为：海藻酸钠浓度2.0%、明胶浓度2.0%、硬化时间6h、CaCl2浓度4.0%、戊二醛浓度0.02%,该条件下所得酶活回收率最高为82%,且具有较好的操作稳定性,重复操作7次后酶活损失不到50%。与游离酶相比,固定化酶的最适反应pH及反应温度没有变化,但pH稳定性和耐热性都有所提高。     

新型甜味剂塔格糖的应用及生产

D-塔格糖是一种新型低热量甜味剂,用于食品和药品领域,有抗高血糖和益生保健等多种功能.本文综述了其基本性质和功能,抗高血糖的机制,食品和药品应用情况及生产技术开发的进展,重点阐述了塔格糖生产的工具酶--L-阿拉伯糖异构酶的基因克隆与筛选、酶工程化改造及固定化等方面的研究进展.     

新型甜味剂塔格糖的应用及生产

D-塔格糖是一种新型低热量甜味剂,用于食品和药品领域,有抗高血糖和益生保健等多种功能。本文综述了其基本性质和功能,抗高血糖的机制,食品和药品应用情况及生产技术开发的进展,重点阐述了塔格糖生产的工具酶——L-阿拉伯糖异构酶的基因克隆与筛选、酶工程化改造及固定化等方面的研究进展。     

乳酸菌SK1.002产L-阿拉伯糖异构酶培养基优化及发酵条件

L-阿拉伯糖异构酶能将D-半乳糖异构成D-塔格糖。通过单因素试验和快速登高法对乳酸菌SK1.002产L-阿拉伯糖异构酶的培养基进行优化,确定发酵优化条件为（组分g/L）：麦芽糊精28,酵母膏10,玉米粉浆22,无水乙酸钠10,K3PO40.2,NaCl 0.01,FeSO4.7H2O 0.01,Mg-SO4.7H2O 0.2,MnSO4.2H2O 0.05,L-阿拉伯糖2.5。发酵初始pH 8.4,培养温度37℃,接种体积分数3%,培养时间12 h。在此发酵条件下,酶活达到了7.28 U/mL。     

D-甘露糖异构酶的克隆表达及酶法制备D-甘露糖

将大肠杆菌BL21中的D-甘露糖异构酶(MIase)基因yihS克隆到表达载体pET-28a(+)中,并转入大肠杆菌BL21(DE3)感受态细胞中表达。重组菌株经IPTG诱导培养6 h后MIase发酵酶活可达4.2 U/mL。重组MIase生产D-甘露糖时不需要金属离子的参与。该酶在40℃和pH 7.5条件下表现出催化D-果糖的最高活性,转化率为25%左右;通过研究底物浓度对酶活性的影响,得出该酶的活性不受底物浓度的抑制,以D-果糖为底物时,动力学参数Km为123.32mmol/L,Vmax为113.64μmol/(min·mg),催化效率kcat/Km为0.691 (s·mmol/L)。     

Direct production of D-arabinose from D-xylose by a coupling reaction using D-xylose isomerase, D-tagatose 3-epimerase and D-arabinose isomerase

Klebsiella pneumoniae 40bXX, a mutant strain that constitutively produces D-arabinose isomerase (D-AI), was isolated through a series of repeated subcultures from the parent strain on a mineral salt medium supplemented with L-Xylose as the sole carbon source. D-AI could be efficiently immobilized on chitopearl beads. The optimum temperature for the activity of the immobilized enzyme was 40 degrees C and the enzyme was stable up to 50 degrees C. The D-Al was active at pH 10.0 and was stable in the range of pH 6.0-11.0. The enzyme required manganese ions for maximum activity. Three immobilized enzymes, D-xylose isomerase (D-XI), D-tagatose 3-epimerase (D-TE and D-AI were used for the preparation of D-arabinose from D-xylose in a coupling reaction. After completion of the reaction, degradation of D-xylulose was carried out by Saccharomyces cerevisiae. The reaction mixture containing D-Xylose, D-ribulose and the product was then separated by ion exchange column chromatography. After crystallization, the product was checked by HPLC, IR spectroscopy, NMR spectroscopy and optical rotation measurements. Finally, 2.0 g of D-arabinose could be obtained from 5 g of the substrate.     

Efficient expression of myo-inositol oxygenase in Escherichia coli and application for conversion of myo-inositol to glucuronic acid

Glucuronic acid is an important biochemical with wide applications in the food and medical industries. The myo-inositol oxygenase (MIOX) gene was synthesized and expressed in Escherichia coli BL 21(DE3). After optimization of induction conditions and the culture temperature, the highest MIOX activity (45.46 kU/L) was achieved when 0.1 mM isopropyl-thio-β-d-galactoside (IPTG) was added to cell cultures with an OD₆₀₀ value 1.0 followed by induction at 26°C for 8 hours. The purified MIOX enzyme exerted characteristics similar to the native form. Conversion of myo-inositol to glucuronic acid was performed using whole cells in a pH 8.5 buffer system. Whole cells harboring myo-inositol oxygenase were used as a biocatalyst to produce 2.13 g/L of glucuronic acid with a conversion rate of 99%. A promising novel process for glucuronic acid production from abundant agricultural byproducts is presented.     

