pH对克雷伯氏菌1,3-丙二醇合成关键酶酶活及发酵特性的影响

在5 L发酵罐进行甘油脉冲流加发酵,分析了不同pH值对克雷伯氏肺炎杆菌发酵特性的影响,pH 6.5为菌体最佳生长条件,克雷伯氏肺炎杆菌合成1,3-丙二醇的产量最高。在1,3-丙二醇合成速率较大的对数中前期,进行甘油脉冲流加发酵,提高甘油浓度促进甘油脱水酶、1,3-丙二醇氧化还原酶和甘油脱氢酶活性。不同pH值的脉冲试验表明,甘油脱水酶,2,3-丁二醇脱氢酶比酶活随着pH值的升高而升高,1,3-丙二醇氧化还原酶,乳酸脱氢酶比酶活在pH6.5最高,因此偏酸性的发酵条件和对数期维持一定的甘油浓度能够促进1,3-丙二醇的合成。     

D-泛解酸内酯水解酶的固定化及酶学性质

为有效提高D-泛解酸内酯水解酶的利用效率,笔者选择合适的载体对酶进行固定化,在优化固定化条件的同时研究固定化酶的最佳反应条件和酶学性质。结果表明,选择的最佳固定化载体为树脂D380,最佳固定化条件为:酶的吸附添加量为30 U(以1 g湿树脂计),吸附pH 7.0,吸附温度30℃,吸附时间5 h,戊二醛体积分数0.1%,交联时间1 h。在最优条件下得到的固定化酶的比酶活为(11.5±0.12) U/g。固定化酶的最适反应温度为37℃,最适反应pH为7.5。游离酶和固定化酶的动力学常数K_m分别为170.25和207.60 mmol/L。Ca~(2+)对酶促反应有激活作用,Mn~(2+)对该酶有较强的抑制作用。     

酶电极法快速测定甘油含量的研究

利用酶固定化技术,以甘油激酶（GK）、甘油-3-磷酸氧化酶（GPO）为反应酶,研究GK、GPO的固定化方法及固定化模式,制备甘油酶膜、甘油酶电极,并利用其测定甘油含量。结果表明,GK、GPO按1：1比例固定化时,酶电极电流信号最高;最高效固定模式为：GK固定于核微孔膜,共价偶联GPO固定于Biodyne膜,形成共价双酶膜,进而组装为甘油酶电极。性能研究表明,甘油酶电极最适pH值为7.0,最佳温度为28~32℃;最佳实验条件下,线性范围为0.05~9.00 g/L;回收率为98.4%~102.4%,稳定性高,相对标准偏差（RSD）<5%;测定结果与高效液相色谱法、高碘酸氧化法比较,无明显差异（P>0.05）,且该方法操作简单,专一性强,检测快速,适于实际生产中甘油的实时定量及监控。     

dhaBCE基因与yqhD基因串联表达载体的构建及其生物转化甘油

将来自于肺炎克雷伯氏杆菌的甘油脱水酶基因插入到质粒pET28(a+) -yqhD的上游,并用SD序列隔开,串联构建重组质粒pET28(a+)dhaBCE-yqhD,转化到大肠杆菌E.coli novablue中进行共表达。结果显示：含有pET28(a+) dhaBCE-yqhD的重组菌在28℃条件下,IPTG诱导16h后,甘油脱水酶和yqhD氧化还原酶的酶活力分别达到35 U/ mg和 82 U/ mg ,而对照组检测不到甘油脱水酶酶活;当甘油浓度为55g/L,产物1,3-PD的产量可达39g/L;甘油浓度过量不利于产物合成,且产物1,3-丙二醇对合成反应具有一定的抑制作用。     

半夏内生真菌所产β-葡萄糖苷酶的酶学特性研究

从半夏中分离筛选得到一株植物内生真菌,提取培养液中的β-葡萄糖苷酶,研究其酶学特性。结果表明:该菌种所产β-葡萄糖苷酶的最适反应温度在45~50℃之间,最适p H在7~8之间;在低于40℃条件下,p H为6~9时,酶活较稳定;其最适反应时间为40 min,金属离子和有机溶剂对酶活性影响都很大,在最适反应条件下其酶活为(77.83±0.42)U/m L。     

双歧杆菌属特征性酶F6PPK测定条件的优化

以两歧双歧杆菌为材料,进行了双歧杆菌属特征性酶F6PPK测定条件的优化。实验首先改良了原初文献(Scardovi法)F6PPK酶活测定中对照反应管的设置,又对双岐杆菌培养时间、细胞破碎方法、底物浓度、比色波长、反应温度等条件进行了优化。实验表明,双岐杆菌培养18 h,收集菌体,以超声波法破碎细胞制备粗酶液,采用4%的底物浓度,将酶液与底物于40℃保温30 min,最终反应物于500 nm比色,所得F6PPK酶活测定结果更可靠。     

