Mn~(2+)对肠膜状明串珠菌产右旋糖酐的影响

肠膜状明串珠菌在其产生的右旋糖酐蔗糖酶的作用下,以蔗糖为原料转化合成右旋糖酐和产生果糖。着重进行了Mn~(2+)对肠膜状明串珠菌Lm-1226发酵产右旋糖酐影响的初步探索。对Mn~(2+)对肠膜状明串珠菌Lm-1226的生长,产果糖、右旋糖酐和右旋糖酐蔗糖酶,右旋糖酐蔗糖酶作用影响进行了研究。一定浓度的Mn~(2+)对肠膜状明串珠菌Lm-1226的生长具有促进作用; Mn~(2+)抑制肠膜状明串珠菌Lm-1226发酵产果糖和右旋糖酐,且Mn~(2+)浓度越高,抑制性越强; Mn~(2+)对肠膜状明串珠菌Lm-1226发酵产右旋糖酐蔗糖酶有抑制作用; Mn~(2+)对右旋糖酐蔗糖酶具有较强的激活作用,激活作用可达158%,最适Mn~(2+)浓度为5. 0 mmol/L。     

右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究

[目的]右旋糖酐蔗糖酶是一种以蔗糖为底物,催化转移D-葡萄糖基生成α-葡聚糖或低聚糖的葡萄糖基转移酶.[方法]利用PCR扩增技术,将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a( )上,转化E.coli BL21(DE3),经过卡那霉素抗性筛选和酶切验证后,得到右旋糖酐蔗糖酶工程菌株BL21(DE3)/pET28-dexYG.[结果]经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制.通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察,得到最佳培养条件为:培养时间5h、IPTG浓度0.5mmol/L、25℃、OD600值1.0和pH6.0.酶活力由最初的5.39U/mL提高到35.62U/mL,其中pH值对产酶活力影响最大,在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍,并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一.[结论]酶的表达和酶活的研究结果表明,构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶,并且表现出较高的酶活力.     

淡紫拟青霉右旋糖酐酶的形成条件

比较了各种碳水化合物对淡紫拟青霉(Paecilomyces lilacinus)右旋糖酐酶形成的影响,右旋糖酐是最好的碳源,也是最佳诱导物。不同分子量(17．2—1000kD)的右旋糖酐对酶形成的诱导作用不同,酶的产生随右旋糖酐分子量的增大而增加。用分子量为1000kD的右旋糖酐作碳源时比用17.2kD的右旋糖酐作碳源时的产酶量高40％以上。用右旋糖酐和其它糖的混合物作碳源时,酶的形成受到不同程度的抑制。右旋糖酐酶形成的其它适宜条件：氮源为牛肉蛋白胨,培养基初始pH6．0—7.0.种龄为48小时,在250ml三角瓶中装50ml培养基,于28℃在200r／min摇床上培养6天。     

氧化葡萄糖杆菌合成右旋糖酐糊精酶的pH两阶段控制策略

为了进一步提高氧化葡萄糖杆菌右旋糖酐糊精酶的产量,在3 L发酵罐水平上考察了pH(3.5?6.0)对菌体生长和产酶的影响。基于不同pH发酵过程中菌体生长及产物合成的变化,确定了pH两阶段控制策略,即0?6 h时控制pH 5.0,6 h后将pH调至4.0。通过采用这一优化策略,右旋糖酐糊精酶酶活有了较大的提高,可达4.03 U/mL,比不控制pH模式下提高了38.5%,是摇瓶水平的12.5倍,同时发酵时间从47 h缩短为15 h。     

重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

[目的]研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质.[方法]工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究.[结果]经过SDS-PAGE测得该酶的分子量约为170 kDa,与理论推测值基本相同.以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43 mmol/L;酶活在pH 5.0～8.0较为稳定,在室温(25 ℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2 对催化作用有较大的促进,Mg2 有微弱的促进作用,K 对催化反应无影响,Cu2 的抑制作用最强.其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强.[结论]研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数.     

