青霉素酰化酶:固定化及其应用

青霉素酰化酶广泛应用于β-内酰胺类抗生素中间体和半合成β-内酰胺类抗生素的合成。虽然游离酶具有极高的催化活性,但其对温度、pH值、溶剂极性等的耐受范围极窄,极易失活,而且在有机溶剂中发生的失活是不可逆的。为了提高其各方面稳定性,不断有新的固定化方法提出。本文综述了青霉素酰化酶的固定化方法及其应用。     

纳米级磁性微粒的制备及固定化纤维素酶的研究

研究了明胶改性新途径,制备了核壳式纳米级磁性明胶微粒.以此为载体对纤维素酶进行固定化,确定了固定化工艺条件,研究了磁性固定化酶的最适温度、最适pH,使磁性固定化酶的热稳定性、贮存稳定性及操作稳定性均显著提高.     

纳米二氧化硅固定化酶的研究进展

酶是活细胞产生的具有催化功能的蛋白质,人们可以利用酶来酿酒、制作食物以及生产各种工业用品,还可以诊断和治疗各种疾病。游离酶在工业生产和其他实际应用中存在许多局限性,如性能不稳定、易失活、耐热性低等。固定化游离酶是克服上述问题的有效手段之一。在众多的固定化酶载体中,纳米二氧化硅因其具有高表面积、强机械稳定性、制备简单、无化学反应和绿色环保等特点而被认为是固定化酶的优良基质。文章主要阐述了纳米二氧化硅固定化酶的不同方法,总结了不同类别的纳米二氧化硅固定化酶的固定效果,同时对复合纳米二氧化硅材料对固定化酶的应用前景及其存在的挑战进行了探讨。     

纳米级磁性微粒的制备及固定化纤维毒酶的研究

研究了明胶改性新途径,制备了核壳式纳米级磁性明胶微粒。以此为载体对纤维素酶进行固定化,确定了固定化工艺条件,研究了磁性固定化酶的最适温度,最适pH,使磁性固定化酶的热稳定性、贮存稳定性及操作稳定性均显著提高。     

青霉素酰化酶的固定化与应用新进展

青霉素酰化酶被广泛应用于半合成抗生素及中间体的制备、手性药物的拆分和多肽合成等方面。高效固定青霉素酰化酶能提高酶对温度、pH值、溶剂极性等方面的适用性和反复使用的稳定性，将成为拓宽青霉素酰化酶在工业中应用的必然选择和关键。本文主要介绍了青霉素酰化酶固定化技术的进展，讨论了不同固定化技术的特点和固定化酶在非水相体系中的催化作用，并展望了固定化青霉素酰化酶的发展前景。     

壳聚糖微球固定化重组TEM-116型ESBL的研究

目的 利用固定化酶技术,使重组TEM-116型超广谱β-内酰胺酶(ESBL)在清除环境中残留抗生素的应用中可重复使用,并改善其稳定性.方法 以戊二醛为交联剂将重组TEM-116型ESBL固定于壳聚糖微球载体,并采用单因素轮换法和响应曲面法对固定化条件进行优化.结果 4℃条件下,当戊二醛浓度0.54%、交联pH5.4、给酶量0.58IU/g壳聚糖微球、固定化pH6.2时,同定化酶的酶活和回收率分别可达到0.49IU/g和84%(0.49IU/0.58IU).与游离酶相比,固定化酶的酸碱稳定性和可重复利用性得到显著提高,在pH5.8～7.4范围内酶活性保持在73%～100%的水平,经7次重复性使用,每次1h,酶活力保留高达80%.结论 通过固定化酶技术,以重组TEM-116型ESBL为代表的耐药酶能够开发为具有广泛应用前景的环保制剂,以消除外环境中残留的抗生素.     

