右旋糖酐对Savinase蛋白酶的化学修饰及酶学性质研究

采用经高碘酸钠活化的右旋糖酐修饰Savinase蛋白酶,通过凝胶过滤层析(GPC)和圆二色性光谱(CD)表征了修饰后蛋白酶分子量和结构的变化,测试了修饰酶的反应动力学参数,并考察了温度及pH对修饰酶活力的影响。凝胶过滤层析结果证明修饰后蛋白酶分子量明显提高,圆二色光谱分析表明修饰后蛋白酶的结构有所改变,进一步验证了右旋糖酐和蛋白酶发生了反应。与原酶相比,修饰酶对底物的亲和力增加。原酶和修饰酶的最适温度均为40℃,在30℃~50℃之间修饰酶表现出优于原酶的热稳定性。在pH8.5~9.5之间,修饰酶的稳定性高于原酶。     

几种蛋白酶对文蛤肉的酶解工艺条件研究

以文蛤为原料,水解度为指标,从胰蛋白酶,木瓜蛋白酶,胃蛋白酶和风味蛋白酶中选出水解效果较好的胰蛋白酶和木瓜蛋白酶.并通过实验确定了胰蛋白酶,木瓜蛋白酶单酶水解及两者组合复合酶解文蛤肉的最佳工艺.结果表明:复合水解效果最佳.最佳工艺:先添加胰酶6000 u/g(原料),水解温度50℃,固液比1:3(V/W),pH 8.0,在此条件下水解6 h;然后改变条件,温度55℃,pH 5.5,底物浓度1:3(V/W),添加木瓜蛋白酶2000 u/g(原料),在此条件下水解2 h.通过实验验证,胰蛋白酶和木瓜蛋白酶组合在该条件下对文蛤肉蛋白具有较好的水解效果,其水解度为28.15%.     

巨大芽孢杆菌青霉素G酰化酶在新型环氧载体ZH-HA上的固定化

巨大芽孢杆菌青霉素G酰化酶共价结合在新型环氧-氨基型载体ZH-HA 上,通过对酶浓度、固定化时间、pH以及缓冲液浓度等条件的考察,确定了最优固定化条件:50 mg比活力6000 U/g的巨大芽孢杆菌青霉素G酰化酶蛋白和1g ZH-HA悬浮于pH 9.01 mol/L磷酸缓冲液,室温搅拌6 h,制得固定化巨大芽孢杆菌青霉素G酰化酶,活力2126 U/g湿载体,活力回收率7.67%.比较研究了固定化酶与原酶性质,原酶最适温度45℃,最适pH为8.0.固定化酶则分别是50℃和9.0,分别比溶液酶偏移5℃、1.0个pH单位.经过40批连续水解青霉素G钾盐,固定化巨大芽孢杆菌青霉素酰化酶仍保持80%的活力,显示出良好的工作稳定性.     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

短小芽孢杆菌(Bacillus pumilus) WHK4以羽毛粉为底物产蛋白酶条件的优化

探索Bacillus pumilusWHK4以羽毛粉为底物产酶的最佳条件和最佳培养基组成。以羽毛粉发酵培养基为基础,首先采用单因子试验考察底物浓度、初始pH、接种量、外加碳源、外加氮源对WHK4产酶活力的影响。在单因子试验的基础上采用正交试验设计对底物浓度、温度、初始pH、接种量、外加(NH4)2SO4、外加麦芽糖进行优化。结果显示:Bacillus pumilusWHK4最佳的产酶条件为初始pH7.38,菌龄16 h,接种量5%,37℃。最佳的培养基组成为:1 L基础发酵培养基,40.0 g羽毛粉,10.0 g(NH4)2SO4和10.0 g麦芽糖。在优化的条件下Bacillus pumilusWHK4 24 h产蛋白酶活力为每毫升90 U。对培养条件和培养基的优化为Bacillus pumilusWHK4产蛋白酶的分离纯化奠定了基础。     

以Fe(Ⅲ)改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上.制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％.研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性.结果表明:过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25℃;但固定化酶的热稳定性显著提高,在75℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失活;此外,固定化过氧化氢酶还表现出了良好的操作稳定性,在室温下连续反应26次后,相对活力为57％.该研究表明胶原纤维可作为固定化过氧化     

N-乙酰神经氨酸醛缩酶的固定化及固定化酶性质研究

使用LX-1000HFA氨基树脂对N-乙酰神经氨酸醛缩酶(NAL)进行固定化,并对游离酶与固定化酶的酶学性质及稳定性进行了对比研究。结果显示,最佳固定化条件为载体投放量5.0 g,固定化时间12 h,缓冲液浓度1.0 mol/L,pH7.5,温度25℃。在此条件下制备的固定化NAL活力最高,比酶活可达200 U/g湿载体。与游离酶相比,最适反应温度提高了5℃,最适反应pH没有变化,温度和pH耐受性明显提升。同时固定化酶储存稳定性和操作稳定性也显著增强,在4℃条件下储存10 d后其酶活仅损失6%,重复使用10次后仍保持初始酶活的80%。因此,该固定化酶具有良好的温度稳定性、pH稳定性、储存稳定性和操作稳定性,为酶法工业化生产N-乙酰神经氨酸研究提供了理论依据。     

