镁离子对枯草杆菌碱性磷酸酶提取条件的研究

一、前言碱性磷酸酶(EC3.1.3.1.)是一种水解酶,在碱性条件下,能催化磷酸单酯水解,生成无机磷酸。碱性磷酸酶在医学上对某些疾病的诊断有重要意义,在核酸等研究工作中是一种重要的工具酶。目前,碱性磷酸酶多从动物内脏或大肠     

微机在酶促反应动力学中的应用

介绍了微机程序在判断酶促反应抑制类型和计算有关参数方面的应用。     

热动力学法研究间歇反应器中酶促反应动力学

用热动力学法研究了间歇反应器中单底物酶促反应动力学,并详细讨论了产物竞争性抑制作用对反应速率的影响.用微量热法研究了过氧化氢酶催化分解过氧化氢和精氨酸酶催化水解L-精氨酸的反应,实验验证了本文热动力学法研究间歇反应器中单底物酶促反应动力学的正确性.     

枯草杆菌溶栓酶恒溶氧发酵动力学参数估计

用10 L自控搅拌式反应器对枯草杆菌(Bacillus subtilis HL-986)发酵生产溶栓酶的动力学进行了研究，建立了恒溶氧分批培养条件下细胞生长、还原糖消耗及溶栓酶积累的动力学模型.     

蛋白质酶促水解过程集总动力学研究

针对蛋白质酶促水解反应特点，根据水解反应机理，对水解过程进行合理的假设，将水解产物按分子量分段集总，构建出集总反应网络，用包含酶失活，产物抑制和底物抑制的本征动力学方程描述集总组分的反应行为，以牛血清白蛋白－胰蛋白酶反应体系为具体研究对象，建立了4集总动力学模型，根据分层估算的原理，进行了动力学参数的测定，经不同原料组成和反应条件的实验验证，所建立的集总动力学模型具有良好的拟合性和外推性。     

微乳液体系中酶促反应的研究

综述了在微乳液体系中的酶促反应。讨论了表面活性剂、溶剂和助表面活性剂的选择对微乳液中酶促反应的影响及酶在反胶束中的增溶位置。庚烷、辛烷、壬烷是较好的溶剂 ,酶分子可增溶于反胶束中的水池 ,或吸附于油—水界面层极性头微乳液膜附近。描述了微乳液中水含量和缓冲溶液 pH对酶活力的影响 ,酶活力与水含量一般呈钟形曲线 ,与 pH可呈钟形或S形曲线。     

酶促反应结晶研究进展

在“双碳”背景下,绿色可持续的酶促反应正受到工业界的广泛关注,但在实际应用中仍面临着诸多挑战,如反应平衡的限制、不稳定产物的分解、酶的产物抑制等。结晶作为一种高效成熟的分离技术,可通过移除液相产物的方式有效解决上述问题。同时,结晶也是晶体产品的“生成”过程,其与酶促反应耦合可一步实现晶体产品的高效、绿色、可控制备。综述了近年来酶促反应结晶的研究进展,介绍了原位产物结晶（ISPC）技术的发展历程,并讨论了结晶与酶促反应耦合时的相互影响关系;从结晶方式和过程控制角度阐述了酶促反应结晶的实现形式和连续化过程;最后,对酶促反应结晶这一耦合过程的发展和应用进行了总结和展望。     

噬菌体单链抗体碱性磷酸酶细胞ELISA检测方法的建立

目的建立噬菌体单链抗体碱性磷酸酶(alkaline phosphatase,ALP)细胞ELISA检测方法,以期排除外周血中白细胞的干扰,将筛选到的噬菌体抗体用于临床样本的检测。方法从大容量噬菌体抗体库中筛选与食管癌细胞结合的噬菌体单链抗体,制备食管癌细胞KYSE-170和EC109鉴定板及白细胞鉴定板,采用ALP细胞ELISA法分析筛选到的噬菌体抗体与两种食管癌细胞及白细胞的结合活性,同时与辣根过氧化物酶(horse radish peroxidase,HRP)细胞ELISA法进行比较,以抗卵清蛋白(ovalbumin,OA)为阴性对照。结果经ALP细胞ELISA法检测,筛选到的噬菌体抗体1、2、3均可与KYSE-170和EC109细胞结合,A405值在检测范围内;3个噬菌体抗体与不同食管癌细胞系的结合活性差异有统计学意义(P<0.001);抗OA抗体与两种食管癌细胞的结合活性明显低于3个噬菌体抗体,且差异有统计学意义(P<0.001);3个噬菌体抗体与白细胞结合的A405值在检测范围内,与食管癌细胞系的结合活性相比,差异有统计学意义(P<0.01)。结论 ALP细胞ELISA法可鉴定噬菌体单链抗体,并可排除白细胞的干扰,有望应用于临床样本的检测。     

