甲醇对胰蛋白酶催化活性影响的机理研究

为了探索甲醇影响胰蛋白酶催化活性的作用机理,将胰蛋白酶纯化到电泳纯的水平,用纯酶进行了催化动力学研究;测定了酶分子的紫外吸收光谱、紫外差示光谱和荧光发射光谱的变化.试验结果表明:胰蛋白酶经7%甲醇处理时,其比活力比对照提高了17.97%.经甲醇处理后的胰蛋白酶,其动力学参数Km值及Vmax值均得到提高,且Vmax提高幅度比较大.甲醇处理后,酶的紫外吸收光谱基本没有变化,其差示光谱出现明显的正峰和负峰,而其荧光发射光谱也基本不变,只是荧光强度有所增加.实验结果证明,在7%甲醇存在下,胰蛋白酶分子构型不变,酶活性的变化是甲醇引起酶分子构象改变的结果.     

砷对土壤脲酶活性影响的研究

采用模拟方法对As污染土壤脲酶特征进行了研究．结果表明,土壤中As浓度在0～200mg·kg^-1浓度范围内,反应初期脲酶活性变化无明显规律;一年后砷激活土壤脲酶活性,二者达到显著或极显著正相关．随As浓度增加,土壤脲酶Km值基本不变或略有增加,Vmax增大,从机制上揭示出As加速脲酶-尿素复合物的解离．厩肥和无肥土样脲酶对As的反应类似,只是变化幅度有所差异．     

汞、豆磺隆复合污染对土壤脲酶活性及动力学特征的影响

通过室内模拟研究了重金属汞(Hg)和除草剂豆磺隆单一和复合污染条件下对草甸棕壤和黑土4个土样脲酶活性和动力学参数的影响.结果发现:汞对脲酶活性、Km、Vmax、Vmax/Km均有负效应,其影响幅度分别为39%～98%、46%～74%、90%～98%、20%～68%;HgCl2浓度与脲酶活性之间符合对数方程,与3动力学参数之间呈显著(P＜0.05)或极显著(P＜0.01)线性负相关;豆磺隆对脲酶有激活作用,除2号土样脲酶活性对不同浓度豆磺隆影响差异不明显外,其余3土样最大增幅分别为17%、18%和15%,原因可能是豆磺隆的加入提高了脲酶酶促反应的初速度;汞、豆磺隆复合污染对脲酶活性和3特征参数均产生负效应,其影响幅度与汞单一污染情况相似,但与污染物浓度之间的相关性较差,豆磺隆的加入使汞与脲酶之间的相互作用趋于复杂.     

氮添加对杉木林土壤有机碳矿化速率及酶动力学参数温度敏感性的影响

为探讨氮添加对亚热带森林土壤有机碳矿化速率(Cmin)及酶动力学参数温度敏感性(Q10)的影响,选择亚热带杉木林土壤为研究对象,采用野外长期氮添加与室内控温培养试验,分析土壤Cmin及β~(-1),4-葡萄糖苷酶(βG)动力学参数温度敏感性。野外试验设置对照(N0)、低氮(N1:50 kg N hm~(-2)a~(-1))、高氮(N2:100 kg N hm~(-2)a~(-1)) 3种处理,每种处理3次重复,室内培养设置10—40℃。结果表明:(1)氮添加增加土壤Cmin,为N2N1N0,但其Q10(Cmin)差异不显著。(2)氮添加增加βG的潜在最大反应速率(Vmax)和催化效率(Vmax/Km),且Vmax和Vmax/Km均为N2N1N0,而氮添加对半饱和常数(Km)影响不显著。Q10(Vmax)和Q10(Km)大小为N2N1N0且差异显著,但是Q10(Vmax/Km)无显著差异。(3)相关分析表明,30℃培养温度下,Cmin和全磷(TP)、硝态氮(NO-3-N)、有效磷(a P)、Vmax正相关; Vmax和TP、NO-3-N正相关,和p H负相关; Km和全氮(TN)负相关; Vmax/Km和p H负相关,和TP正相关。30—40℃培养温度下,Q10(Vmax)和p H负相关,Q10(Vmax/Km)和TN负相关。研究可为氮沉降背景下土壤碳素循环的生物化学过程对增温响应的模型提供重要参数。     

