Mass production of D-psicose from d-fructose by a continuous bioreactor system using immobilized D-tagatose 3-epimerase

An improved process for the mass production of D-psicose from D-fructose was developed. A D-fructose solution (60%, pH 7.0) was passed at 45 degrees C through a column filled with immobilized D-tagatose 3-epimerase (D-TE) which was produced using recombinant Escherichia coli, and 25% of the substrate was converted to D-psicose. After epimerization, the substrate, D-fructose, was removed by treatment with baker's yeast. The supernatant was concentrated to a syrup by evaporation under vacuum and D-psicose was crystallized with ethanol. Approximately 20 kg of pure crystal D-psicose was obtained in 60 d.     

Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization

Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.     

Efficient conversion of allitol to D-psicose by Bacillus pallidus Y25

An efficient method for conversion of allitol to D-psicose was achieved by a resting cell reaction of Bacillus pallidus Y25 for the first time. Notably, it was possible to produce D-allose and D-altrose from allitol directly via D-psicose by prolonging the reaction time. This method was applied for the preparation of D-psicose using the extract of Itea virginica as a starting material in this study. D-Psicose which is the absolutely key precursor for the production of other six carbon sugars could be obtained as the sole product at high yield.     

生物转化生成D-阿洛酮糖的类球红细菌的筛选

对鱼塘淤泥、水样中富集分离的27株光合细菌的静止细胞反应产物,采用高效液相色谱进行分析,从中筛选到1株D-阿洛酮糖产率较高的菌株,在形态和常规生理生化方面鉴定的基础上,结合16S rDNA序列分析鉴定为类球红细菌(Rhodobacter sphaeroides),定名为类球红细菌SK011。SK011利用D-果糖(36 g/L,pH7.5)为底物进行静止细胞转化,45℃,5 h,D-阿洛酮糖产率达到6.54%。     

弗吉尼亚鼠刺叶片中稀少糖醇生物转化及分离制备

为探讨使用鼠刺植物制备稀少糖醇的可能性,以弗吉尼亚鼠刺叶片为原料,热水提取其所含的稀少糖及糖醇,使用产酸克雷伯氏菌静息细胞对提取物的单糖类物质进行生物转化,再利用絮凝、离子树脂色谱分离以及乙醇结晶连用手段对转化的提取物进行纯化,最后使用核磁共振谱对获得的稀少糖醇进行分析鉴定。液相色谱分析结果显示弗吉尼亚鼠刺叶片提取物含有稀少D-阿洛酮糖和蒜糖醇,其质量分数分别为27.86%和7.86%;利用制备的产酸克雷伯氏菌的静息细胞进行60 h催化反应后,提取物中86%的D-阿洛酮糖转化为蒜糖醇,其余被细胞代谢消耗。多种分离纯化手段的联合使用,能有效去除杂质和色素,获得蒜糖醇纯品。     

D-Psicose,a Sweet Monosaccharide,Ameliorate Hyperglycemia,and Dyslipidemia in C57BL/6J db/db Mice

ABSTRACT: D-psicose has been implicated in glycemic control in recent animal and human studies. In this study, the effects of D-psicose on glycemic responses, insulin release, and lipid profiles were compared with those of D-glucose and D-fructose in a genetic diabetes model. C57BL/6J db/db mice were orally supplemented with 200 mg/kg BW of D-psicose, D-glucose, or D-fructose, respectively, while diabetes control or wild type mice were supplemented with water instead. D-psicose sustained weight gain by about 10% compared to other groups. The initial blood glucose level maintained from 276 to 305 mg/dL during 28 d in the D-psicose group, whereas a 2-fold increase was found in other groups (P < 0.05) among diabetic mice. D-psicose significantly improved glucose tolerance and the areas under the curve (AUC) for glucose among diabetes (P < 0.05), but had no effect on serum insulin concentration. The plasma lipid profile was not changed by supplemental monosacchrides, although the ratio of LDL-cholesterol/HDL-cholesterol was ameliorated by D-psicose. The administration of D-psicose reversed hepatic concentrations of triglyceride (TG) and total cholesterol (TC) by 37.88% and 62.89%, respectively, compared to the diabetes control (P < 0.05). The current findings suggest that D-psicose shows promise as an antidiabetic and may have antidyslipidemic effects in type 2 diabetes.     

