嗜热革节孢多糖单加氧酶氧化方式

多糖单加氧酶(polysaccharidemonooxygenase,PMO)是一种铜离子依赖的氧化酶,属于辅助活性酶类第九家族(auxiliary activity 9,AA9),在存在电子供体维生素C(vitamin C,Vc)的情况下,可以氧化裂解纤维素的多糖链,显著提高纤维素的酶解效率。本文克隆了嗜热革节孢Scytalidium thermophilumAA9家族的一个编码基因pmo7651,并在毕赤酵母GS115进行诱导表达,通过His标签获得了重组蛋白PMO7651-His。以磷酸膨胀纤维素(PASC)为底物进行酶促反应,薄层层析法(TLC)结果显示PMO7651酶解产物主要为纤维二糖至纤维五糖;飞行时间质谱法(MALDI-TOF-MS)和溴氧化法确定PMO7651具有C1、C4、C6位的氧化活性;底物结合平面的3个芳香族氨基酸位点突变对酶的活性具有不同程度的影响;在PMO7651帮助下,纤维素酶的降解效率均具有不同程度的提高。     

链霉菌GXT6 β-葡萄糖苷酶的酶学性质及葡萄糖耐受性分子改造

【目的】从经过全基因组测序的链霉菌GXT6中克隆、表达一个编码糖基水解酶家族3的新β-葡萄糖苷酶基因,研究重组酶的酶学性质并进行相关葡萄糖耐受性的氨基酸残基的分子改造,提高其对葡萄糖的耐受性。【方法】根据链霉菌GXT6的全基因测序结果,对其中一个注释为糖基水解酶的基因设计引物,PCR扩增目的基因,以p SE380为表达载体构建重组质粒,转化至大肠杆菌中诱导表达;采用镍亲和层析技术纯化重组蛋白质,对目的蛋白质进行酶学性质研究;采用定点饱和突变的方法对重组酶进行相关氨基酸残基的分子改造。【结果】从链霉菌GXT6中克隆到一个编码糖基水解酶家族3的新β-葡萄糖苷酶基因,并在大肠杆菌中表达。酶学性质研究结果表明该β-葡萄糖苷酶的最适温度为40°C,最适p H为6.0,Km值为(0.4712±0.0180) mmol/L,Vmax值为(128.000±1.741)μmol/(min·mg),葡萄糖抑制常数Ki值为(1.8880±0.1307)mmol/L。该BGL3-GXT6能够水解黄豆苷、染料木苷、甜茶苷、虎杖苷、淫羊藿苷。还对BGL3-GXT6中与葡萄糖耐受性可能相关的氨基酸残基位点81-Trp和233-Trp进行了定点饱和突变,获得了25个具有酶活的突变酶并对其进行酶学性质研究。其中W233位点饱和突变后获得的突变酶的Km和葡萄糖抑制常数Ki值与重组酶BGL3-GXT6相比均发生明显变化,葡萄糖耐受性有不同程度的提高,最高的提高了209倍。【结论】本研究获得的BGL3-GXT6对天然底物甜茶苷、黄豆苷、染料木苷、虎杖苷和淫羊藿苷具有水解功能,这些特性表明该β-葡萄糖苷酶在理论研究及在工业中有一定的应用价值。     

黄翅大白蚁来源β-葡萄糖苷酶的分子改造

纤维素水解成为葡萄糖需要一系列纤维素酶的作用,其中β-葡萄糖苷酶(β-glucosidases)起着至关重要的作用。来自于培菌白蚁中肠的β-葡萄糖苷酶(MbmgBG1)具有较高的葡萄糖耐受性(1.5 mol/L的葡萄糖,保持60%以上的酶活力),但是,酶活力低和热稳定性差限制了β-葡萄糖苷酶(MbmgBG1)在食品以及工业领域中的应用。因此通过对保守氨基酸附近的非保守氨基酸定点突变,获得点突变体(F167L、T176C、E347I、R354K、N393G和V425M),其中突变体F167L、R354K的比活力(底物pNPG)比MbmgBG1分别高出约2倍和4倍。突变体的K_(cat)/K_m值比野生型大,反映了突变体对底物的亲和力以及催化能力比MbmgBG1强。当酶活力保留60%以上时,MbmgBG1所耐受的葡萄糖浓度为1.5 mol/L,而F167L为2.0 mol/L,R354K为3.0 mol/L。这些特性的增强表明,对活性中心附近保守区域内的非保守氨基酸突变,可以较大程度地影响活性,因此需要更深入地研究β-葡萄糖苷酶的活性中心位点,进行改造以提高催化效率。     