与氮转化有关的土壤酶活性对抑制剂施用的响应

利用室内模拟培养试验,研究好气条件下施用尿素后土壤脲酶、脲酸还原酶、亚硝酸还原酶和羟胺还原酶活性对脲酶抑制剂氢醌（HQ）与硝化抑制剂包被碳化钙（ECC）和双氰胺（DCD）组合（HQ ECC、HQ DCD）的响应、结果表明,HQ DCD组合与其它抑制剂处理相比能更有效地降低土壤脲酶活性,增加硝酸还原酶、亚硝酸还原酶、羟胺还原酶活性,不同处理土壤脲酶、亚硝酸还原酶和羟胺还原酶活性与土壤NH4^ 、NO3^-、NH3挥发和N2O排放速率间存在不同形式的显著相关关系：土壤脲酶、亚硝酸还原酶和羟胺还原酶活性之间存在不同形式的显著正相关关系。     

镉、汞复合污染对土壤脲酶和酸性磷酸酶活性的影响

通过土壤培养试验, 研究了重金属镉（Cd）、汞（Hg）复合污染对小粉土和黄红壤脲酶和酸性磷酸酶活性的影响.结果表明：整个培养过程中,Cd、Hg单一及复合污染都对两种土壤中脲酶和酸性磷酸酶的活性具有明显抑制作用,且随重金属浓度增强而增强（浓度≤1 mg·kg^-1Cd除外）.与单一Cd或Hg污染相比,同剂量Cd和Hg复合污染时两种酶活性的净变化量均大于0,表明Cd和Hg复合污染在两种土壤中对土壤脲酶和酸性磷酸酶活性的抑制作用均表现为一定的协同作用.所有处理黄红壤脲酶和酸性磷酸酶活性均高于小粉土,这可能与黄红壤有机质和粘粒含量相对较高有关.     

超声波对猪胰脂肪酶催化活性影响的机理研究

猪胰脂肪酶(porcine pancreas Lipase,PPL)反应系统经适宜参数的超声波处理,催化活性提高了11.22%～24.42%。PPL经超声波处理后,其动力学参数Km和Vmax都变小,酶分子的紫外吸收光谱不变,荧光发射光谱不变。而酶分子经超声波处理后,其紫外差示光谱出现了明显的正峰或负峰。探讨了超声波影响PPL活性的作用机理。     

元宝枫叶蛋白酶的动力学特征

酶促动力学是研究酶促反应的速度及各种因素如底物浓度、酶浓度、抑制剂、激活剂、温度、pH等对酶促反应速度影响的科学,是酶学研究中重要的内容。在一定条件下,酶促反应都有其特定的动力学参数,如Km值（米氏常数）和Vmax值（最大反应速率）等。Km是反映酶动力学性质的重要特征性常数,对某一酶促反应而言,在一定条件下都有特定的Km值,可以用来鉴别酶。Km值还可以判断酶的专一性和天然底物,     

毛苔草湿地土壤酶活性及活性有机碳组分对水分梯度的响应

通过设置的W1（15cm）、W2（-5cm）、W3（-5～5cm）、W4（淹没）4种水分梯度的毛苔草（Carex lasiocarpa）盆栽培养实验,研究了湿地土壤酶活性、微生物量碳（MBC）、可溶性有机碳（DOC）及毛苔草地上生物量对水分梯度的响应及土壤酶活性与MBC、DOC、地上生物量的关系。土壤酸性磷酸酶、蔗糖酶和脲酶活性随着土壤水分增加而降低,但过氧化氢酶活性随着土壤水分增加而增加。与持续淹水相比,于湿交替（W3）增加了土壤酸性磷酸酶、蔗糖酶、脲酶和过氧化氢酶活性。土壤微生物量碳（MBC）含量表现为W3〉W1〉W2〉W4,蔗糖酶、脲酶、过氧化氢酶活性与MBC呈显著正相关（P〈0．05）。土壤可溶性有机碳（DOC）含量表现为W4〉W1〉W3〉W2,脲酶和酸性磷酸酶活性与DOC呈极显著负相关（P〈0．01）。毛苔草地上生物量与土壤酶活性呈正相关,其中,蔗糖酶、过氧化氢酶、脲酶活性与毛苔草生长状况密切相关。     

外源亚精胺可缓解荇菜镉毒害

研究了外施浓度为0．01mmol·L^-1的亚精胺（Spd）对不同浓度镉（Cd2＋）胁迫下荇菜（Nymphoides peltatum）叶片的叶绿体结构、叶绿素含量、可溶性蛋白含量、超氧阴离子（O2）产生速率和丙二醛（MDA）含量,以及保护酶——超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性的影响。结果表明,（1）Cd2＋胁迫可使荇菜细胞的叶绿体结构遭到破坏,叶绿素含量减少。外施Spd则可有效地保护叶绿体结构,减少叶绿素的流失。（2）在单一Cd2＋处理条件下,随着Cd2＋浓度的升高,叶绿素含量呈现先升后降的趋势,可溶性蛋白含量则逐渐下降。外源Spd处理显著提高了二者的含量,并延缓了它们的下降速度。（3）在单一Cd^2＋处理条件下,SOD、POD和CAT活性分别在Cd2＋浓度为1、1和2mg·L1时达到最高值,而后随着Cd^2＋浓度的增加其活性逐渐下降。外施Spd使它们的活性分别提高了5．8％、37．5％和3．3％,并降低了O2^-产生速率和MDA的含量。上述结果表明,Spd增强了荇菜对Cd^2＋毒害的抗性,并在一定程度上缓解了Cd^2＋对荇菜的毒害。     

Preparation and characterization of kappa-carrageenan immobilized urease

Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity.     

