醇腈酶:生物与化学的结合点

辞腈酶是用途较广的一类酶,在植物体内抵御微生物侵袭方面起着重要作用。在化学工业,醇腈酶用于合成手性氰醇的生物催化剂。氰醇是合成β-氨基醇类,α-羟基酮类及酸类化合物的重要生物活性物质,这些化合物又是精细化工、制药和农用化学品领域的重要原料和中间体。研究人员已经利用蛋白质工程和定点突变技术来改变各种醇腈酶的活性位点和底物特异性等。许多重组醇腈酶业已在大肠杆菌,啤酒酵母和甲醇酵母中成功表达。     

耦合末端盐桥与定点突变的重组腈水合酶催化动力学

双亚基腈水合酶是催化丙烯腈水合生产丙烯酰胺的重要工业酶。通过Discovery Studio 2.5软件对C-末端盐桥耦合定点突变改造的耐热型基因重组腈水合酶NHaseM-TH-SBM(S344K-S346K-L347E-N362S-435DT436(+))进行盐桥网络分析,发现其全局盐桥总数有所下降,但双亚基界面间盐桥数量提高到7个。在重组大肠杆菌中表达了原腈水合酶NHaseM-TH(TH)、盐桥突变酶NHaseM-TH-SB(SB)和盐桥耦合定点突变的重组酶NHaseM-TH-SBM(SBM),研究了3种酶的催化反应动力学和失活动力学。结果表明,SBM的腈水合酶活性为543.9 U·mg?1,比TH提高了31.0%;其米氏方程催化速率常数Kcat比TH提高了20%。SBM的表观失活常数KD在25~42℃范围内均明显低于对照TH,在42℃时为TH的68.0%,显示了良好的热稳定性。SBM是活性和稳定性同时提高的重组腈水合酶。     

醇腈酶在不对称催化合成氰醇中的应用

醇腈酶在化学合成中催化碳碳键的形成,其产物氰醇可以转化成多种重要的手性医药和农药中间体.从天然产物中可以提取能产生R和S两种构型氰醇的醇腈酶.通过基因重组和在微生物宿主体内过量表达,实现了醇腈酶的工业化生产.对醇腈酶作为催化剂用于不对称催化氢氰酸和羰基化合物反应生成氰醇类手性化合物的工业化应用进行了介绍.     

耦合末端盐桥与定点突变的重组腈水合酶催化动力学

双亚基腈水合酶是催化丙烯腈水合生产丙烯酰胺的重要工业酶。通过Discovery Studio 2.5软件对C-末端盐桥耦合定点突变改造的耐热型基因重组腈水合酶NHase^M-TH-SB^M（S344K-S346K-L347E-N362S-435DT436（+））进行盐桥网络分析,发现其全局盐桥总数有所下降,但双亚基界面间盐桥数量提高到7个。在重组大肠杆菌中表达了原腈水合酶NHase^M-TH（TH）、盐桥突变酶NHase^M-TH-SB（SB）和盐桥耦合定点突变的重组酶NHase^M-TH-SB^M（SB^M）,研究了3种酶的催化反应动力学和失活动力学。结果表明,SB^M的腈水合酶活性为543.9 U·mg^-1,比TH提高了31.0%;其米氏方程催化速率常数K_cat比TH提高了20%。SB^M的表观失活常数K_D在25～42℃范围内均明显低于对照TH,在42℃时为TH的68.0%,显示了良好的热稳定性。SB^M是活性和稳定性同时提高的重组腈水合酶。     

微生物法生产丙烯酰胺的生物催化剂——腈水合酶研究进展

回顾了近年来国内外在微生物法生产丙烯酰胺的生物催化剂腈水合酶的结构、催化机理、光活性、热稳定性等方面的研究进展 .腈水合酶的活性部位含有螯合的金属离子作为辅助因子 ;结合金属离子的活性中心在腈水合酶的催化反应中起着重要的作用 ;其中铁型腈水合酶具有光活性 ,其活性是通过NO调节的 :在非活性腈水合酶的铁中心上连有一个内生的NO分子 ,在光的作用下 ,NO释放出来 ,使酶的活性恢复 .进一步探讨了温度对不同菌株酶活稳定性以及氨基化合物等因素对酶活性的影响 ,并对研究的发展方向提出了一些设想     