酶促吉西他滨乙酰化反应的研究

探讨了八种不同来源水解酶催化吉西他滨乙酰化反应的催化活性,结果发现,有四种脂肪酶能够催化该酰化反应,且它们的产物只有一种,其中脂肪酶Novozyme435的催化活性最高。此外,通过13C-NMR和MS等技术手段对酶促反应产物的结构进行鉴定,发现它们以催化吉西他滨5’-羟基酰化反应为主,且反应选择性均高于99%。     

次磷酸钠制备及反应动力学研究

探讨了一步法生产次磷酸钠的方法，可间歇生产，也可连续生产，并将有毒的副产物PH3转变成目的产品次磷酸钠。最佳反应条件：反应温度70℃；黄磷，氢氧化钠和过氧化氢的摩尔比为2：2：1，反应时间为5h。此法可将产的收率从两步法的30％提高到71％以上。通过反应动力学研究可知，一步法生产次磷酸钠的总反应为一级反应，反应速率常数为k=111519exp(-40697.3/RT)。     

Cross-Flow Microfiltration of Bacillus Subtilis Broths under Various Culture Times

Bacillus subtilis broths under different culture times are filtered in a cross-flow microfilter. The operating condition effects, such as cross-flow velocity, transmembrane pressure, and broth culture time, on the filtration flux, cake properties, and extracellular polymeric substances (EPS) transmissions are discussed thoroughly. The culture broths contain B. subtilis cells and EPS which is characterized as polysaccharides (hydrocarbons) and proteins. An increase in broth culture time leads to higher concentrations of cells, soluble and extractable EPS. The total protein to polysaccharide concentration ratio in the broths is ca 0.2. However, the soluble polysaccharide concentration is 10-fold higher than that of soluble proteins. The filtration flux increases with increasing cross-flow velocity or transmembrane pressure. However, the impact of cross-flow velocity is more significant. The filter cake resistance formed by B. subtilis cells and EPS flocs plays the most important role in determining the overall filtration resistance. The mass and average specific filtration resistance of cake can be estimated using a force balance model and empirical equations. The cake structure and thickness are analyzed using SEM. A thicker and more compact cake may be formed under longer broth culture time. Most soluble polysaccharide and protein molecules have the opportunity to penetrate through the cake and membrane into the filtrate because the solute transmissions are measured as high as 0.75–1.0. The influences of operating conditions on the polysaccharide and protein transmissions are negligible. Therefore, to enhance filtration flux by increasing transmembrane pressure or cross-flow velocity is beneficial to improve separation efficiency, especially by increasing cross-flow velocity.     

基于脯氨酸理论提高枯草芽孢杆菌脂肪酶A的热稳定性

以枯草芽孢杆菌脂肪酶A(LipA)为研究对象,根据从RCSB数据库中获取的晶体结构,采用分子动力学模拟和分子生物学实验相结合的方法进行脂肪酶热稳定性位点突变的理性设计。首先,利用分子动力学模拟获得晶体结构中柔性较高的Loop区域;进而,结合"脯氨酸理论",将位于该区域附近的Gly残基突变为Pro,分析引入Pro突变对LipA热稳定性的影响,筛选得到Gly52和Gly158两个突变位点;最后,通过定点突变操作对突变株LipAG52P和LipAG158P进行热稳定性实验验证。结果显示,突变株LipAG52P、LipAG158P的比活力分别是野生型LipA的5.6倍和2.7倍,Tm值分别提高了15℃和7℃,催化效率分别提高了85%和22%。     

Purification and Characterization of Highly Alkaline Lipase from Bacillus licheniformis MTCC 2465: and Study of its Detergent Compatibility and Applicability

An extracellular alkaline lipase from Bacillus licheniformis MTCC 2465 has been studied and analyzed for its applicability as a detergent additive. The lipase obtained from Bacillus licheniformis MTCC 2465 was purified by ammonium sulfate fractionation and gel filtration chromatography. The enzyme was precipitated with a 60 % saturated ammonium sulfate fraction showed 6.73 fold purification with the fold purification of 10.4 and specific activity of 398 U/mg of protein with gel filtration chromatography. The optimal pH and temperature for activity were 10 and 60 °C respectively. The enzyme was found to be stable in the pH range of 8–11 with 90 % retention of activity at pH 11. The enzyme retained 90 % activity at 60 °C and 70 % of activity at 70 °C for 1 h. The enzyme activity was maximally enhanced by Hg²⁺ followed by Co²⁺ and Fe²⁺. The lipase was found to be stable in organic solvents with maximum stability in acetone followed by ethanol. The lipase exhibited remarkable stability in the presence of commercial detergents and found to be stable in bleaching agents. Wash performance analysis resulted in improvement of 10 % more grease removal ability of the present lipase in comparison to commercially available lipase.     