Characteristics and antioxidant activity of Maillard reaction products from psicose-lysine and fructose-lysine model systems

D-Psicose, an epimer of D-fructose isomerized at C-3 position, is a rare ketohexose that is thought to be beneficial for obese people and diabetic patients as a noncaloric sweetener. In the present study, model Maillard reaction products were obtained from D-psicose (or D-fructose) and L-lysine heating at 120 °C up to 8 h with the initial pH 9.0. The changes in pH, UV-vis absorbance, and free amino groups during the reaction were detected. Moreover, the antioxidant potential of the Maillard reaction products at different intervals was investigated. Although there was almost no difference in the oxygen radical absorbance capacity, the Maillard reaction products from psicose performed better than that from fructose in the radical-scavenging activity of 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 1, 1,-diphenyl-2-picryl-hydrazyl. The reducing power of the Maillard reaction products from psicose was also stronger than that from fructose. These results indicated that psicose played an effective role in the Maillard reaction and its Maillard reaction products could act as potential antioxidants in food industry.     

Bioproduction of D-allulose: Properties,applications, purification,and future perspectives

D-allulose is the C-3 epimer of D-fructose, which rarely exists in nature, and can be biosynthesized from D-fructose by the catalysis of D-psicose 3-epimerase. D-allulose is safe for human consumption and was recently approved by the United States Food and Drug Administration for food applications. It is not only able be used in food and dietary supplements as a low-calorie sweetener, but also modulates a variety of physiological functions. D-allulose has gained increasing attention owing to its excellent properties. This article presents a review of recent progress on the properties, applications, and bioproduction progress of D-allulose.     

Dietary D-psicose reduced visceral fat mass in high-fat diet-induced obese rats

D-Psicose, a C-3 epimer of D-fructose, has shown promise in reducing body fat accumulation in normal rats and plasma glucose level in genetic diabetic mice. Effects of D-psicose on diet-induced obesity are not clearly elucidated, and we investigated food intake, body weight, and fat accumulation in rats fed high-fat (HF) diet. Sprague-Dawley rats became obese by feeding HF diet for 4 wk, and were assigned either to normal or HF diet supplemented with or without D-psicose, sucrose, or erythritol for 8 wk. Changing HF to normal diet gained less body weight and adipose tissue due to different energy intake. D-psicose-fed rats exhibited lower weight gain, food efficiency ratio, and fat accumulation than erythritol- and sucrose-fed rats. This effect was more prominent in D-psicose-fed rats with normal diet than with HF diet, suggesting combination of psicose and calorie restriction further reduced obesity. There was no difference in serum cholesterol/high-density lipoprotein (HDL)-C and low-density lipoprotein (LDL)-C/HDL-C ratios between D-psicose group and other groups. Liver weight in 5% psicose group with normal diet was higher than in other groups, but histopathological examination did not reveal any psicose-related change. D-Psicose inhibited the differentiation of mesenchymal stem cell (MSC) to adipose tissue in a concentration-dependent manner. These results demonstrate that D-psicose produces a marked decrease, greater than erythritol, in weight gain and visceral fat in an established obesity model by inhibiting MSC differentiation to adipocyte. Thus, D-psicose can be useful in preventing and reducing obesity as a sugar substitute and food ingredient. PRACTICAL APPLICATION: We can develop D-psicose as a sugar substitute and food ingredient since it can prevent obesity in normal people, but also suppress adiposity as a sugar substitute or food ingredients with antiobesity effect in obese people. D-psicose can be unique functional sweetener because of its function of reducing visceral fat mass and weight gain.     