Cloning and sequencing of the histidine decarboxylase gene from Photobacterium phosphoreum and its functional expression in Escherichia coli

The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. We reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 10 degrees C. Moreover, histamine-forming ability was affected by two histidine decarboxylases (HDCs): constitutive and inducible enzymes. In this study, the gene encoding P. phosphoreum HDC was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA corresponding to the hdc gene revealed an open reading frame of 1,140 bp coding for a pyridoxal-5'-phosphate-dependent HDC of 380 amino acid residues with a predicted molecular mass of 42.6 kDa. The HDC amino acid sequences formed a phylogenetic clade with strong bootstrap support and revealed high sequence similarities among the P. phosphoreum isolate and species of the family Enterobacteriaceae and a separate phylogenetic branch with the lowest sequence similarity between the isolate and the taxonomically closer Listonella anguillarum. The T7 promoter was used to overexpress the hdc gene in E. coli cells. The recombinant clone, E. coli BL21(DE3), displayed significant levels of HDC activity. The recombinant hdc gene was suggested to code the inducible HDC; therefore, the optimum reaction conditions of the recombinant HDC were similar to those of the inducible HDC in the P. phosphoreum isolate. In addition, a putative catabolite-repressor protein binding site, amino acid permease gene, and histidine-tRNA synthetase gene were found in flanking regions of the hdc gene.     

Acidothermus cellulolyticus 11B葡萄糖异构酶基因的克隆、表达及酶活性研究

葡萄糖异构酶是生物法转化葡萄糖为果糖制备果葡糖浆的关键酶。本文克隆到一种葡萄糖异构酶基因xyl,来源于嗜酸耐热型微生物Acidothermus cellulolyticus 11B(ATCC43068)。以pET-22b(+)为载体质粒,E.coliBL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的重组葡萄糖异构酶过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE分析,在约43kDa处出现显著的特征蛋白条带;活性检测结果表明该重组葡萄糖异构酶具有较高的果糖转化活性。     

L-阿拉伯糖的生理功能及市场应用进展

L-阿拉伯糖是一种天然五碳醛糖,可以发生美拉德反应和焦糖化反应,在食品加工中具有很好的特性。L-阿拉伯糖几乎不被人体利用,其被证明可以与小肠中的蔗糖酶结合降低酶活性,减少蔗糖的消化吸收,进而降低由蔗糖引起的血糖和胰岛素水平的升高,另外一些研究数据表明L-阿拉伯糖也具有肠道调节功能。目前L-阿拉伯糖已经被广泛应用到各类食品中。本文对L-阿拉伯糖的生理功能及市场应用等方面的进展进行综述。     

Cloning of a thermostable xylanase from Actinomadura sp. S14 and its expression in Escherichia coli and Pichia pastoris

A thermophilic xylan-degrading Actinomadura sp. S14 was isolated from compost in Thailand. Hemicellulase activities such as endo-1,4-β-xylanase, β-xylosidase and α-arabinofuranosidase were induced with xylan-containing agriculture wastes and oat spelt xylan. The gene encoding xylanase consisting of 687bp was cloned from Actinomadura sp. S14. The deduced amino acid sequence contained a signal peptide of 41 amino acids and a probable mature xylanase of 188 amino acids. An open reading frame (xynS14) corresponding to a mature xylanase was expressed in Escherichia coli and Pichia pastoris. The specific activity of purified XynS14 (P. pastoris) was 2.4-fold higher than XynS14 (E. coli). Both XynS14s showed the same basic properties such as optimal pH and temperature (pH 6.0 and 80°C) and stability in a broad pH range (pH 5.0-11.0) and at high temperatures up to 80°C. Both XynS14s showed approximately the same substrate specificity and K(m) values toward various xylans, but XynS14 (P. pastoris) showed higher V(max) and K(cat) than XynS14 (E. coli). Higher specific activities of XynS14 (P. pastoris) may be due to protein-folding in the host. Purified XynS14 showed more endo-1,4-β-xylanase activity on xylan and xylooligosaccharides than on xylotriose.     

Cloning and heterologous expression of the Candida albicans gene PMI 1 encoding phosphomannose isomerase

Using a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI-deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64·1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D-mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells. The nucleotide sequence data reported here will appear in the EMBL database under Accession Number X82024.