生物法生产丙烯酰胺过程中腈水合酶的抑制和失活

为改进生物法生产丙烯酰胺工艺,研究了腈水合酶催化丙烯腈水合生产丙烯酰胺过程中影响腈水合酶反应速率和酶失活的因素. 实验证明,水合过程中体系pH值的变化基本不影响酶反应速率;底物丙烯腈的浓度低于10 g/L时,酶反应速率与底物浓度成正比,大于75 g/L后,对酶有抑制作用;产物丙烯酰胺显著抑制腈水合酶的活性;菌体细胞内可能存在可以稳定腈水合酶的物质,胞内的腈水合酶在40℃下的半衰期可以达到59.9 h;丙烯酰胺与温度的协同作用是腈水合酶失活的重要原因.     

重组降血压肽在大肠杆菌中的高效表达

目的获得具有高活性的重组降血压多肽。方法人工合成高活性降血压肽单体,经酶切位点串联,克隆至表达载体,并转化宿主菌BL21进行诱导表达。结果经酶切、PCR和测序均证明,串联的基因ACEIP已成功克隆至pGEX-4T-2表达载体中,融合蛋白GST-ACEIP的表达水平达24·6%,表达产物的融合蛋白和多肽含量分别为1·1g/L和0·14g/L,其抑制活性达85%。结论已成功制备出高活性的重组降血压多肽,为进一步开发降血压药物奠定基础。     

翻译调控肿瘤蛋白的原核表达及纯化

目的原核表达并纯化翻译调控肿瘤蛋白(Translationally controlled tumor protein,TCTP)。方法以人乳腺癌细胞系MCF-7总RNA为模板,PCR扩增TCTP基因,克隆至原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达。His Trap FF层析柱纯化重组蛋白后,进行Western blot鉴定。结果克隆的目的基因序列正确,未发生碱基突变;重组表达质粒经双酶切鉴定构建正确;表达的重组蛋白相对分子质量约为20 000,主要以包涵体形式表达;纯化后纯度为80%,可与兔抗人TCTP多克隆抗体特异性结合。结论已成功在大肠杆菌BL21(DE3)中表达并纯化了TCTP,为进一步研究其在肿瘤等疾病发生、发展及治疗中的作用奠定了基础。     

人溶菌酶-木聚糖酶融合基因在毕赤酵母中的表达

目的在毕赤酵母中表达人溶菌酶(Human lysozyme,hLY)-木聚糖酶(Xylanases,XynⅡ)融合基因。方法通过PCR技术将hLY基因与XynⅡ基因连接,中间插入肠激酶识别位点序列;将其克隆至载体pPIC9K上,构建重组表达质粒pPIC9K-XynⅡ-EKsite-hLY,经SacⅠ线性化后,电转化毕赤酵母GS115,通过G418抗性筛选得到高拷贝转化子,PCR鉴定为阳性的克隆用甲醇进行诱导;表达的融合蛋白经Sephadex G-75凝胶柱层析纯化后,用肠激酶酶切,分别采用改良舒加法和DNS法测定hLY和XynⅡ的活性。结果获得的融合基因序列与理论序列完全一致;重组表达质粒构建正确;融合蛋白的hLY和XynⅡ活性分别为170和158 U/ml,经肠激酶酶切后,hLY的活性为520 U/ml,XynⅡ的活性达244 U/ml。结论已在毕赤酵母中成功表达了XynⅡ-EKsite-hLY融合基因,经肠激酶酶切后的目的蛋白活性均有较大提高。     