固载化木聚糖酶交联酶聚集体的制备及性质D

目的 提高木聚糖酶YS1069的稳定性、重复利用率以及便于从反应体系中分离。方法 本文将交联酶聚集体CLEAs技术与载体固定化技术相结合,用LKZ-128氨基型树脂对木聚糖酶CLEAs进行固定化,并对其性质进行初步研究。结果及结论 采用单因素法和响应面法对影响固定化酶活性的因素进行分析和优化,获得最佳固定化条件为: pH 9.0的酶液,以2 mL的异丙醇为沉淀剂,沉淀1 h,以1.59 g/L的京尼平为交联剂,交联2 h,20 ℃的环境中固定化9.25 h,加酶量为8 mg时回收率最高,达到65.63%,加酶量为60 mg时酶活性最高,达到190 U/g。重复使用10次后,其相对酶活仍为42.3%,说明具有良好的批次操作稳定性。该酶经过固定化后热稳定性和pH稳定性均得到了提高。     

青霉素酰化酶提取及其与固定化酶的动力学参数测定和比较

采用冻融-溶媒法从大肠杆菌中提取青霉素酰化酶(经硫酸铵沉淀、透析、冷冻干燥),制得酶粉,活力为1100.37u/g,经六批提取试验,平均提取收率47.93％。分别测定游离酶及其固定化酶米氏常数Km值。游离酶的Km值为2.39mM,固定化酶按其颗粒大小不同,Km值分别为5.62、8.46、8.42mM。 在对pH稳定性测定中,游离酶最适pH为7～8,而固定化后在pH5～9之间都较稳定。对温度的稳定性测定中,游离酶与固定化酶在40℃以下都较稳定,但在45℃保温一小时后,游离酶只存留活力39.51％,而固定化酶却存留活力86.92％,二者有显著差异、温度再高相差更大。因此酶经固定化后,对pH和温度的稳定性都有明显提高。金属离子对青霉素酰化酶的活力有所影响。特别是铁离子和铜离子影响较大,故酶反应容器不能采用铁和铜的材料。     

抑肽酶的亲和层析——Tosy1（对甲苯磺酰氯）在固定化酶上的应用

以4%琼脂糖为载体，对甲苯磺酰氯为活化剂，对胰蛋白酶的固定化条件及固定化酶的性质进行了研究。其固定化条件为pH7.4，温度15℃，时间8hr，酶量20mg/g(wet-gel）。同时研究了温度、pH值、时间以及载体与酶的比例等因素对胰蛋白酶固定化的影响。最后报道了该固定化酶的最适pH，热稳定性，pH贮存稳定性和操作稳定性。用这种固定化胰蛋白酶纯化抑肽酶，抑肽酶纯度提高50倍，回收率在85%-95%。     

温敏载体固定化木瓜蛋白酶的特性及其消化HCG抗体的应用

以温敏性材料N-羟基琥珀酰亚胺活化的聚(N-异丙基丙烯酰胺)为载体制备固定化木瓜蛋白酶,对其酶学性质进行了研究。结果表明:固定化酶的最适pH值为7.5,最适温度为65℃,米氏常数为2.72 mg/mL;经过24次温敏循环操作,剩余相对活力86.6%;在4℃条件下放置60 d,剩余相对活力为71.5%。与游离酶相比,固定化酶pH值稳定性、热稳定性、操作稳定性和储存稳定性都有显著提高。将此固定化酶应用于HCG单克隆抗体消化,与纤维素固定化酶相比,水解能力更强,更适合于大分子的酶解,特别是抗体片段的制备。     

微乳和基于微乳的经皮给药制剂的研究进展

近年来,微乳作为一种具有粒径小、渗透性强、溶解度大、易于制备的新型递药载体,在药物制剂的开发过程中得到广泛研究。本文总结了近年来国内外微乳和基于微乳的经皮给药制剂在不同种类药物中应用的相关文献,介绍了微乳的组成、形成机制、优缺点及在各治疗领域的研究实例。微乳具有促进药物透皮吸收、提高稳定性、延长作用时间和降低皮肤刺激性的优势,因此在经皮给药制剂领域具有广阔的应用前景。     