黄杆菌肝素酶Ⅱ重组菌培养条件的优化及高密度发酵

黄杆菌肝素酶Ⅱ(HepⅡ)是一类可特异性切割肝素、硫酸乙酰肝素类分子内连接键的酶。文中对黄杆菌肝素酶Ⅱ重组菌的诱导时机、诱导剂添加量、诱导温度、诱导时间等诱导产酶条件进行优化。经过优化最佳摇瓶发酵产酶条件为:37℃培养重组菌至对数生长前期,添加诱导剂IPTG至终浓度为0.3 g/L,20℃下诱导10 h,酶活达到最高,为570 U/L。在此基础上通过发酵罐高密度培养手段将菌体浓度OD600进一步提高到98,酶活大幅度提高到9 436 U/L,该研究结果为HepⅡ的工业化生产与应用奠定了良好的基础。     

基于右旋糖酐蔗糖酶转糖基作用的槲皮素葡萄糖苷的合成研究

右旋糖酐蔗糖酶是一种以蔗糖为唯一底物,将蔗糖分子中D-葡萄糖基催化转移到受体分子上的葡萄糖基转移酶。利用右旋糖酐蔗糖酶的转糖基作用,以蔗糖为葡萄糖糖基供体,槲皮素为糖基受体,对槲皮素糖苷的酶法合成进行了探索。通过对该酶催化反应体系、催化反应条件及产物分析的研究,结果表明：在25℃下,右旋糖酐蔗糖酶能够在30％DMSO-70％乙酸-乙酸钙（0.02 mol/L,pH值5.4）的反应体系中催化合成一种槲皮素葡萄糖苷,在这个反应体系下,以10％的蔗糖作为糖基供体,槲皮素为糖基受体,右旋糖酐蔗糖酶活力为40 U/mL,转速为150 r/min,槲皮素糖苷的转化率最高,可达39.5％。通过质谱分析确定是一种槲皮素单糖苷,分子量为464。该研究结果为黄酮类物质的糖基化修饰奠定了基础。     

南极交替单胞菌R11-5产卡拉胶酶的发酵条件优化

【背景】极地寒冷环境中发现了大量具有潜在应用前景的冷适应酶,同时也存在种类繁多的海藻多糖降解菌,因此极端环境微生物是筛选获得新颖、高效多糖降解酶的重要新源泉。由于筛选培养基通常并非野生菌发酵产酶的最优条件,为了使野生菌的产酶效率达到最高,需要对其培养条件进行优化,从而为其深入研究及开发利用提供依据。【目的】对一株产卡拉胶酶的南极菌株进行种属鉴定,并采用响应面法对该菌的发酵产酶条件进行优化。【方法】通过16SrRNA基因对产卡拉胶酶的南极菌株进行种属鉴定,采用响应面法优化南极菌株产酶发酵条件。【结果】该南极菌属于交替单胞菌属(Alteromonas),命名为交替单胞菌R11-5。发酵条件优化结果显示,7个环境因子影响交替单胞菌R11-5的产酶量。利用Design-Expert软件中的Plackett-Burman设计实验,筛选出影响交替单胞菌R11-5产酶量的4个主要因素分别为培养温度、牛肉膏浓度、卡拉胶浓度和Ca~(2+)浓度。通过Box-Behnken设计和响应面分析得到交替单胞菌R11-5最佳产酶发酵条件为:温度15.0°C,牛肉膏浓度11.0 g/L,卡拉胶浓度3.0 g/L,Ca~(2+)浓度5.0 mmol/L。优化后发酵上清液酶产量达到87.193 U/mL,与优化前相比提高了1.8倍。【结论】响应面法提高了南极交替单胞菌R11-5卡拉胶酶的产量,为其开发应用提供了科学依据。     

腈水解酶psn基因工程菌的发酵及产酶条件优化

来自恶臭假单胞菌的腈水解酶具有高效催化3-氰基吡啶产烟酸的能力,对表达该酶的基因psn进行发酵和产酶条件优化,通过对C源、N源、磷酸盐、金属离子、温度、诱导剂浓度和诱导时间进行单因素考察,获得最适培养基条件(g/L):葡萄糖5、蛋白胨15、酵母粉5、(NH4)2SO45、K2HPO424.5、KH2PO45.76、MgSO40.48;最佳诱导条件:培养2.5 h后添加IPTG诱导,浓度0.2 mmol/L,诱导温度30℃。在该条件下培养,重组大肠杆菌的腈水解酶比酶活可达到45.67 U/mL,比优化前提高了2.26倍。在此基础上,于5 L发酵罐上进行C、N源的补料研究,获得最适分批补料策略,发现其腈水解酶活力可达到75.40 U/mL,是优化前的3.74倍。     

右旋糖酐酶的底物吸附

用35％乙醇保护,底物占旋糖酐能对右旋糖酐酶有效地吸附。采用0．2mol／L pH8．0的KzHPO4-KH2PO4缓冲液(内含30％乙醇)进行解吸,总同收率在80％以上。粗酶液经此过程提纯了9倍。低温对吸附有利。 在酶稳定范围内,pH对吸附影响不大。酶浓度过高,吸附效率下降。对稀酶液可连续多次吸附以达到浓缩目的。1．5％(W／V)的右旋糖酐使可得到满意的吸附效果,对五种不同来原的右旋糖酐酶吸附率都很高,但不同来源的底物适用情况差别很大。     

