以纤维二糖为底物利用重组大肠杆菌合成海藻糖

探讨以纤维二糖为底物合成海藻糖的可行性。首先克隆来自施氏假单胞菌A1501的otsA/otsB基因,外源导入Escherichia coli BL21（DE3）构建以葡萄糖为底物合成海藻糖的OtsAB途径。继而通过过表达E. coli本身的galU基因增加海藻糖合成前体物质尿苷二磷酸（uridine diphosphate,UDP）-葡萄糖的含量,使海藻糖产量提高了3 倍。通过添加井冈霉素抑制海藻糖的降解,进一步提高海藻糖的产量。在此基础上,过表达来自天然纤维素分解细菌Saccharophagus degradans的纤维二糖磷酸化酶基因cepA,使重组大肠杆菌具有利用纤维二糖产海藻糖的能力。利用该重组大肠杆菌全细胞催化生产海藻糖,底物为20 g/L纤维二糖,并添加0.05 mmol/L的井冈霉素抑制海藻糖降解时,48 h后可生成1.3 g/L的海藻糖。本研究利用重组大肠杆菌以纤维二糖为底物合成海藻糖,证实了以纤维二糖为底物合成海藻糖的可行性,为纤维二糖来源精细化学品的生产提供了新的思路。     

山葡萄UDP-葡萄糖:类黄酮5-O-葡萄糖基转移酶(5GT)等位基因的克隆与分析

山葡萄中大量的双糖苷花色苷严重影响山葡萄酒品质,而5GT是合成双糖苷花色苷的关键酶。研究不同葡萄品种间5GT等位基因的差异,为抑制双糖苷花色苷的合成奠定基础,对提高山葡萄酒的品质有重要意义。本研究克隆了‘赤霞珠’、‘左山一’、‘哈桑’和‘左红一’中的5GT等位基因,并用软件对其进行了序列分析和生物信息学分析,共获得了7个5GT等位基因,均位于葡萄9号染色体上,分别编码297~464个氨基酸。序列分析结果表明,4个5GT等位基因由于基因突变可能丧失了5GT功能;7个5GT等位基因均没有信号肽,属于GT1家族中的5GT亚家族。不同葡萄品种中的5GT等位基因存在一定差异,推测这7个5GT等位基因中可能只有3个5GT可以合成双糖苷花色苷。     

Mechanism of inactivation of UDP-glucose 4-epimerase from Saccharomyces cerevisiae by D-xylose and L-arabinose

In a previous paper (Cármenes et al., 1984) we reported that UDP-glucose 4-epimerase from Saccharomyces was inactivated both in vivo and in vitro (crude extracts) by L-arabinose or D-xylose. In this paper, we report that pure epimerase requires the presence of UMP or UDP to be inactivated by sugars and that the inactivation is due to the reduction of the epimerase NAD+, which is essential for epimerase activity. The inactivation rate is directly proportional to epimerase and sugar concentrations and hyperbolically proportional to UMP concentration. In situ experiments made with permeabilized cells showed that epimerase is inactivated in the same way when it is inside the cell. In vivo studies showed that epimerase is inactivated to a smaller extent when 1% D-galactose is present in the culture medium than when 1% ethanol is the main carbon source.     

Thermophilic Inorganic Pyrophosphatase Ton1914 from Thermococcus onnurineus NA1 Removes the Inhibitory Effect of Pyrophosphate

Pyrophosphate (PP_i) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PP_i can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg²⁺ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32–41%, while that of short-chain DNA was only improved by 9.5–15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.     

A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes.     