纳米药物载体在经皮给药系统中应用的研究进展

目的介绍纳米药物载体在经皮给药系统中的应用。方法查阅国内外文献共31篇,从纳米药物载体在经皮给药系统中的应用及各自的优势和不足等方面进行综述。结果纳米药物载体具有提高药物的化学稳定性、促进药物经皮吸收、控制药物释放以及定位给药等优点,在药物的经皮吸收方面具有广阔应用前景。结论纳米药物载体为药物的经皮通透提供了新的途径和方法,但是其安全性和有效性仍需进一步研究。     

Chemistry and mechanism of urease inhibition

Studies on enzyme inhibition remain an important area of pharmaceutical research since these studies have led to the discoveries of drugs useful in a variety of physiological conditions. The enzyme inhibitors can interact with enzymes and block their activity towards natural substrates. Urease inhibitors have recently attracted much attention as potential new anti-ulcer drugs. Ironically, urease was the first enzyme crystallized but its mechanism of action is still largely misunderstood. This chapter therefore reviews comprehensive developments in the field of urease inhibitors. Inhibitors of urease can be broadly classified into two categories: (1) active site directed (substrate-like), (2) mechanism-based directed. We present here the examples of selected inhibitors along with their mechanisms of action to characterize their mode of urease inhibition. The observations that urease due to its high substrate (urea) specificity can only bind to a few inhibitors with a similar binding mode as urea is also discussed. Several non-covalent interactions including hydrogen bonds and hydrophobic contacts stabilize the enzyme-inhibitor complex. Regardless of the class of compound, it is reported that only a few functional groups with electronegative atoms such as oxygen, nitrogen and sulfur act either as bidentate (mostly), tridentate (rarely), or as ligand-chelator to form octahedral complexes with two slightly distorted octahedral Ni ions of the enzyme. Bulky groups attached to the pharmacophore were found to decrease the activity of inhibitors, since the lack of a bulky attachment makes it easier for urease inhibitors to enter the substrate-binding pocket as well as avoid unfavorable steric interactions with amino acid residues in its vicinity. This review is intended to provide highlights of the inhibition of urease by hydroxamic acids (HXAs), phosphorodiamidates (PPDs), imidazoles, phosphazene and related compounds. These compounds are compared to previously reported urease inhibitors for the catalytic models proposed for urease activity. The differences in inhibition of urease activities from plants and of bacterial origin by various inhibitors and physiological implications of urease inhibition are discussed.     

Discovery, structure-activity relationship, and biological evaluation of noninhibitory small molecule chaperones of glucocerebrosidase

A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease.     

Catalytic nucleic acids: from lab to applications

Since the discovery of self-cleavage and ligation activity of the group I intron, the expansion of research interest in catalytic nucleic acids has provided a valuable nonprotein resource for manipulating biomolecules. Although a multitude of reactions can be enhanced by this class of catalyst, including trans-splicing activity of the group I intron (which could be applied to gene correction), RNA-cleaving RNA enzymes or "ribozymes" hold center stage because of their tremendous potential for mediating gene inactivation. This application has been driven predominantly by the "hammerhead" and "hairpin" ribozymes as they induce specific RNA cleavage from a very small catalytic domain, allowing delivery either as a transgene expression product or directly as a synthetic oligonucleotide. Although advances in the development of RNA modifications have improved the biological half-life of synthetic ribozymes, their use is restricted by the mechanistic dependence on conserved 2'OH-moieties. Recently a new class of catalytic nucleic acid made entirely of DNA has emerged through in vitro selection. DNA enzymes or deoxyribozyme with extraordinary RNA cleavage activity has already demonstrated their capacity for gene suppression both in vitro and in vivo. These new molecules, although rivaling the activity and stability of synthetic ribozymes, are limited equally by inefficient delivery to the intracellular target RNA. The challenge of in vivo delivery is being addressed with the assessment of a variety of approaches in animal models with the aim of bringing these compounds closer to the clinic.     