重组根瘤农杆菌葡萄糖耐受型β-葡萄糖苷酶性质

目前已发现的葡萄糖耐受型β-葡萄糖苷酶均来源于真菌, 尚无原核细胞来源的相关报道。从根瘤农杆菌LBA4404中克隆β-葡萄糖苷酶基因bg1, 将其构建在表达载体pET-28b上, 转化Escherichia coli RP (DE3), IPTG诱导表达。重组β-葡萄糖苷酶的比活高达36.7 μmol/(min·mg)。对经过Ni柱纯化的重组酶进行酶学分析发现: 该酶是糖基水解酶家族1的成员, 底物亲和力高, 专一性低, 在温度为40°C和pH在5-8之间时具有较高的酶活, 在低于40°C和pH 5-10之间时可稳定保存。以pNP-β-Glc为底物, 该酶的最适pH为6.4, 最适温度为60°C, 在37°C和pH 6.4的反应体系中, 该酶的Km为0.09 mmol/L, 竞争性抑制剂葡萄糖酸-δ-内酯和葡萄糖的Ki分别为0.03 mmol/L和75 mmol/L, 具有很高的葡萄糖耐受性, 当金属离子Ag+和Zn2+存在时, 酶活被明显抑制。该酶对pNP-β-Gal和pNP-α-Glc的Km分别为3.61 mmol/L和14.31 mmol/L。     

嗜冷德沃斯氏菌来源GH1家族β-葡萄糖苷酶Bgl59的原核表达及酶学性质分析

葡萄糖苷酶在食品、医学以及生物能源等多个领域有着广泛的应用,因此挖掘新型高效的β-葡萄糖苷酶是十分必要的。【目的】对嗜冷德沃斯氏菌（Devosia psychrophile）来源的GH1家族的葡萄糖苷酶原核表达并测定其酶学性质。【方法】通过人工合成技术得到D. psychrophila来源的β-葡萄糖苷酶的编码基因,命名为bgl59。将该基因转化到大肠杆菌（Escherichiacoli）BL21（DE3）中,诱导基因表达,对得到的蛋白进行纯化并测定其酶学性质。【结果】Bgl59的分子量为48.8 kDa,最适温度为55℃,最适pH为6.0;Bgl59在pH 5.0-8.5范围内处理1 h后仍保持80%以上酶活;在8种供试底物中,Bgl59对4-硝基苯基-β-D-葡萄糖苷（4-nitrophenyl-β-D-glucopyranoside,p NPG）有着最高的水解能力,其K_m为3.090 mmol/L,V_max为194μmol/（min·mg）,k_cat为159 s^-1;1 mmol/L的Ca²⁺、Co²⁺对Bgl59具有明显的激活作用,0.1%的SDS会使酶活全部丧失;0.10 mol/L葡萄糖和0.30 mol/L木糖具有促酶活作用,可分别使Bgl59酶活提高74%和91%;在1.25 mol/L葡萄糖或2.00 mol/L木糖存在的条件下,仍可保持50%以上酶活。【结论】Bgl59的酶学性质优异,具有良好的pH稳定性,对金属离子或化学试剂都具有一定耐受能力,是少见的葡萄糖激活型β-葡萄糖苷酶,具有优良的糖促活性和耐受性,在未来的工业生产以及应用中具有潜在研究价值。     

扣囊复膜孢酵母BGL1基因在工业酒精酵母中的整合表达

构建了含有工业酿酒酵母自身GPD2启动子和终止子、扣囊复膜孢酵母b-葡萄糖苷酶基因(BGL1)和潮霉素选择性标记hyg的重组质粒pPIC-gpd-bgl-hyg, 通过酵母染色体同源重组, 将BGL1基因整合进入工业酒精酵母的染色体上。重组酵母可以在以纤维二糖为唯一碳源的培养基上生长, 48 h时b-葡萄糖苷酶酶活达到0.764 U/mL。在玉米浓醪酒精发酵实验中, 与宿主菌株相比, 重组酵母醪液中纤维二糖含量减少约80％, 达到了消耗醪液中纤维二糖含量的目的。     