粪产碱杆菌胞内核苷酸的反相高效色谱法测定

建立了用反相离子对色谱法同时精确测定产热凝胶粪产碱杆菌胞内中ATP、ADP、AMP、UTP、UDP、UMP、NADH、NAD+及UDP-glucose含量的方法。采用AgilentZorbaxSB-AqC18反相色谱柱(5μm,4.6×250nm),紫外检测波长为254nm,以乙腈-含10mmol/L四正丁基溴化铵的0.2mol/LNa2HPO4-NaH2PO4缓冲液(体积比10.5∶89.5)为流动相,流速为1.0mL/min时,成功分离了上述九种核苷酸,分离效果良好。且九种核苷酸的线性范围为10～100mg/L,回收率均在97%～104%之间,说明其准确度较好。相对标准偏差均在3%之内,说明本方法精密度和重现性均较好,且其最低检出限均在0.1～0.3mg/L之间。该方法为研究热凝胶发酵过程中粪产碱杆菌胞内的核苷酸变化和氧化-还原态趋势及其胞内的核苷酸水平和热凝胶生物合成的关系提供了基础。     

Unlocking the Potential of Leloir Glycosyltransferases for Applied Biocatalysis: Efficient Synthesis of Uridine 5′‐Diphosphate‐Glucose by Sucrose Synthase

Despite the unsurpassed selectivity that enzymes usually offer, biocatalytic transformations repeatedly fall short of the robustness and process efficiency demanded for production‐scale chemical synthesis. Nucleotide sugar‐dependent “Leloir” glycosyltransferases (GTs) are fine catalysts of glycosylation but there is concern as to whether reactions from this enzyme class are fit for industrial process development. We demonstrate in this study of sucrose synthase (SuSy; EC 2.4.1.13) that, in order to unlock the synthetic potential of the GT reaction, it was vital to combine a focused, kinetic characteristics‐based enzyme selection with a reaction design properly aligned to thermodynamic constraints. The equilibrium constant (K_eq) for the conversion of sucrose and uridine 5′‐diphosphate (UDP) into the target product UDP‐α‐d ‐glucose and d ‐fructose decreased with increasing pH due to deprotonation of the β‐phosphate group of UDP above the pK_a of ∼6.0. Proton uptake coupled to the glucosyl transfer made it essential that the pH was carefully controlled throughout the reaction. Comparing two SuSys from Acidithiobacillus caldus and Glycine max (soybean), substrate inhibition by UDP superseded catalytic efficiency as the prior selection criterion, demanding choice of the bacterial GT for use at high UDP concentrations. Reaction at the operational pH optimum, determined as 5.0, gave 255 mM (144 g L⁻¹) of UDP‐glucose in 85% yield from UDP. Using an enzyme concentration of only 0.1 g L⁻¹, a space‐time yield of 25 g L⁻¹ h⁻¹ was obtained. The mass‐based turnover number (g product formed per g enzyme added) reached a value of 1440 from a single batch conversion. Therefore, these parameters of the UDP‐glucose synthesis show that the reaction of a GT can be pushed to a process efficiency typically required for implementation in fine chemicals production.

     

Resistance of cereals to aphids: The interaction between hydroxamic acids and UDP-glucose transferases in the aphidSitobion avenue (Homoptera: Aphididae)

UPD-glucose transferases are found in the cytosolic and microsomal fractions of the grain aphidSitobion avenae F. Gel filtration and SDSPAGE revealed that the microsomal fraction contained several forms of the enzyme. The molecular weights of the three most active fractions might be 68,000, 66,000, and 36,500. There was a negative correlation between the enzymes' activity in extracts of aphids and the concentration of DIMBOAaglucone in the winter wheat variety fed on by the aphid. A strong inhibition of the activity of the UPD-glucose transferases was observedin vitro at a concentration of DIMBOA as low as 0.01 mM. There was a greater activity of the enzymes in aphids fed on seedlings of susceptible than on moderately resistant wheat cultivars. Prolonged feeding on resistant cultivars resulted in a further reduction in the activity of the aphid's enzymes. The significance for cereal aphids of the role of their UDP-glucose tranferases in the detoxification of plant allelochemicals and adaptation to resistant varieties of cereals is discussed.