来源于P.bacterium 1109的甘露醇脱氢酶的重组纯化及酶学性质研究

本文将来自P.bacterium 1109的甘露糖醇脱氢酶(MDH)表达并提取纯化,研究了该重组酶的酶学性质及其在甘露醇生产中的工艺条件。结果显示重组MDH是一个相对分子量为37 kDa的四聚体。氨基酸序列比对发现其与大多数MDHs的同源性小于40%。该酶的最适pH和温度分别为8.5和80℃,且当金属离子Zn2+存在时,重组MDH的活力提高到对照组的260%。此外,重组MDH在75℃孵育6 h后仍可保留超过85%的残留活性,热稳定性较高,比大多数MDHs的活性高。底物特异性研究表明其对D-果糖具有较高的专一性。重组MDH催化D-果糖的米氏常数(K_m)和催化效率(k_cat/K_m)分别为20 mmol/L和7.5 L/(mmol·min)。重组MDH在以400 mmol/L的D-果糖为底物的反应系统中,可将80%以上的D-果糖转化为甘露糖醇。通过对反应条件的优化,为后续工业化生产制备甘露醇奠定了基础。     

Clostridium bolteae ATCC BAA-613 D-塔格糖3-差向异构酶的诱导表达、纯化及活性研究

D-塔格糖3-差向异构酶是生物法生产新型功能性因子D-阿洛酮糖最为有效的酶。一种新型的能够编码D-塔格糖3-差向异构酶的基因CLOBOL₀0069被克隆,它来源于Clostridium bolteae ATCC BAA-613。以pUC57为克隆载体,以pET-22b（+）为载体质粒,E.coli BL21（DE3）为宿主细胞,构建了基因重组工程菌。IPTG诱导剂诱导目的蛋白的表达;通过镍柱亲和层析,杂蛋白与目的蛋白得到了很好的分离。对纯化的重组蛋白样品进行SDS-PAGE分析,在约32ku处出现明显的特征条带。通过活性研究表明,Clostridium bolteae ATCC BAA-613 DTEase属于DTEase家族,并具有较高的生物转化率,反应10h后转化率达到20%。     

Clostridium scindens ATCC35704中的D-塔格糖-3-差向异构酶的克隆表达、纯化及活性研究

D-阿洛酮糖作为一种新型低热量功能性甜味剂,可以通过D-塔格糖-3-差向异构酶家族,以D-果糖为底物C-3位异构化得到。一个新的来源于微生物Clostridium scindens ATCC 35704中ZP 0243228的D-塔格糖-3-差向异构酶基因(CS-DTE),通过克隆并成功导入E.coli BL21(DE3)中,构建了基因重组菌,诱导目的重组基因过量表达;经亲和层析纯化的重组蛋白样品进行SDS-PAGE分析,在约31 ku处出现显著的特征蛋白条带;通过对其活性检测表明,该重组酶分别以D-塔格糖和D-果糖为底物,可以生成D-山梨糖和D-阿洛酮糖,转化率分别为8.6%和27.9%。     

CRISPR/Cas9介导的果糖低消耗酿酒酵母工程菌的构建

利用酿酒酵母为宿主,以果糖为原料合成D-阿洛酮糖具有食品方面的先天优势。为了减少酿酒酵母宿主本身对果糖的消耗,对酿酒酵母的己糖激酶同工酶2(Hexokinaseisoenzyme2,hxk2)基因进行编辑。本研究采用CRISPR/Cas9技术,构建了Cas9和gRNA共表达质粒p YES2-CG-Δhxk2,以酿酒酵母BY4741为出发菌株,URA3为筛选标记,采用高效电击转化法和胞内同源重组技术,获得hxk2基因缺陷株BY4741-Δhxk2。在此基础上,进行果糖发酵实验以评估突变菌株的果糖消耗速率。实验结果显示,发酵培养14 h时,缺陷株BY4741-Δhxk2果糖消耗速率为3.35 mg/h;与野生型菌株相比其下降了6.42%。此外,发酵培养22 h,BY4741-Δhxk2的OD600nm值为8.65,相比于野生型提高了6.40%。研究表明,己糖激酶hxk2基因的缺陷编辑可以一定程度降低酿酒酵母对果糖的利用,同时缺陷株较野生型表现出一定的生长优势,这为后续以酿酒酵母为宿主生产D-阿洛酮糖奠定了初步基础。     