小鼠脱氧核糖核酸酶Ⅱα融合基因的原核表达及多克隆抗体的制备

目的原核表达LHRH-TAT-DNaseⅡα融合基因(LTD),并制备多克隆抗体。方法采用PCR法克隆LTD基因片段,插入p ET-28a载体,构建重组原核表达质粒p ET-28a-LTD,转化大肠埃希菌BL21(Rossetta),经IPTG诱导表达。融合蛋白经电洗脱分离纯化后,免疫獭兔,制备LTD蛋白多克隆抗体,采用ELISA法检测抗血清效价。结果构建的重组原核表达质粒经PCR、双酶切及测序鉴定证明构建正确;纯化得到的融合蛋白相对分子质量为39 270,浓度为0.5 mg/ml,可被兔抗LTD抗体识别。血清抗体效价可达1∶6 400。结论成功原核表达了DNaseⅡ融合蛋白LTD,并制备了多克隆抗体,为进一步研究DNaseⅡ蛋白体内外抗肿瘤活性及作用机制奠定了基础。     

Many Drugs of Abuse May Be Acutely Transformed to Dopamine,Norepinephrine and Epinephrine In Vivo

It is well established that a wide range of drugs of abuse acutely boost the signaling of the sympathetic nervous system and the hypothalamic–pituitary–adrenal (HPA) axis, where norepinephrine and epinephrine are major output molecules. This stimulatory effect is accompanied by such symptoms as elevated heart rate and blood pressure, more rapid breathing, increased body temperature and sweating, and pupillary dilation, as well as the intoxicating or euphoric subjective properties of the drug. While many drugs of abuse are thought to achieve their intoxicating effects by modulating the monoaminergic neurotransmitter systems (i.e., serotonin, norepinephrine, dopamine) by binding to these receptors or otherwise affecting their synaptic signaling, this paper puts forth the hypothesis that many of these drugs are actually acutely converted to catecholamines (dopamine, norepinephrine, epinephrine) in vivo, in addition to transformation to their known metabolites. In this manner, a range of stimulants, opioids, and psychedelics (as well as alcohol) may partially achieve their intoxicating properties, as well as side effects, due to this putative transformation to catecholamines. If this hypothesis is correct, it would alter our understanding of the basic biosynthetic pathways for generating these important signaling molecules, while also modifying our view of the neural substrates underlying substance abuse and dependence, including psychological stress-induced relapse. Importantly, there is a direct way to test the overarching hypothesis: administer (either centrally or peripherally) stable isotope versions of these drugs to model organisms such as rodents (or even to humans) and then use liquid chromatography-mass spectrometry to determine if the labeled drug is converted to labeled catecholamines in brain, blood plasma, or urine samples.     

Insect antifeedant properties of anthranoids from the genusVismia

Vismiones and ferruginins, representatives of a new class of lypophilic anthranoids from the genusVismia were found to inhibit feeding in larvae of species ofSpodoptera, Heliothis, and inLocusta migratoria.     

ICP-MS法测定地球化学样品中As、Cr、Ge、V等18种微量痕量元素的研究

建立了测定地球化学样品中包括As、Cr、Ge、V等18种微量、痕量元素的ICP-MS方法。地化试样用HF-HNO3混酸分解后,以1 1 HNO3溶解干渣。由于制样不使用盐酸,避免了Cl对As、Cr、Ge、V的质谱干扰。用国家一级地球化学标准物质GBW 07309制备溶液优化仪器工作参数,并用于校准。方法测定限(6s)为:0.007~6.4μg/g,精密度(RSD%,n=12)为:29%~9.4%,经过国家一级地球化学标准物质的分析验证,结果与标准值吻合。方法已应用于国土资源调查的试样分析。     

Direct observation of freeze-thaw instability of latex coatings via high pressure freezing and cryogenic SEM