对高效石油降解菌株C_(230)酶活的最适pH和温度的探究

以中国甘肃华庆油田附近被石油污染的土壤中分离得到的六株高效石油降解菌A6、L5、L3、M4、B1、B3为菌种材料。通过对六种菌株进行整细胞破碎处理,研究了在不同的pH值和不同温度条件下的各菌株C23O(邻苯二酚2,3-双加氧酶)酶活。实验结果表明:C230酶的最佳反应pH值为7.5;最适反应温度为35℃。     

一株高产纤维素酶菌的筛选与鉴定

利用刚果红脱色从森林腐木中筛选到一株高产纤维素酶菌,结合18S rRNA基因序列分析和菌株形态特性分析,确定该菌株为爪哇正青霉(Eupenicillium javanicum).通过研究粗酶提取时不同离心时间和离心转速对酶活的影响,确定采用3000r/min,15min离心条件下获得的粗酶测定CMC酶活,采用4000r/min,10min的离心条件下获得的粗酶测定FPA酶活和β-葡萄糖苷酶活.酶学性质初步研究显示,纤维素酶反应的最适pH值以5.0为宜;CMC酶最适酶解温度和酶解时间为60℃,15min;FPA和β-葡萄糖苷酶最适酶解温度和酶解时间均为50℃,20min.     

重组大肠杆菌产蔗糖磷酸化酶的酶学性质及其催化合成α-熊果苷

利用重组大肠杆菌Escherichia coli Rosetta(DE3)/pET-SPase发酵生产蔗糖磷酸化酶(EC 2.4.1.7,Sucrose phosphorylase,SPase)。收集的菌体经高压破碎后离心得到粗酶液,通过镍NTA亲和层析、超滤除盐后得到电泳纯的SPase,纯化后的SPase的比酶活是原来的2.1倍,酶活回收率达到82.7%。经SDS-PAGE电泳测定,重组SPase的分子量约为59 kDa。该酶在不高于37℃,pH 6.0~6.7的条件下比较稳定,最适催化温度与最适催化pH分别为37℃,pH 6.7,该酶对蔗糖的米氏常数(Km)为7.3 mmol/L,最大反应速率(Vmax)为0.2μmol/(min.mg)。此外文中还以蔗糖和氢醌为底物,利用重组SPase催化合成α-熊果苷。其最佳反应条件为:20%蔗糖,200 U/mL的酶液,1.6%氢醌,pH 6.0~6.5,25℃,反应21 h。α-熊果苷的摩尔产率为78.3%,α-熊果苷的产量为31 g/L。     

几丁质酶产生菌筛选鉴定及产酶性能研究

从土壤样品中筛选得到一株高产几丁质酶菌株C65-2,经形态学观察和18S rDNA序列测定,鉴定为Aspergillus fumigatus,对产酶培养基进行初步优化,测得最高酶活可达6.9U/ml,酶活力较优化之前提高了210%。酶学性质研究表明该几丁质酶分子量约为20kDa,酶在60℃下保温50min酶活降为0,最适酶反应温度是55℃,酶反应最适pH为7.0,Mg2+,Cu2+对酶反应有促进作用,Fe3+对酶反应有抑制作用。     

Enzymes Involved in Anaerobic Polyethylene Glycol Degradation by Pelobacter venetianus and Bacteroides Strain PG1

In extracts of polyethylene glycol (PEG)-grown cells of the strictly anaerobically fermenting bacterium Pelobacter venetianus, two different enzyme activities were detected, a diol dehydratase and a PEG-degrading enzyme which was characterized as a PEG acetaldehyde lyase. Both enzymes were oxygen sensitive and depended on a reductant, such as titanium citrate or sulfhydryl compounds, for optimal activity. The diol dehydratase was inhibited by various corrinoids (adenosylcobalamin, cyanocobalamin, hydroxocobalamin, and methylcobalamin) by up to 37% at a concentration of 100 μM. Changes in ionic strength and the K⁺ ion concentration had only limited effects on this enzyme activity; glycerol inhibited the enzyme by 95%. The PEG-degrading enzyme activity was stimulated by the same corrinoids by up to 80%, exhibited optimal activity in 0.75 M potassium phosphate buffer or in the presence of 4 M KCI, and was only slightly affected by glycerol. Both enzymes were located in the cytoplasmic space. Also, another PEG-degrading bacterium, Bacteroides strain PG1, contained a PEG acetaldehyde lyase activity analogous to the corresponding enzyme of P. venetianus but no diol dehydratase. Our results confirm that corrinoid-influenced PEG degradation analogous to a diol dehydratase reaction is a common strategy among several different strictly anaerobic PEG-degrading bacteria.     