Allitol: production,properties and applications

Allitol is a rare alcohol monosaccharide sweetener containing six carbons atoms. It is a major product of the d ‐psicose reduction pathway that exerts various physiological effects, for example laxative effects for the treatment of constipation and anti‐obesity effects by suppressing lipid accumulation. Allitol can be also used as an anticrystallisation agent. In addition, allitol cross‐links d ‐ and l ‐hexoses, thus contributing to production of l ‐psicose. This is a review of the recent studies on the sources, properties, physiological functions and applications of allitol. In addition, the biochemical properties of ribitol dehydrogenase enzymes and the biotechnological production of allitol via microbial and enzymatic synthesis from d ‐psicose and directly from d ‐fructose are reviewed.     

Purification and characterization of alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from Geotrichum capitatum

(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.     

落叶松RDH硫酸盐法蒸煮及高白度漂白浆的研究

对比研究了落叶松，南方松ＲＤＨ和常规蒸煮硫酸盐浆，采用氧脱木素及低毒性ＥＤ Ｄ漂程，将纸浆漂至８９％以上，并保持较高的物理强度。ＲＤＨ蒸煮较常规蒸煮可获得卡伯值较低的纸浆，氧脱木素降低了用氯量和漂白废水中ＡＯＸ含量。落叶松较南方松浆得率稍低，物理强度性能相近。     

Purification, characterization, and gene cloning of glycerol dehydrogenase from Hansenula ofunaensis, and its expression for production of optically active diol

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols.     

乙醇体系中D-阿洛酮糖的结晶工艺优化

为开发一套D-阿洛酮糖的结晶工艺,本文以乙醇为结晶体系,分别对影响结晶过程的4个主要因素(包括D-阿洛酮糖溶液的密度、乙醇与D-阿洛酮糖的比例、结晶时间和结晶温度)进行了单因素实验,并在单因素实验的基础上,采用响应曲面法对结晶的工艺参数进行优化。结果表明,最优操作条件为:D-阿洛酮糖溶液密度为1.35 g/mL,乙醇与D-阿洛酮糖溶液比例为3.8:1,结晶时间为325 min,结晶温度为25℃。在此条件下,D-阿洛酮糖的初次结晶收率可达71.58%。以上结果可以得出结论:乙醇体系中可获得较高的D-阿洛酮糖结晶收率,本研究获得的模型可以用来优化D-阿洛酮糖在乙醇体系中的结晶过程。     

产酸克雷伯氏菌乳酸脱氢酶基因敲除提高2,3-丁二醇产量

基于传统发酵工艺,通过在发酵阶段进行菌种强化结合在蒸馏烤酒阶段进行工艺优化（头尾酒浸提菌剂后返回地锅蒸馏）提升酱香型白酒基酒中四甲基吡嗪（TTMP）的含量。结果表明,采用菌种强化、工艺优化及二者相结合的方法分别能将酱香型白酒基酒中四甲基吡嗪的含量提高160.71%、85.75%和202.75%。感官评价结果表明,菌种强化结合工艺优化的技术对基酒的感官风味影响不大。该研究提供了一种适合企业落地应用的进一步提高酱香型白酒基酒中四甲基吡嗪含量的方法和思路。     

Formation of α-dicarbonyl fragments from mono- and disaccharides under caramelization and Maillard reaction conditions

Glyoxal, methylglyoxal and diacetyl were identified as reaction products from non-enzymatic browning of D-glucose (Glc), D-fructose (Fru), maltose and maltulose. The α-dicarbonyl compounds were quantified throughout the reaction. Monosaccharides formed more dicarbonyl fragments than disaccharides and Glc formed more than Fru. It is suggested that Fru tends to yield cyclic products rather than fragmentation products under the reaction conditions used. Furthermore, a mechanism for the fragmentation of disaccharides is proposed.