扩环酶的研究进展及其应用前景

扩环酶,也叶脱乙酰氧基头孢菌素Ｃ合成酶,催化青霉素Ｎ扩环生成脱乙酰氧基头孢菌素Ｃ,是头孢菌素生物合成中的关键酶。在真菌中它是一个双功能酶,同时具有扩环酶和羟化酶的活性;而在细菌中扩环和羟化却是由两个独立的酶来承担的。近年来,扩环酶被纯化成均一蛋白,有关它的性质、分子结构以及基因结构等方面的研究都取得了飞速发展,并不断地应用基因工程的技术探索其在抗生素生产上的应用。     

酶法合成羟头孢霉素

本文报道具有青霉素酰化酶的大肠杆菌(E. coli PN-66)细胞酶促合成羟头孢霉素的研究结果。酶反应的最适温度为20℃,最适pH为6．0。羟头孢霉素的合成率随着母核7一氨基脱乙酰氧基头孢烷酸的浓度的增加而提高,随侧链对羟基苯甘氨酸甲酯盐酸盐在与7一氨基脱乙酰氧基头孢烷酸的配比中的增加而提高。苯乙酸和苯氧乙酸对羟头孢霉素的合成有着强烈的抑制作用。在合适的条件下,羟头孢霉素的合成率可达90％以上。     

紫黄质脱环氧化酶的某些特性和研究方法

紫黄质脱环氧化酶是高等植物体内叶黄素循环的关键酶,它催化紫黄质脱环氧化生成花药黄质和玉米黄质,在这个过程中伴随着过剩光能的热耗散.文中主要对此酶的性质、功能、研究方法以及分子生物学等内容作了简要介绍.     

一株产多种β-内酰胺类抗生素酰化酶菌株的筛选

为了从大量的候选菌株中快速筛选头孢菌素酰化酶产生菌,设计并合成了一系列头孢菌素酰化酶的底物类似物。这些酰胺类的底物类似物由二部分组成,一部分为与头孢菌素相同或相似的侧链,另外一部分为发色基团或便于检测的基团。它们被酰化酶水解酰胺键以后可以方便快速的检测,因此用于对大量菌株进行快速筛选。采用这些化合物筛选到6株酰化酶阳性菌株。其中菌株ZH0650能够同时水解GL-7ACA和多个底物类似物。进一步研究表明,该菌至少产生3种酰化酶,AD-NABA酰化酶,青霉素G酰化酶和头孢菌素C酰化酶。我们初步纯化了AD-NABA酰化酶和青霉素G酰化酶,并对头孢菌素C酰化酶的活力进行了鉴定。这是首次报道的可以产生青霉素G酰化酶和头孢菌素酰化酶等多种酰化酶的菌株,具有良好的应用前景。     

一株产多种β-内酰胺类抗生素酰化酶菌株的筛选

为了从大量的候选菌株中快速筛选头孢菌素酰化酶产生菌,设计并合成了一系列头孢菌素酰化酶的底物类似物。这些酰胺类的底物类似物由二部分组成,一部分为与头孢菌素相同或相似的侧链,另外一部分为发色基团或便于检测的基团。它们被酰化酶水解酰胺键以后可以方便快速的检测,因此用于对大量菌株进行快速筛选。采用这些化合物筛选到6株酰化酶阳性菌株。其中菌株ZH0650能够同时水解GL7ACA和多个底物类似物。进一步研究表明,该菌至少产生3种酰化酶,ADNABA酰化酶,青霉素G酰化酶和头孢菌素C酰化酶。我们初步纯化了ADNABA酰化酶和青霉素G酰化酶,并对头孢菌素C酰化酶的活力进行了鉴定。这是首次报道的可以产生青霉素G酰化酶和头孢菌素酰化酶等多种酰化酶的菌株,具有良好的应用前景。     

铜绿假单胞菌完整细胞a-氨基酸酯水解酶的性质

铜绿假单胞菌(Pseudomonas aeruginosa) AS 1.204完整细胞a-氨基酸酯水解酶能催化a-氨基酸酯的水解和转移a-氨基酸酯的酰基到胺亲核试剂上。在水解和转移反应中酶的一般性质相同,最适pH为5．2,最适温度都是40℃。7-氨基脱乙酰氧基头孢烷酸(7一ADCA)抑制苯甘氮酸甲酣(PG-Ome)的水解。因此,该酶催化7一ADCA和PG-Ome转化成相应的半合成头孢菌素。     

真养产碱杆菌112R4的二羧酸单酰胺酰胺水解酶

在一株具有环酰亚胺转化活性的真养产碱杆菌112R4中发现了一种特异性的二羧酸单酰胺酰胺水解酶（半酰胺酶）,它催化环酰亚胺代谢的第二步反应,将二羧酸单酰胺水解为二羧酸和氨。该酶的底物仅限于此代谢途径的第一个酶——酰亚胺酶的产物二羧酸单酰胺,而对其它的酰胺类化合物没有明显水解活性。真养产碱杆菌112R4中的半酰胺酶和酰亚胺酶在表达上具有相关性,环酰亚胺（如琥珀酰亚胺）和二羧酸单酰胺（如琥珀酰胺酸）对它们有正调控作用,游离氨离子显示出负调控作用,琥珀酸则在酶合成和活性两方面均表现出影响作用。对重组大肠杆菌中表达的半酰胺酶粗酶的部分性质进行了研究。钴离子对半酰胺酶的活性表现出促进作用,比活力提高到3.37倍,表明半酰胺酶可能是一种金属结合酶。     