7-木糖紫杉烷糖基水解酶LXYL-P1-1和LXYL-P1-2活性中心预测及初步的功能分析

7-木糖紫杉烷糖基水解酶LXYL-P1-1和LXYL-P1-2是克隆自真菌香菇的两个双功能酶(序列一致性97%),具有β-木糖苷酶/β-葡萄糖苷酶双重活性,能特异性地水解移除7-木糖-10-去乙酰紫杉醇等紫杉烷上的木糖基。采用生物信息学方法对两个酶蛋白进行酶活性中心预测,初步确定Asp300和Glu529分别为亲核试剂和一般酸/碱催化剂,而Asn172-Gly173-Arg174和Lys207-His208为底物结合结构域。以LXYL-P1-2为研究对象,以毕赤酵母细胞为表达宿主,应用定点突变技术获得了N172A、G173A、R174A、K207A、H208A、D300N和E529Q突变体,并进行了酶活性分析。结果显示:在分别以PNP-Xyl、PNP-Glc和7-木糖-10-去乙酰紫杉醇为底物时,N172A、G173A、R174A、K207A、D300N和E529Q的β-木糖苷酶与β-葡萄糖苷酶活性大幅度下降甚至完全消失;H208A的β-木糖苷酶活性也显著下降,但仍保持98%的β-葡萄糖苷酶活性。其结果初步验证了对上述两个酶蛋白的活性中心的预测,为进一步揭示7-木糖紫杉烷糖基水解酶结构与功能的关系提供了实验依据。     

嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点,利用重叠PCR法构建定点突变体,采用薄层层析(TLC)法和3,5-二硝基水杨酸(DNS)法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体,其中第150和159位的氨基酸突变对活性改变甚少或几乎没有,而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失,说明位于β4和β7折叠的C末端的E151和E231的羧基基团作为功能基团参与了催化反应。【结论】E151和E231分别是新型甘露聚糖酶AaManA的酸碱催化位点和亲核催化位点。     

采用随机突变方法对β-琼胶酶YM01-3活性位点的研究

【背景】琼胶酶是一种多糖水解酶,在保健食品、医药、科研及化妆品等行业极具价值。本实验室发现来源自嗜琼胶卵链菌(Catenovulumagarivorans)的β-琼胶酶YM01-3具有较高的酶活性,在最适条件下的比酶活可达到1.14×10~4U/mg。【目的】探讨不同位点的突变对β-琼胶酶YM01-3酶活力的作用,发现影响其酶活力的新位点。【方法】通过易错PCR在短芽孢杆菌(Bacillus brevis)表达系统中构建随机突变文库,从约10 000个克隆中筛选出227株有效突变体,从中选取80株进行测序。【结果】对突变体序列进行分析和定点突变验证发现,137位和237位氨基酸发生突变后酶活力丧失90%以上。【结论】位于催化腔内的137位和237位氨基酸,对于维持β-琼胶酶YM01-3酶活力具有重要作用。该研究结果为β-琼胶酶的催化机理研究及分子改造提供了借鉴。     

Identification of Residues Essential for the Activity and Substrate Affinity of l-Carnitine Dehydrogenase

Recently, two l-carnitine dehydrogenases from soil isolates Rhizobium sp. (Rs-CDH) and Xanthomonas translucens (Xt-CDH) have demonstrated to exhibit mutually differing affinities toward l-carnitine. To identify residues important for affinity to the substrate, we compared the primary structure of Xt-CDH and Rs-CDH with the recognized 3D structure of 3-hydroxyacyl-CoA dehydrogenase (PDB code: 1F0Y). Then, six residues of Xt-CDH (Phe143, Gly188, Ile190, Ala191, Gly223, and Ala224) and the corresponding residues of Rs-CDH (Tyr140, Ala185, Val187, Gly188, Ser220, and Phe221) were selected for further mutagenesis. The residues of Xt-CDH were replaced with that of Rs-CDH at the corresponding position and vice versa. All Rs-CDH mutants exhibited slight effects on substrate affinity, except for the double mutants Rs-V187I/G188A, which was devoid of enzyme activity. All Xt-CDH mutants showed different K _m values. Xt-F143Y caused a higher increase in the K _m value. Furthermore, the kinetic parameters of 10 mutants at Xt-F143 and Rs-Y140 were investigated. All Rs-Y140 mutants, except aromatic residues (Phe, Trp), produced proteins that were almost entirely devoid of enzyme activity and with disrupted affinity to l-carnitine. All Xt-F143 variants showed a marked reduction (P ≤ 0.05) in enzyme activity. Overall, our results suggest that the aromatic rings of Tyr140 in Rs-CDH and Phe143 of Xt-CDH are essential for substrate recognition.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [H]leucine as a marker for the leucine pool and l-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (T(r), percent per hour) and hydrolysis rates of Leu-MCA (H(r), percent per hour), as well as the quotient T(r)/H(r), reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient T(r)/H(r) is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.     