发酵生产魔芋葡甘聚糖酶

目的:探索和了解发酵生产葡甘聚糖酶的最佳条件。方法:在 5L发酵罐上测定不同温度、pH和接种量对发酵的影响,并用正交试验筛选最佳的产酶条件配伍。结果:该菌产生葡甘聚糖酶的条件为接种量 1 0 %,通气量 2 0L h,发酵的前 1 6h温度为 40℃,搅拌速度 2 0 0r min,pH 7 0左右,之后温度调至 5 0℃,搅拌速度 1 0 0r min,pH调至 6. 0左右,添加 0 . 2 5 %的豆油做消泡剂。在这个条件下发酵获得的酶比活力可达 5 0 0 9U mg,总活力达到 1 0 81 9U ml。     

拟南芥叶肉原生质体分离条件的优化研究

以野生型拟南芥(Arabidopsis thaliana,ecotype Columbia)无菌苗为材料,研究了叶肉原生质体分离过程中的预处理条件、酶解方式、酶解温度和离心力大小等因素对产量和活力的影响.结果表明,酶解方式和酶解温度对原生质体产量影响显著,28℃静置酶解14 h能够将原生质体产量提高6.32倍.低温预处理和离心力大小对原生质体活力影响显著,4℃低温预处理24 h能够将原生质体活力提高55%.适宜拟南芥原生质体叶肉细胞分离的最佳条件为:4℃低温预处理24 h,28℃静置酶解14 h,600 r·min-1离心3次,每次10 min,得到的原生质体产量为2.91×106个·g-1,活力为84.03%.     

Maltose uptake in the Zea mays scutellum

Maltose transport in slices of the maize scutellum was demonstrated despite the presence of an active maltase situated at the cell surface. The maltase could be inhibited or destroyed by treatments (neutral pH during uptake, pretreatment in Tris buffer at pH 7·5, or in 0·01 N HCl) that allowed appreciable rates of maltose uptake to occur. Using Tris- and HCl-treated slices, it was found that at disaccharide concentrations of 50 and 100 mM, maltose and sucrose were taken up at very nearly the same rates. At sugar concentrations below 50 mM, sucrose was taken up at greater rates than maltose. The maltose content of the slices was directly proportional to the maltose concentration of the bathing solution, and about 4 hr were required for equilibration. From this, it is concluded that one way maltose enters the slices is by free or facilitated diffusion. However, endogenous maltose is utilized by the slices at rates that are much too low to account for the net rates of maltose uptake. Although the slices contain a high level of surface maltase activity, only a low level of endogenous maltase activity was found. This probably accounts for the slow utilization of endogenous maltose. Therefore, the existence of a specific maltose transport system is proposed; a system that contains a carrier saturable with maltose, but one that does not release free maltose into the cytoplasm.     

Substrate Inhibition of Transglucosyl-Amylase by Maltose

Transglucosyl-amylase was inhibited by maltose when maltose served as a substrate. As a function of substrate concentration, the rates initially rose proportionately with increases of maltose levels until a maximal rate was attained. Further increases of maltose concentration decreased reaction rates. The attainment of a maximal rate with increasing substrate concentration and close correspondence between experimental and calculated rates indicated the involvement of ternary complex formation. Evidence also suggested that substrate inhibition is caused by maltose competing with water for the acceptor sites during complex formation.     

Maltose metabolism by pea chloroplasts

The aim of this work was to investigate the origin of maltose formed during starch breakdown in the dark by chloroplasts of Pisum sativum. The maximum catalytic activities of maltose phosphorylase and maltase in pea leaves were shown to be low, relative to those of enzymes known to be involved in starch breakdown. Fractionation of pea leaves indicated that the chloroplasts lack maltase but have enough maltose phosphorylase to synthesize the amounts of maltose formed when isolated chloroplasts breakdown starch. The absence of exogenous phosphate markedly reduced starch breakdown and maltose accumulation by isolated chloroplasts. When [¹⁴C]glucose was supplied to chloroplasts that were breaking down starch in the dark, maltose was labelled and most of the label was in the glucose moeity. It is suggested that maltose phosphorylase, using glucose-1-phosphate formed from starch by α-glucan phosphorylase, is responsible for, at least some of, the synthesis of maltose during starch breakdown by pea chloroplasts in vitro.     

Enzymatic Synthesis of Trehalose from Maltose

Synthesis of trehalose from maltose by a coupled enzyme system with trehalose Phosphorylase and maltose Phosphorylase has been studied. Trehalose Phosphorylase was partially purified from Euglena gracilis and maltose Phosphorylase was obtained from Lactobacillus brevis. The optimum pH of the reaction was 6.5~7.0 and the reaction rate was faster in the rection mixture containing a low concentration of phosphate. The final ratio of conversion (the ratio of trehalose to maltose) in the pH range between 6.0 and 8.0 was about 60%.

Immobilized maltose and trehalose Phosphorylase in κ-carageenan could be used without any appreciable loss of activity for batch reactions at least 10 times.     

Maltose chemoreceptor of Escherichia coli.

Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.     

Maltose metabolism of Pseudomonas fluorescens.

Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis.     

Partial Conversion of Maltose into Cyclohexane Derivatives

5,6-Unsaturated disaccharide derivative prepared from maltose via its 6-iodo derivative was treated with mercuric chloride, giving α-linked pseudodisaccharide in good yield, which contains cyclohexanone derivatives as constituents. On treatment with an acetic anhydride-pyridine mixture, the cyclohexanone constituents underwent β-elimination to be changed into a sole cyclohexenone moiety. Hydrogenation of the double bond between two carbon atoms and subsequent reductive amination of the carbonyl group gave aminocyclitol-containing pseudodisaccharides.     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.