Allosteric interactions in glycerol dehydratase. Purification of enzyme and effects of positive and negative cooperativity for glycerol

An improved method for the purification of the apoenzyme (AB complex) of glycerol dehydratase from Aerobacter aerogenes is presented. One hundredfold purification Was achieved. This purification was possible due to stabilization of the AB complex by glycerol. Using chromatography on Sephadex G-200, the highest degree of association of AB complex was found in glycine buffer in the presence of glycerol.Potassium ions, in contrast to glycerol, seem to weaken the forces which bind subunits A and B together. This may be the action of potassium necessary for the performance of glycerol dehydratase activity, since potassium is required for holoenzyme activity.From kinetic studies it appears that the enzyme exhibits homotrophic effects with regard to glycerol binding sites, which can he extended from positive (in the presence of glycine buffer) to negative (in the presence of ethanolamine buffer) cooperativity. The high cooperativity between glycerol binding sites on the enzyme, in glycine buffer, can be abolished by the addition of phosphate. By decreasing the value of the Hill coefficient and increasing the V of the enzymatic reaction, phosphate seems to act as an allosteric activator.     

Phage particle-mediated gene transfer to cultured mammalian cells

Recombinant phage particles carrying the thymidine kinase (TK) gene of herpes simplex virus type 1, coprecipitated with calcium phosphate, efficiently transformed mouse Ltk- cells to the TK+ phenotype. The conditions necessary to achieve high efficiency of transfer of the TK gene by phage particle-mediated gene transfer were investigated. Of the parameters examined, the pH of the buffer used for coprecipitation of phage particles with calcium phosphate, the length of time of coprecipitation, and the length of the adsorption period were found to alter the transfer efficiency significantly. The optimal pH was 6.87 at 25 degrees C. The other optimal values for these parameters were as follows: coprecipitation time, 7 to 20 min; adsorption time, 18 to 30 h. Treatment with dimethyl sulfoxide, glycerol, or sucrose did not enhance gene transfer. The optimal conditions yielded about 1 transformant per 10(5) phage particles per 10(6) cells without carrier DNA. An increase in the dosage of phage particles, up to at least 5 x 10(7) phage particles per 100-mm dish, resulted in a linear increase in the number of transformants. Addition of carrier phage, up to 10(10) phage particles per dish, did not significantly affect the number of transformants.     

Determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis combined with online electrochemiluminescence using ultrasonic-assisted extraction

A novel method for determination of pseudolycorine in the bulb of lycoris radiata by capillary electrophoresis coupled with online electrochemiluminescence detection with ultrasonic-assisted extraction has been developed. The effects of several factors such as detection potential, concentration and pH of phosphate buffer, separation voltage, injection time, ultrasonic power and extraction time were investigated. Under optimal conditions, the linear concentration range for pseudolycorine was 0.002-2 μg/mL with a correlation coefficient of 0.9991. Relative standard deviations of migration time and peak areas were 1.4 and 3.2%, respectively. The limit of detection (S/N=3) was 0.46 ng/mL. The proposed method can be successfully applied to the determination of pseudolycorine in the bulb of lycoris radiata.     

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 μA/mM in phosphate buffer), low detection limit (0.8 μM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).     

应用细胞透性化技术快速提取氧化葡萄糖杆菌胞内右旋糖酐糊精酶

将细胞透性化技术应用到右旋糖酐酶的提取检测过程中,根据单因素实验和正交试验设计确定了从氧化葡萄糖杆菌(Gluconobacter oxydans)细胞中提取胞内右旋糖酐糊精酶(DDase)的最佳方法:甘氨酸(Gly)质量分数15%,Triton X-100体积分数1%,菌悬液OD600为0.5~6.0,pH为6.81,冰浴处理时间1 h,透性化试剂的提取效率可达到酶活最大值的90%以上。与超声波细胞破碎法相比,该方法条件温和,酶的释放率较高并易于大量试样的平行实验操作。     

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.     

Quantitative analysis on inactivation and reactivation of recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li

The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H₁₃, S₁₉₃, N₃₅₉, E₄₀₇, and M₅₁₅ of α subunit, N₄₇, L₁₅₀, V₁₈₉ of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg^?1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O₂ could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg²⁺ and coenzyme B₁₂ were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O₂-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.     

Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.

The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively. They were identified by mass spectrometry to correspond to the large, medium and small subunits of the dehydratase encoded by the pduC, pduD and pduE genes, respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC, D, E genes (61, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme, another protein involved in the 1,2-propanediol metabolism pathway.     

Regulation of thymidine metabolism in Escherichia coli K-12: optimal conditions for the assay of 1,5-phosphodeoxyribomutase in ultrasonic extracts

Assay conditions have been established which allow maximum expression of phosphodeoxyribomutase activity in ultrasonic extracts of Escherichia coli K-12. The enzyme requires 2-mercaptoethanol, manganous ions, and glucose 1,6-diphosphate for optimal activity. When cells are grown in minimal medium with glycerol as the carbon source and supplemented with 10(-3)m thymidine, phosphodeoxyribomutase is induced three- to four-fold. Cell pellets may be frozen for 24 hr before sonic disruption without loss of enzyme activity. The assay is highly reproducible.