球面设计优化金线莲黄酮类物质提取工艺及糖耐受性的研究(英文)

以金线莲为材料,利用统计学实验设计方法对黄酮类物质的提取工艺进行了优化。选定液料比、乙醇浓度和时间三个因素的五个水平进行球面设计实验,建立黄酮提取率的二次回归方程,通过回归分析及岭脊分析得到优化提取组合条件。研究结果表明,当提取工艺条件为:液料比24,乙醇浓度63%,提取时间48 h时,黄酮提取率的最大预测值为1.107%,验证值为1.05%。葡萄糖耐受性试验结果表明:金线莲黄酮类萃取物能够提高正常小鼠对糖的耐受性。     

The Association between HbA1c,Fasting Glucose, 1-Hour Glucose and 2-Hour Glucose during an Oral Glucose Tolerance Test and Cardiovascular Disease in Individuals with Elevated Risk for Diabetes

Objective

To determine the association between HbA1c, fasting plasma glucose (FPG), 1-hour (1 hPG) and 2-hour (2 hPG) glucose after an oral glucose tolerance test (OGTT) and cardiovascular disease in individuals with elevated risk for diabetes.

Design

We studied the relationship between baseline, updated mean and updated (last) value of HbA1c, FPG, 1 hPG and 2 hPG after an oral 75 g glucose tolerance test (OGTT) and acute CVD events in 504 individuals with impaired glucose tolerance (IGT) at baseline enrolled in the Finnish Diabetes Prevention Study.

Setting

Follow-up of clinical trial.

Participants

504 individuals with IGT were followed with yearly evaluations with OGTT, FPG and HbA1c.

Main Outcome Measure

Relative risk of CVD.

Results

Over a median follow-up of 9.0 years 34 (6.7%) participants had a CVD event, which increased to 52 (10.3%) over a median follow-up of 13.0 years when including events that occurred among participants following a diagnosis of diabetes. Updated mean HbA1c, 1 hPG and 2 hPG, HR per 1 unit SD of 1.57 (95% CI 1.16 to 2.11), p = 0.0032, 1.51 (1.03 to 2.23), p = 0.036 and 1.60 (1.10 to 2.34), p = 0.014, respectively, but not FPG (p = 0.11), were related to CVD. In analyses of the last value prior to the CVD event the same three glycaemic measurements were associated with the CVD events, with HRs per 1 unit SD of 1.45 (1.06 to 1.98), p = 0.020, 1.55 (1.04 to 2.29), p = 0.030 and 2.19 (1.51 to 3.18), p<0.0001, respectively but only 2 hPG remained significant in pairwise comparisons. Including the follow-up period after diabetes onset updated 2 hPG (p = 0.003) but not updated mean HbA1c (p = 0.08) was related to CVD.

Conclusions and Relevance

Current 2 hPG level in people with IGT is associated with increased risk of CVD. This supports its use in screening for prediabetes and monitoring glycaemic levels of people with prediabetes.     

Maize Root Phytase (Purification,Characterization, and Localization of Enzyme Activity and Its Putative Substrate)

Three phytase (EC 3.1.3.26) isoforms from the roots of 8-d-old maize (Zea mays L. var Consul) seedlings were separated from phosphatases and purified to near homogeneity. The molecular mass of the native protein was 71 kD, and the isoelectric points of the three isoforms were pH 5.0, 4.9, and 4.8. Each of the three isoforms consisted of two subunits with a molecular mass of 38 kD. The temperature and pH optima (40[deg]C, pH 5.0) of these three isoforms, as well as the apparent Michaelis constants for sodium inositol hexakisphosphate (phytate) (43, 25, and 24 [mu]M) as determined by the release of inorganic phosphate, were only slightly different. Phytate concentrations higher than 300 [mu]M were inhibitory to all three isoforms. In contrast, the dephosphorylation of 4-nitrophenyl phosphate was not inhibited by any substrate concentration, but the Michaelis constants for this substrate were considerably higher (137-157 [mu]M). Hydrolysis of phytate by the phytase isoforms is a nonrandom reaction. D/L-Inositol-1,2,3,4,5- pentakisphosphate was identified as the first and D/L-inositol-1,2,5,6-tetrakisphosphate as the second intermediate in phytate hydrolysis. Phytase activity was localized in root slices. Although phosphatase activity was present in the stele and the cortex of the primary root, phytase activity was confined to the endodermis. Phytate was identified as the putative native substrate in maize roots (45 [mu]g P g-1 dry matter). It was readily labeled upon supplying [32P]phosphate to the roots.     

RNAi-Based Transcriptome Suggests Candidate Genes Regulated by ZxNHX1 to Affect The Salt Tolerance of Zygophyllum xanthoxylum

Journal of Plant Growth Regulation - Zygophyllum xanthoxylum, a succulent xerophyte, possesses excellent salt tolerance which is closely associated with vacuolar Na+ compartmentation via ZxNHX1....