Despite its industrial importance, the subject of freeze-thaw (F/T) stability of latex coatings has not been studied extensively. There is also a lack of fundamental understanding about the process and the mechanisms through which a coating becomes destabilized. High pressure (2100 bar) freezing fixes the state of water-suspended particles of polymer binder and inorganic pigments without the growth of ice crystals during freezing that produce artifacts in direct imaging scanning electron microscopy (SEM) of fracture surfaces of frozen coatings. We show that by incorporating copolymerizable functional monomers, it is possible to achieve F/T stability in polymer latexes and in low-VOC paints, as judged by the microstructures revealed by the cryogenic SEM technique. Particle coalescence as well as pigment segregation in F/T unstable systems are visualized. In order to achieve F/T stability in paints, latex particles must not flocculate and should provide protection to inorganic pigment and extender particles. Because of the unique capabilities of the cryogenic SEM, we are able to separate the effects of freezing and thawing, and study the influence of the rate of freezing and thawing on F/T stability. Destabilization can be caused by either freezing or thawing. A slow freezing process is more detrimental to F/T stability than a fast freezing process; the latter actually preserves suspension stability during freezing. Presented at the 82nd Annual Meeting of the Federation of Societies for Coatings Technology, October 27–29, 2004 in Chicago, IL. Tied for first place in The John A. Gordon Best Paper Competition.     

Ethanol and (−)-α-Pinene: Attractant Kairomones for Bark and Ambrosia Beetles in the Southeastern US

In 2002–2004, we examined the flight responses of 49 species of native and exotic bark and ambrosia beetles (Coleoptera: Scolytidae and Platypodidae) to traps baited with ethanol and/or (−)-α-pinene in the southeastern US. Eight field trials were conducted in mature pine stands in Alabama, Florida, Georgia, North Carolina, and South Carolina. Funnel traps baited with ethanol lures (release rate, about 0.6 g/day at 25–28°C) were attractive to ten species of ambrosia beetles (Ambrosiodmus tachygraphus, Anisandrus sayi, Dryoxylon onoharaensum, Monarthrum mali, Xyleborinus saxesenii, Xyleborus affinis, Xyleborus ferrugineus, Xylosandrus compactus, Xylosandrus crassiusculus, and Xylosandrus germanus) and two species of bark beetles (Cryptocarenus heveae and Hypothenemus sp.). Traps baited with (−)-α-pinene lures (release rate, 2–6 g/day at 25–28°C) were attractive to five bark beetle species (Dendroctonus terebrans, Hylastes porculus, Hylastes salebrosus, Hylastes tenuis, and Ips grandicollis) and one platypodid ambrosia beetle species (Myoplatypus flavicornis). Ethanol enhanced responses of some species (Xyleborus pubescens, H. porculus, H. salebrosus, H. tenuis, and Pityophthorus cariniceps) to traps baited with (−)-α-pinene in some locations. (−)-α-Pinene interrupted the response of some ambrosia beetle species to traps baited with ethanol, but only the response of D. onoharaensum was interrupted consistently at most locations. Of 23 species of ambrosia beetles captured in our field trials, nine were exotic and accounted for 70–97% of total catches of ambrosia beetles. Our results provide support for the continued use of separate traps baited with ethanol alone and ethanol with (−)-α-pinene to detect and monitor common bark and ambrosia beetles from the southeastern region of the US.     

基于ATRP法制备的触角状亲水性羟胺树脂的吸硼性能的研究

以大分子引发剂氯乙酰化聚苯乙烯微球(PS-acyl-Cl)经原子转移自由基聚合(ATRP)法引发丙烯酰胺(AM)和甲基丙烯酸缩水甘油酯(GMA)单体的共聚接枝,制得一种触角状亲水性环氧载体(PS-acyl-g-P(AM-co-GMA)),再经二乙醇胺(DEA)的环氧基开环胺化反应,得到一种含多个-NCH2CH2OH螯合配基的多齿-五元螯合环的触角状亲水性羟胺树脂(PS-acyl-g-P(AM-co-GMA)-DEA)。将此树脂用于硼吸附研究,结果表明,PS-acyl-g-P(AM-co-GMA)-DEA树脂对硼的吸附满足Langmuir方程,为单分子层吸附;饱和吸附量约为37.7 mg·g-1,且树脂5 min即可达到吸附平衡,与其它已报道的吸硼树脂相比,该树脂具有更高的吸附量和吸硼速率。吸附动力学研究表明,树脂吸附硼的过程主要由颗粒扩散过程控制。重复使用5次后该树脂的吸附量基本不变,解吸率均在90%以上,重复使用性能良好。