Dextransucrase constitutive mutants of Leuconostoc mesenteroides B-1299

Leuconostoc mesenteroides B-1299 dextrans are separated into two kinds: fraction L, which is precipitated by an ethanol concentration of 38%, and fraction S, which is precipitated at an ethanol concentration of 40%. Fraction S dextran contained 35% of -1,2 branch linkages, and fraction L contained 27% -1,2 branch linkage with 1% -1,3 branch linkages. We have isolated mutants constitutive for dextransucrase from L. mesenteroides NRRL B-1299 using ethyl methane sulfonate. The mutants produced extracellular as well as cell-associated dextransucrases on glucose media with higher activities (2.5–4.5 times) than what the parental strain produced on sucrose. Based on Penicillium endo-dextranase hydrolysis, mutant B-1299C dextransucrases produced slightly different dextrans when they were elaborated on a glucose medium and on a sucrose medium. Mutant B-1299CA dextransucrase elaborated on a glucose medium and on a sucrose medium synthesized the same dextran, although the dextran was different from those of other mutants and the parental strain. Mutant B-1299CB dextransucrase, elaborated on a glucose medium and on a sucrose medium, formed different dextrans. Differences in water solubility, susceptibility to endo-dextranase hydrolysis, and the physical appearance of the ethanol precipitated dextrans elaborated by different mutants grown on glucose media and sucrose media were found. All mutant dextransucrases elaborated on a glucose medium bound to Sephadex G-200. After activity staining of nondenaturing sodium dodecyl sulfate—polyacrylamide gel electrophoresis activity bands, 184 and 240 Kd for each enzyme preparation, although each dextransucrase formed different dextran(s).     

分散泛菌蔗糖异构酶在大肠杆菌中的表达及发酵优化

为了提高分散泛菌Pantoea dispersa UQ68J来源的蔗糖异构酶产量,研究了不同信号肽及发酵条件对蔗糖异构酶在大肠杆菌中重组表达的影响。将携带天然信号肽的蔗糖异构酶基因优化后,转入大肠杆菌Escherichia coli BL21(DE3)构建重组表达菌株——ORI菌株,摇瓶发酵总酶活和胞外酶活分别为85 U/m L、65 U/m L。从天然信号肽开始第22位氨基酸作为成熟蛋白的起始,连接Pel B或Omp A信号肽构建P22和O22菌株,其中P22菌株发酵总酶活提高至138 U/m L,是ORI菌株总酶活的1.6倍;而O22菌株发酵总酶活和ORI菌株无明显差别。采用3.0 g/L的乳糖诱导,P22菌株的蔗糖异构酶总酶活提高至168 U/m L。在3 L发酵罐中,研究甘氨酸浓度和诱导时间对蔗糖异构酶分泌的影响,当补加0.5%甘氨酸,DCW为18 g/L(OD_(600)=30)开始诱导,P22菌株的蔗糖异构酶胞外酶活最高达1 981 U/m L,同时蔗糖异构酶总酶活达到2 640 U/m L,是已报道大肠杆菌重组表达蔗糖异构酶的最高水平。     

Streptococcus mutans Dextransucrase: Requirement for Primer Dextran

Dextran stimulation (priming) of the dextransucrase (EC 2.4.1.5) from Streptococcus mutans strain 6715 was studied. The dextransucrase activity in supernatant fluids from glucose-grown cultures was shown to be partially primer dependent. During extended storage at 4 C the enzyme retained its activity. However, the ability to make dextran became increasingly primer dependent. Hydroxylapatite-chromatographed enzyme preparations were completely dependent upon added dextran for rapid synthesis of methanol-insoluble glucan from sucrose. Half-maximal stimulation of new dextran synthesis occurred with dextran at a concentration of 2 to 3 muM and with a molecular weight of about 2,600. Neither glycogen, amylose, inulin, nor isomaltose functioned as primer. Studies with the dextransucrase activities detectable by in situ assay in polyacrylamide gels subjected to electrophoresis under nondenaturing conditions revealed that the major activity was detectable in the presence of sucrose alone and was stimulated by addition of primer dextran. The minor activity was only detected when primer dextran was present. Homogeneous preparations of both enzymes contained 30 to 40% carbohydrate.     