化学抑制剂Woodward's Reagent K对来源于Erwinia rhapontici NX-5蔗糖异构酶的抑制动力学

从重组大肠杆菌E.coli BL21(p ET22b-palⅠ)中纯化得到来源于Erwinia rhapontici NX-5的蔗糖异构酶(sucrose isomerase,SIase,EC 5.4.99.11),以纯酶为对象考察其酶活力抑制动力学。结果表明:SIase纯比酶活1 512.77 U/mg,动力学常数Km=260 mmol/L,Vmax=39.41μmol/(L·s)。以化学抑制剂Woodward's Reagent K(WRK)对重组蔗糖异构酶进行抑制反应,反应体系中随着WRK浓度的升高,SIase与底物蔗糖的亲和力常数Km增大,最大反应速度Vmax在一定范围内保持稳定。通过对SIase的抑制动力学分析可得到,WRK对SIase的抑制类型为可逆的竞争性抑制,这可能与WRK与蔗糖的结构类似,与可竞争性的结合SIase的活性中心有关。     

Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport.

Cd2+ and Mn2+ accumulation was studied with wild-type Bacillus subtilis 168 and a Cd2+-resistant mutant. After 5 min of incubation in the presence of 0.1 microM 109Cd2+ or 54Mn2+, both strains accumulated comparable amounts of 54Mn2+, while the sensitive cells accumulated three times more 109Cd2+ than the Cd2+-resistant cells did. Both 54Mn2+ and 109Cd2+ uptake, which apparently occur by the same transport system, demonstrated cation specificity; 20 microM Mn2+ or Cd2+ (but not Zn2+) inhibited the uptake of 0.1 microM 109Cd2+ or 54Mn2+. 54Mn2+ and 109Cd2+ uptake was energy dependent and temperature sensitive, but 109Cd2+ uptake in the Cd2+-resistant strain was only partially inhibited by an uncoupler or by a decrease in temperature. 109Cd2+ uptake in the sensitive strain followed Michaelis-Menten kinetics with a Km of 1.8 microM Cd2+ and a Vmax of 1.5 mumol/min X g (dry weight); 109Cd2+ uptake in the Cd2+-resistant strain was not saturable. The apparent Km value for the saturable component of 109Cd2+ uptake by the Cd2+-resistant strain was very similar to that of the sensitive strain, but the Vmax was 25 times lower than the Vmax for the sensitive strain. The Km and Vmax for 54Mn2+ uptake by both strains were very similar. Cd2+ inhibition of 54Mn2+ uptake had an apparent Ki of 3.4 and 21.5 microM Cd2+ for the sensitive and Cd2+-resistant strains, respectively. Mn2+ had an apparent Ki of 1.2 microM Mn2+ for inhibition of 109Cd2+ uptake by the sensitive strain, but the Cd2+-resistant strain had no defined Ki value for inhibition of Cd2+ uptake by Mn2+.     

甲醇溶液对木瓜蛋白酶催化活性的影响

试验结果表明,木瓜蛋白酶在一定浓度甲醇溶液中水解酪蛋白的活性显著上升;动力学测定表明,该酶在甲醇溶液中Km下降;在甲醇溶液中木瓜蛋白酶催化酪蛋白水解反应的最适pH为8.5,最适温度为70℃;紫外差示光谱研究表明,在甲醇溶液作用下木瓜蛋白酶的二级结构发生了变化;荧光发射光谱研究表明,木瓜蛋白酶在甲醇溶液中发射峰位向低波长移动,荧光峰值明显增高。     