The therapeutic potential for catalytic antibodies: from a concept to a promise

More than ten years have now elapsed since the first reports confirmed that antibodies not only label antigenic targets but can also perform catalytic functions. Much of the initial research in this area focussed on exploring the scope and utility of these biocatalysts both as enzyme mimics and as programmable protein catalysts. However, their potential in the biomedical field has also been probed. This review details the present perspective of catalytic antibodies as new tools for immunotherapy and specifically focuses on their application to prodrug activation and drug inactivation.     

青蒿素及其衍生物的研究进展

青蒿素类化合物是含过氧桥的化合物,在治疗多药抗药性恶性疟疾方面卓有成效。除此之外,该类化合物还具有抗肿瘤、抗真菌、抗心律失常、抗寄生虫等活性。本文就近年来国内外学者对青蒿素及其衍生物的结构改造及生物活性等方面的研究进行概述。     

Antimicrobial β-peptides and α-peptoids

The field of drug discovery and development has seen tremendous activity over the past decade to better tackle the increasing occurrence of drug-resistant bacterial infections and to alleviate some of the pressure we put on the last-resort drugs on the market. One of the new and promising drug candidates is derived from naturally occurring antimicrobial peptides. However, despite promising results in early-stage clinical trials, these molecules have faced some difficulties securing FDA approval, which can be linked to their poor metabolic stability. Hence, mimetics of these antimicrobial peptides have been suggested as new templates for antibacterial compound design, because these mimetics are resistant against degradation by proteases. This review will discuss the structural features of two different types of mimetics, β-peptides and α-peptoids, in relation to their antibacterial activity and conclude on their potential as new candidates for bacterial intervention.     

Engineering magnetic nanobiocatalytic systems with multipurpose functionalities for biocatalysis,biotechnology and bioprocess applications

Enzymes are high-performance natural biological catalysts with wide-ranging applications in agricultural, medical, food and environmental sectors. Nevertheless, lack of efficient recovery, reusability, and high cost of the soluble form of enzymes are the most daunting challenges rendering biocatalytic systems inadequate for industrial exploitation. In order to deal with these inadequacies, immobilization appears to be a prodigious approach for enhancing the stability and catalytic efficiency of enzymes, as well as enabling their separation and reusability in continuous reaction batches. Among different nanostructures, magnetic nanomaterials have garnered supreme interest as support matrices for biomolecules and enzymes immobilization because of their substantial surface area, larger surface-to-volume ratio, modifiable surface, and adjustable surface particle size, stability, and high mass transferring ability. In addition, they can be quickly recovered from the complex reaction system by a simple external magnetic field. Magnetic nanoparticles incorporated biocatalysts demonstrated a broad-working temperature and pH profile and augmented storage and thermal stabilities compared to their native derivatives. This paper provides a recent and state-of-the-art overview of the development and application of multifunctional magnetic nanobiocatalytic systems for an array of biotechnological purposes. In the first half, the development, functionalization, and use of nanostructured magnetic materials as enzyme immobilization supports are delineated. Then the prospective applications of magnetic nanobiocatalytic systems in different industrial sectors, including wastewater treatment, biodiesel and butanol production, hydrolysis of lignocellulosic biomass, glucose monitoring, fruit juice extraction and clarification, and synthesis of non-natural benzylisoquinoline alkaloid are comprehensively vetted with representative examples. Finally, the conclusive points and future prospects in this evolving field are also directed.     

Human beta-defensin-3: a promising antimicrobial peptide

The field of naturally occurring antimicrobial peptides is a research area rapidly expanding due to the high potential of such molecules as new antimicrobial drugs. In this regard, the human beta-defensin-3 is particularly attractive because of its strong antibacterial activity, relative salt-insensitiveness and low toxicity for host cells.