金属有机框架材料固定化酶的研究进展

酶催化由于具有特异性强、催化效率高和环境友好等优点,已被广泛应用于多种化学和生物转化过程。传统的固定化酶技术可以扩大生物酶在工业生产中的应用范围,但通常存在载体负载效率低、催化活性低及酶的构象易改变等问题。近年来,具有高比表面积、高孔隙率、高稳定性和功能多样的金属有机框架材料(MOFs)作为新型的酶固定化材料引起了研究人员的广泛兴趣,因为固定后的酶@MOFs复合物比游离酶具有更好的催化性、稳定性、选择性及可回收性。为了对此新兴领域进行系统化梳理,本文从基于MOFs的酶固定化方法、影响酶固定化效果的主要因素、MOFs在酶固定化中的应用以及MOFs的毒性等方面对MOFs固定化酶领域的最新研究进展进行了综述,并分析了MOFs固定化酶领域现存的问题和与未来的发展前景。     

多级孔金属-有机框架固定化酶的研究进展

金属-有机框架(metal-organic frameworks, MOFs)作为酶固定化的优良载体,为生物催化反应提供优越的物理和化学保护。近年来,多级孔金属-有机框架(hierarchical porous metal-organic frameworks, HP-MOFs)由于其独特的结构优势,在固定化酶方面显示出更大的潜力。到目前为止,已经开发了各类具有原生多级孔或缺陷多级孔的HP-MOFs用于酶的固定化研究,并且使得固定化酶在催化活性、稳定性和重复利用性等方面得到了显著增强。本文系统总结了HP-MOFs用于固定化酶的各种策略,介绍了HP-MOFs固定化酶(enzyme@HP-MOFs)在催化合成、生物传感、生物医药等领域的最新应用进展。最后,讨论并展望了HP-MOFs固定化酶这一领域所面临的挑战和机遇。     

固定化细胞生物催化制备(S)-苯基乙二醇的研究

采用海藻酸钙固定化细胞方法能有效提高近平滑假丝酵母催化(RS)-苯基乙二醇(S-PED)去消旋化反应的稳定性,在优化反应条件下,固定化细胞催化批次由游离细胞的2次提高至6次,产物可保持高光学纯度(>95%)、高产率(>85%)。与化学合成液晶掺杂剂S-1011的理化特性对比结果表明,生物催化法制备获得的(S)-PED能够替代化学法制备的相同产品。     

金属有机框架纳米酶的研究进展

纳米酶作为一种具有类酶活性的纳米材料,与天然酶相比,具有制备过程简单、受外界环境干扰小、对酸碱和温度具有较好的耐受性等优点.金属有机框架(metal-organic frameworks,MOFs),即多孔配位聚合物,具有结构多样性、高比表面积、孔隙率可控等独特性质.因有序框架的保护以及结构可调控的性质,基于MOFs构建的纳米酶受到研究人员的广泛关注.本文综述不同类型MOFs基的纳米酶,主要从原始MOFs、化学修饰的MOFs、MOFs基复合材料和MOFs衍生物等四大方面进行论述;随后,对4种类型MOFs基纳米酶的构建特点和生化分析应用进行归纳和比较;最后对其当前面临的挑战和未来的发展趋势进行讨论.     

乙醇脱氢酶与葡萄糖脱氢酶偶联催化制备(S)-1-(2,6-二氯-3-氟苯基)乙醇

(S)-1-(2,6-二氯-3-氟苯基)乙醇是抗癌药物克唑替尼的手性合成前体,可由2,6-二氯-3-氟苯乙酮经乙醇脱氢酶催化还原制备,还原中所需的还原型辅酶Ⅱ再生是该反应的技术瓶颈.本研究构建重组大肠杆菌E.coli BL21-ADH和E.coli BL21-GDH,实现了葡萄糖脱氢酶和乙醇脱氢酶的共表达,并进行偶联转化.结果表明,当在反应温度为30℃,pH为7的条件下,(S)-l-(2,6-二氯-3-氟苯基)乙醇的产量达到最高,在投料量为6％时,该体系转化率为93.75％.     

半胱氨酸辅助的酶@ZIF-8固定化酶制备及其特性研究

以共沉淀包埋的策略将酶固定在ZIF-8金属有机骨架中能显著提高酶的催化稳定性,但是,所制备的酶@ZIF-8固定化酶的固定化效率和酶活回收率却很低.为了解决这一问题,本研究以半胱氨酸(cysteine,Cys)为辅助剂,采用共沉淀包埋的策略将苯丙氨酸解氨酶(PAL)固定在ZIF-8载体中,制备出PAL@ZIF-8固定化酶...     