盐生植物盐地碱蓬酪氨酸酶的酶学特性

酪氨酸酶是植物甜菜素生物合成的限速酶,但是,其酶学特性尚不了解。以黑暗培养3d的盐地碱蓬幼苗为材料,采用NaF抽提、饱和硫酸铵沉淀法提取盐地碱蓬中的酪氨酸酶,以研究其酶学特性。结果表明,酪氨酸酶氧化活性的最适温度为35℃,最适pH值为6.6,最适条件下Km=1.09mmol·L-1,Vmax=71.43μmol·g-1（FW）·min-1;酪氨酸酶羟化活性的最适温度为40℃,最适pH值为6.6,最适条件下Km=3.16mmol·L-1,Vmax=0.645μmol·g-1（FW）·min-1。Na2S2O3是盐地碱蓬酪氨酸酶的强效抑制剂,0.05mol·L-1Na2S2O3几乎完全抑制酪氨酸酶氧化及羟化活性。而0.01mmol·L-1的Cu2＋可以显著激活酪氨酸酶的氧化及羟化活性,分别为对照的126%和128.2%。这些结果表明盐地碱蓬中酪氨酸酶的羟化活性是影响甜菜红素合成速率的关键,也为深入研究盐地碱蓬酪氨酸酶在甜菜素合成中的作用及其与环境之间的关系奠定了基础。     

汞、镉对土壤脲酶活性影响的研究Ⅰ.尿素浓度

对不同尿素浓度条件下重金属与土壤脲酶活性关系进行了研究。结果表明,尿素浓度对脲酶活性具有显著的影响。在供试浓度范围内可采用线性和Langmuir模型较好地表征二者关系。并得到脲酶活性的尿浓贡献率,尿浓贡献变化率和最大表观脲酶活性等参数;Hg,Cd明显降低了前述参数值,其中Hg Cd复合污染的抑制作用最强,Hg的生态毒性最大;同时初步获得土壤酶促反应过程中存在吸附机制。     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Differential response of cadmium toxicity towards Mg2(+)-ATPase activity in hepatic and renal nuclear membrane in rats

Treatment with cadmium chloride (40mg/kg body wt/day) for three days led to a marked inhibition of Mg2(+)-ATPase activity in rat liver nuclear membrane, whereas it stimulated the enzyme in renal nuclear membrane. On 30 days treatment (15 mg/kg body wt/day) the effect was totally different i.e. stimulation of enzyme activity in the liver and inhibition in the kidney tissue. Arrhenius plot analysis of the enzyme activity showed significant increase in phase transition temperature only in liver tissue of rats subjected to acute treatment. Lineweaver Burk plots also showed differential effect of cadmium toxicity on the enzyme activity, i.e. while both Km and Vmax were changed in the liver, there was change only in Km of enzyme from kidney.     

谷氨酸棒杆菌YILW苏氨酸脱水酶基因的克隆表达及酶学性质

将L-异亮氨酸生产菌谷氨酸棒杆菌(Corynebacterium glutamicum YILW)苏氨酸脱水酶(threonine de-hydratase,TD)的编码基因ilvA在大肠杆菌中进行异源表达及进行初步的酶学性质研究。分别以C.glutamicum ATCC13032、YILW的基因组DNA为模板,利用PCR技术扩增出苏氨酸脱水酶的编码基因ilvA,测序获得编码序列。利用质粒PET-His将该基因在大肠杆菌BL21(DE3)中进行重组表达、金属螯合纯化,对其酶学性质进行初步研究。结果显示C.glutamicum YILW编码基因序列与已报道的ilvA序列相差5个碱基,相似度为99.6%,第383位氨基酸由苯丙氨酸突变为缬氨酸。酶学性质研究表明:重组酶YilwTD最适反应温度为32℃,在20~55℃范围内该酶较稳定,最适pH为6.7,该酶底物专一性强,对最适底物苏氨酸的米氏常数Km=8.32 mmol/L,最大反应速度Vmax=3.18×104U/mg,与野生型酶相比,突变(F383V)后可显著降低终产物对酶的反馈抑制作用。为揭示突变对苏氨酸脱水酶活性的影响及进一步利用基因工程技术改造L-异亮氨酸生产菌,提高L-异亮氨酸产量奠定了基础。     

枯草芽孢杆菌TM903嘌呤核苷磷酸化酶的纯化及酶学性质研究

目的：对枯草芽孢杆菌TM903嘌呤核苷磷酸化酶进行分离纯化及酶学性质研究。方法：经加热、硫酸铵盐析和SephadexG-100凝胶过滤,对枯草芽孢杆菌TM903中的嘌呤核苷磷酸化酶进行分离纯化,并对其酶学性质进行研究。结果：酶的最适反应温度为65℃,最适反应pH值为7．5,在30-50℃时热稳定性较好;K^＋对该酶有激活作用,而Na^＋、ca^＋、Mg^＋、Mn^＋等金属离子对该酶有抑制作用;Km值为2．11mmol／L,Vmax值为0．84mmol／（min·L）。结论：分离纯化了枯草芽孢杆菌TM903嘌呤核苷磷酸化酶,并研究了其酶学性质,为利巴韦林的发酵工艺优化提供了重要的酶学理论基础。