枯草芽孢杆菌BS-2018在海参养殖中的应用效果

在东经122°21′,北纬39°34′大连黄海海域的底泥中分离得到一株海洋细菌,经系统的微生物学鉴定及16SrDNA序列测定,定名为枯草芽孢杆菌BS-2018(Bacillus subtilis)。将该菌制成微生物制剂(200×108 cfu/mL),在海参浮游期(10～15 mL/m3水体/d泼洒)、稚幼参期(饵料中1%～2%添加和5～10 mL/m3水体/d泼洒)、越冬苗保苗期(饵料中1%～2%添加和5～10 m L/m3水体/d泼洒)、成参养殖期(腐植酸钠5～10 g和10～15 m L/m3水体/15 d泼洒)进行应用试验。结果表明：该菌株安全可靠,能使育苗浮游期海参成活率分别提高到22.3%、35.3%,发病率分别降低到18.0%、26.0%;稚幼参期增产15.25%～18.14%;越冬苗保苗期增产11.18%～16.77%;成参养殖期增产11.32%～17.82%。枯草芽孢杆菌BS-2018生物制剂在海参育苗和养殖中应用具有广阔的前景。     

Kinetic Studies of Site-Specifically and Randomly Immobilized Alkaline Phosphatase on Functionalized Membranes

A comparison of enzyme activities has been made between a site-specifically immobilized and a randomly immobilized bacterial alkaline phosphatase (BAP) on macroporous membranes. An octapeptide tag (FLAG™) was attached at the N-terminus of alkaline phosphatase by recombinant DNA techniques (gene fusion) to yield BAP that is modified in a site-directed fashion (SDBAP). The corresponding antibody (antiFLAG™) was immobilized on an aldehyde-modified polyethersulfone (MPS) membrane via protein A. Immobilization of SDBAP on this membrane result in a membrane–protein A–antiFLAG–SDBAP linkage. This site-specifically immobilized enzyme demonstrated a relative activity (RA), defined as the ratio of immobilized activity (V_max) to the corresponding homogeneous enzymatic activity, of 85% as compared with the randomly immobilized BAP which had an RA of 0·8%. BAP, when chemically conjugated to the FLAG peptide and immobilized via antiFLAG and protein A on the MPS membrane, showed an RA of only 1·9%, demonstrating the effectiveness of site-directed immobilization. SDBAP was also immobilized on the MPS membrane in the absence of protein A. In this case, the RA dropped to 22%, further explaining the effectiveness of ordered immobilizations as compared with random immobilizations. The ratio of immobilized enzyme activity to the activity in the absence of added phosphate inhibitor for the immobilized BAP was three-fold higher than the corresponding homogeneous ratio, showing a reduction in product inhibition for the immobilized enzyme. © 1997 SCI.     

Alkaline Hydrolysis of an Aromatic Ester: Kinetic Studies Using Thermal Power Profiles

The reaction kinetics of ethyl benzoate saponification with sodium hydroxide in a heterogeneous reaction medium was investigated. The rate of reaction in a heterogeneous liquid‐liquid system incorporates a chemical kinetics term as well as the mass transfer between both phases. The reaction was carried out in a reaction calorimeter and the overall reaction rate was determined from the obtained thermal profiles. The mass transfer parameters were calculated numerically, taking into account those reaction rate constants determined in our previous study, where the above‐mentioned reaction was conducted in a homogeneous reaction medium. The dependence of the mass transfer parameters and reaction rates on different heterogeneity stages and temperatures is described.     

Bacillus Mycoides磷脂酶C部分特性研究

对一种纯化自Bacillus mycoides 970发酵上清液的磷脂酶C的部分特性进行了研究.SDS-PAGE检测表明该纯化磷脂酶C由一条多肽链组成,分子量为75.1 kDa.热稳定性实验表明该纯化磷脂酶C对温度敏感,具有热不稳定性.金属离子依赖性检测表明该纯化磷脂酶C属于金属磷脂酶C家族,Ca2 、Mg2 、Zn2 和Ba2 对该磷脂酶C催化活性的发挥必不可少.血平板及卵黄平板检测表明该磷脂酶C没有溶血活性和卵磷脂酶活性.     

辉锑矿碱性湿法制备焦锑酸钠的研究

联系我国锑矿资源的特点,对过氧化氢在碱性介质中直接氧化辉锑矿硫化碱的浸出液制取焦锑酸钠的工艺作了重点的研究     

枯草芽孢杆菌BSD-2菌株喷雾干燥工艺优化

为研制生物农药新剂型,利用喷雾干燥法制备浓缩枯草芽孢杆菌BSD-2菌粉。以集粉率和抑菌活性为指标,确定最佳喷雾干燥工艺条件。在发酵液中加入15%β-环糊精作为填料,最佳入口温度为170℃,料液温度为30℃,热风量为20 m3/h,入料流量为15 m L/min。此条件下喷雾集粉率达到25.4%,效价保留率为66%,BSD-2菌体存活率为75%。