基于金属有机框架材料酶的固定化策略及其应用

金属有机骨架(MOFs)是由有机配体和金属离子或团簇通过配位键自组装形成的具有分子内孔隙的有机无机杂化材料,由于其比表面积大和化学稳定性、可调控的孔隙以及多样性被越来越多地用于生物分子的装载尤其是酶的固定化领域.本文综述了近年来国内外制备MOFs以及MOFs基的复合材料固定化酶的制备方法、改进策略和应用,并对MOFs基...     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

固定化β-葡萄糖苷酶催化合成红景天甙的研究

目的:利用海藻酸钠和壳聚糖固定β-葡萄糖苷酶,催化合成红景天甙,并对固定化条件的选择、固定化酶催化合成红景天甙的条件优化进行研究.方法:采用正交实验方法寻求最佳固定化条件,以转化率为指标对合成条件进行优化.结果:固定β-葡萄糖苷酶的最佳条件为:壳聚糖浓度1.5%,吸附时间9h,交联时间12h,戊二醛浓度1.0%,吸附温度O℃,酶活力回收率达74.38%.催化合成红景天甙的最佳条件为:反应介质pH 5.8醋酸缓冲液/叔丁醇(1:9),底物浓度酩醇5g/L,D-葡萄糖与酪醇摩尔比为1:1,反应时间50h,反应温度50℃,红景天甙的转化率最高可达到71.9%.结论:固定化酶催化合成红景天甙的转化率得到较大的提高,为工业化生产提供了可靠的理论依据.     

基于多酶级联协调表达策略高效催化合成(S)-2-羟基丁酸

2-羟基丁酸(2-hydroxybutyric acid,2-HBA)是合成生物可降解材料和各种药物的重要中间体,化学法合成的外消旋2-HBA需要去消旋才能获得光学纯对映异构体,应用于工业.文中通过在大肠杆菌Escherichia coli BL21(DE3)中共表达苏氨酸脱氨酶(Threonine deaminase...     

Synthesis of enantiopure (S)-6-chlorochroman-4-ol using whole-cell Lactobacillus paracasei biotransformation

Chromane, which has a fused cyclic structure, is a significant molecule that can be found in the structure of many important compounds. Lactobacillus paracasei BD101 was demonstrated as whole-cell biocatalyst for the synthesis of (S)-6-chlorochroman-4-ol with immense enantioselectivity. The conditions of asymmetric reduction were optimized one factor by one factor using L paracasei BD101 to achieve enantiomerically pure product and complete conversion. Using these obtained optimization conditions, asymmetric reduction of 6-chlorochroman-4-one was performed under environmentally friendly conditions; 6-chlorochroman-4-one, having a fused cyclic structure as previously noted to be difficult to asymmetric reduction with biocatalysts, was enantiomerically reduced to (S)-6-chlorochroman-4-ol with an enantiomeric excess >99% on a high gram scale. This study is the first example in the literature for the enantiopure synthesis of (S)-6-chlorochroman-4-ol using a biocatalyst. Also notably, the optical purity of (S)-6-chlorochroman-4-ol obtained in this study through asymmetric bioreduction using whole-cell biocatalyst is the highest value in the literature. In this study, (S)-6-chlorochroman-4-ol was produced on a gram scale by an easy, inexpensive, and environmentally friendly method, which has shown the production of valuable chiral precursors for drug synthesis and other industrial applications. This study provides a convenient method for the production of (S)-6-chlorochroman-4-ol, which can meet the industrial green production demand of this chiral secondary alcohol.     

（S）-（-）-β-羟基苯丙酸乙酯的微生物法合成

采用酿酒酵母CGMCC No．2266菌体,不对称还原β-羰基苯丙酸乙酯制备光学纯（S）-（-）-β-羟基苯丙酸乙酯。结果表明：采用初始pH为8．0的液体发酵培养基培养的CGMCCNo．2266菌体经过50℃预热处理30min后用于生物转化获得的（S）-（-）-G-羟基苯丙酸乙酯对映体过剩值可以达到100％ee。确定了合成（S）-（-）-β-羟基苯丙酸乙酯的较佳转化条件为pH7．0,温度30℃,转化时间24h,底物浓度为3．63mmol／L,菌体用量为86g/L（干重／反应体积）。以10％葡萄糖为辅助底物,产率比不加辅助底物时提高了75．4％。在最佳转化条件下反应转化率及（S）-（-）-β-羟基苯丙酸乙酯对映体过剩值可分别达到98．4％和100％ee。     

微生物酶不对称水解合成S（＋）—布洛芬的研究：Ⅱ.反应条件和产物提取

皮状丝孢酵母具有较强不对称水解底物专一性。在试验的五种布洛芬消旋酯中,水解甲酯和异丙酯生成Ｓ（＋）－布洛芬ｅｅ可达９７％,乙酯为９３％以上;而水解活性以乙酯最强。转化率高于３０５。不对称水解最适ｐＨ６．５－７．０;温度在２８－３７℃范围内拆分能力无明显差别。该酵母的水解酶为胞内酶,将酵母细胞制成丙酮干粉进行水解可提高立体专一性。产物Ｓ（＋）－布洛芬可借助于酸碱反应和有机溶剂提取得到,同时回收未水解     