Screening and optimization of nutritional factors for higher dextransucrase production by Leuconostocmesenteroides NRRL B-640 using statistical approach

To improve dextransucrase production from Leuconostocmesenteroides NRRL B-640 culture medium was screened and optimized using the statistical design techniques of Plackett-Burman and response surface methodology (RSM). Plackett-Burman design with six variables viz. sucrose, yeast extract, K2HPO4, peptone, beef extract and Tween 80 was performed to screen the nutrients that were significantly affecting dextransucrase production. The variables sucrose, K2HPO4, yeast extract and beef extract showed above 90% confidence levels for dextransucrase production and were considered as significant factors for optimization using response surface methodology. 2(4)-central composite design was used for RSM optimization. The experimental results were fitted to a second-order polynomial model which gave a coefficient of determination R2=0.95. The optimized composition of 30g/l sucrose, 18.9g/l yeast extract, 19.4g/l K2HPO4 and 15g/l beef extract gave an experimental value of dextransucrase activity of 10.7U/ml which corresponded well with the predicted value of 10.9U/ml by the model.     

Factors affecting alpha,-1,2 glucooligosaccharide synthesis by Leuconostoc mesenteroides NRRL B-1299 dextransucrase

The optimization of alpha-1,2 glucooligosaccharide (GOS) synthesis from maltose and sucrose by Leuconostoc mesenteroides NRRL B-1299 dextransucrase was achieved using experimental design and consecutive analysis of the key parameters. An increase of the pH of the reaction from 5.4 to 6.7 and of the temperature from 25 to 40 degrees C significantly favored alpha-1,2 GOS synthesis, thanks to a significant decrease of the side reactions, i.e., dextran and leucrose synthesis. These positive effects were not sufficient to compensate for the decrease of enzyme stability caused by the use of high pH and temperature. However, the critical parameters were the sucrose to maltose concentration ratio (S/M) and the total sugar concentration (TSC). Alpha1,2 GOS synthesis was favored at high S/M ratios. But using these conditions also led to an increase of side reactions which could be modulated by choosing the appropriate TSC. Finally, with S/M = 4 and TSC = 45% w/v, dextran and leucrose productions were limited and the final alpha-1,2 GOS yield reached 56.7%, the total GOS yield being 88%.     

Cloning, sequencing and expression of a dextransucrase gene (dexYG) from Leuconostoc mesenteroides

The gene dexYG encoding the dextransucrase from an industrial strain of Leuconostoc mesenteroides 0326 was isolated by PCR. The nucleotide sequence of the dexYG gene consists of an open reading frame (ORF) of 4,584 bp, coding for a 1,527 aa protein with a Mr of 170 kDa. The results were analysed by a BLAST similarity search of the GenBank database, which revealed the amino acid sequence was similiar to dsrD derived from L. mesenteroides Lcc4. The dexYG gene was subcloned into the plasmid pET28a(+) and was expressed in E. coli BL21 (DE3) by IPTG induction. The pH value was one of the main reasons which caused the degradation of enzyme activity in the later stage of induction. The highest activity was reached 36 U/ml after 5 h induction in medium at pH 6.0. Biotransformation yield of the enzyme reached 65% and the molecular weight of transformed dextran was more than 68 kDa in 2 h.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

Growth and energetics of Leuconostoc mesenteroides NRRL B-1299 during metabolism of various sugars and their consequences for dextransucrase production.

The metabolic and energetic properties of Leuconostoc mesenteroides have been examined with the goal of better understanding the parameters which affect dextransucrase activity and hence allowing the development of strategies for improved dextransucrase production. Glucose and fructose support equivalent specific growth rates (0.6 h-1) under aerobic conditions, but glucose leads to a better biomass yield in anaerobiosis. Both sugars are phosphorylated by specific hexokinases and catabolized through the heterofermentative phosphoketolase pathway. During sucrose-grown cultures, a large fraction of sucrose is converted outside the cell by dextransucrase into dextran and fructose and does not support growth. The other fraction enters the cell, where it is phosphorylated by an inducible sucrose phosphorylase and converted to glucose-6-phosphate (G-6-P) by a constitutive phosphoglucomutase and to heterofermentative products (lactate, acetate, and ethanol). Sucrose supports a higher growth rate (0.98 h-1) than the monosaccharides. When fructose is not consumed simultaneously with G-1-P, the biomass yield relative to ATP is high (16.8 mol of ATP.mol of sucrose-1), and dextransucrase production is directly proportional to growth. However, when the fructose moiety is used, a sink of energy is observed, and dextransucrase production is no longer correlated with growth. As a consequence, fructose catabolism must be avoided to improve the amount of dextransucrase synthesized.     

Origin and function of the multiple extracellular glucosyltransferase species from cultures of a serotype c strain of Streptococcus mutans

Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.