微生物酶不对称水解合成S(+)-布洛芬的研究 Ⅰ.高度立体选择性菌株的筛选

从361株细菌和酵母菌中分别筛选到12株可不对称水解布洛芬乙酯生成R(-)-布洛芬的细菌(5株菌对映体过量(ee)可达85%)和15株具有不对称水解布洛芬乙酯活性的酵母菌,其中皮状丝孢酵母T158生成S( )-布洛芬,ee可超过92%.该酵母菌最适碳源为葡萄糖,浓度以1.0—1.5%适宜,蛋白胨浓度低于0.3%或高于0.5%对水解拆分均不利.酵母膏的加入显著提高水解活性,最适浓度0.3%.在培养基中添加表面活性剂吐温80(0.2%)既可提高拆分专一性,又能增强水解能力.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.     

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

Pharmacokinetic data of propranolol enantiomers in a comparative human study with (S)- and (R,S)-propranolol

The pharmacokinetics of (S)-propranolol were compared after the oral administration of a 40 mg dose of the pure enantiomer and an 80 mg dose of a racemic mixture of (R,S)-propranolol. The results of this study indicate that the bioavailability of (S)-propranolol, as expressed by the mean area under the concentration-time curve (AUC) and maximum serum concentration, is lower after 40 mg of the optically pure drug than after the racemic drug.     

Synthesis and characterization of a novel photoluminescent three-dimensional metal-organic framework

A novel metal-organic framework containing one-dimensional channels of formula [Zn₃(Aco)₂(H₂O)₆]_n (H₃Aco = aconitic acid) has been synthesized and characterized by FT-IR spectroscopy, thermogravimetric analysis (TG), X-ray analysis, and solid state photoluminescence spectra. X-ray crystallographic studies reveal that there are two kinds of crystallographically independent Zn atoms in the title complex. The most interesting feature of the structure is an unprecedented 3D MOF containing infinite Zn(1) linear chains and heterochiral Zn(2) single-stranded helices. The linear chains and helices happen to be perpendicular to each other. Photoluminescence properties of the title compound have been examined in the solid state at room temperature.     

In Vitro Evaluation of 11C-Labeled (S)-Nicotine, (S)-3-Methyl-5-(1-Methyl-2-Pyrrolidinyl)isoxazole, and (R,S)-1-Methyl-2-(3-Pyridyl)azetidine as Nicotinic Receptor Ligands for Positron Emission Tomography Studies

Abstract: The binding characteristics of the novel ¹¹C-labeled nicotinic ligands (R,S)-1-methyl-2-(3-pyridyl) azetidine (MPA) and (S)-3-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole (ABT-418) were investigated in comparison with those of (S)-[¹¹C]nicotine in vitro in the rat brain to be able to predict the binding properties of the new ligands for positron emission tomography studies in vivo. The data from time-resolved experiments for all ligands indicated fast binding kinetics, with the exception of a slower dissociation of [¹¹C]MPA in comparison with (S)-[¹¹C]nicotine and [¹¹C]ABT-418. Saturation experiments revealed for all ligands two nicotinic receptor binding sites with affinity constants (K_D values) of 2.4 and 560 nM and binding site densities (B_max values) of 65.5 and 223 fmol/mg of protein for (S)-[¹¹C]nicotine, K_D values of 0.011 and 2.2 nM and B_max values of 4.4 and 70.7 fmol/mg of protein for [¹¹C]MPA, and K_D values of 1.3 and 33.4 nM and B_max values of 8.8 and 69.2 fmol/mg of protein for [¹¹C]ABT-418. In competing with the ¹¹C-ligands, epibatidine was most potent, followed by cytisine. A different rank order of potencies was found for (?)-nicotine, (+)-nicotine, MPA, and ABT-418 displacing each of the ¹¹C-ligands. Autoradiograms displayed a similar pattern of receptor binding for all ligands, whereby [¹¹C]MPA showed the most distinct binding pattern and the lowest nonspecific binding. We conclude that the three ¹¹C-labeled nicotinic ligands were suitable for characterizing nicotinic receptors in vitro. The very high affinity of [¹¹C]MPA to nicotinic acetylcholine receptors, its low nonspecific binding, and especially the slower dissociation kinetics of the [¹¹C]MPA from the putative high-affinity nicotinic acetylcholine receptor binding site compared with (S)-[¹¹C]nicotine and [¹¹C]ABT-418 raise the level of interest in [¹¹C]MPA for application in positron emission tomography.     

Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino) Cyclohexanol (AH5183)

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)