叶黄素循环酶

依赖叶黄素循环的热耗散是一种主要防御光破坏的机制。参与叶黄素循环的酶是紫黄质脱环氧化酶和玉米黄质环氧化酶 ,紫黄质脱环氧化酶已分离纯化 ,其 c DNA已被克隆 ,其活性主要受跨类囊体膜的 p H梯度和抗坏血酸浓度的调节 ;玉米黄质环氧化酶还没有被分离出来 ,但其 c DNA也已被克隆 ;其活性主要与NADPH的浓度、O2 及光等有关。     

环氧化酶—2及其研究进展

环氧化酶有两种同工酶，环氧化酶－２（ＣＯＸ－２）是受细胞内外相应刺激如细胞脂多糖、细胞因子作用合成的诱导型酶。ＣＯＸ－２在基因结构、表达调控、编码蛋白、定位分布上均与ＣＯＸ－１不完全相同。ＣＯＸ－２是炎症过程中一个重要的诱导酶，在肿瘤的形成与发展过程中也有一定作用。     

无载体酶稳定化——交联酶晶体

本文涉及到无载体稳定化问题,介绍无载体稳定化新方法－交联酶晶体（Ｃｒｏｓｓ－ＬｉｎｋｅｄＥｎｚｙｍｅＣｒｙｓｔａｌｓ,ＣＬＥＣｓ）它是近年发展起来的新型酶晶体催化剂,具有酶的一般特性,催化活性和选择性高,在温和条件下反应等,又具有非均相化学催化剂的操作稳定性高,易回收利用的特点,ＣＬＥＣｓ技术是将酶结晶技术和化学交联技术结合,提高酶抵抗极端条件以及有机溶剂中的稳定性,并广泛适应于各种各样的蛋白质及     

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium.

Deacetoxycephalosporin C synthetase (expandase), which catalyzes ring expansion of penicillin N to deacetoxycephalosporin C (DAOC), has been stabilized in vitro and purified to near homogeneity from the industrially important fungus Cephalosporium acremonium. Throughout the purification, the expandase activity remained physically associated with and in a constant ratio of 7:1 to DAOC hydroxylase activity. The latter activity mediates hydroxylation of DAOC to deacetylcephalosporin C (DAC). The copurified expandase/hydroxylase appeared to be monomeric, with a molecular weight of 41,000 +/- 2,000 and an isoelectric point of 6.3 +/- 0.3. Both catalytic activities required alpha-ketoglutarate, Fe2+, and O2 and were stimulated by ascorbate, dithiothreitol, and ATP. The Fe2+ requirement was specific, and sulfhydryl groups in the purified protein were apparently essential for both ring expansion and hydroxylation. The kinetics and stoichiometry of DAOC/DAC formation from the expandase/hydroxylase-catalyzed reactions suggested that ring expansion of penicillin N preceded hydroxylation of DAOC.     

Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation

Deacetoxycephalosporin C (DAOC) is produced by Acremonium chrysogenum as an intermediate compound in the cephalosporin C biosynthetic pathway, and is present in small quantities in cephalosporin C fermentation broth. This compound forms an undesirable impurity, 7-aminodeacetoxycephalosporanic acid (7-ADCA), when the cephalosporin C is converted chemically or enzymatically to 7-aminocephalosporanic acid (7-ACA). In the cephalosporin C biosynthetic pathway of A. chrysogenum, the bifunctional expandase/hydroxylase enzyme catalyzes the conversion of penicillin N to DAOC and subsequently deacetylcephalosporin C (DAC). By genetically engineering strains for increased copy number of the expandase/hydroxylase gene, we were able to reduce the level of DAOC present in the fermentation broth to 50% of the control. CHEF gel electrophoresis and Southern analysis of DNA from two of the transformants revealed that one copy of the transforming plasmid had integrated into chromosome VIII (ie a heterologous site from the host expandase/hydroxylase gene situated on chromosome II). Northern analysis indicated that the amount of transcribed expandase/hydroxylase mRNA in one of the transformants is increased approximately two-fold over that in the untransformed host. Received 5 January 1998/ Accepted in revised form 29 May 1998     

Cloning and sequencing of the beta-lactam hydroxylase gene (cefF) from Streptomyces clavuligerus: gene duplication may have led to separate hydroxylase and expandase activities in the actinomycetes.

The deacetylcephalosporin C synthetase (hydroxylase) gene from Streptomyces clavuligerus has been cloned and sequenced. The open reading frame codes for a protein with an Mr of 34,584. The hydroxylase gene (cefF) is closely linked to the epimerase gene (cefD) and the expandase gene (cefE) and is transcribed in the opposite orientation. The hydroxylase and expandase genes are 59 and 71% identical at the amino acid and DNA levels, respectively. cefE and cefF may have arisen from a gene duplication in the actinomycetes.     

Purification and initial characterization of an enzyme with deacetoxycephalosporin C synthetase and hydroxylase activities.

Deacetoxycephalosporin C synthetase (expandase) from Cephalosporium acremonium (Acremonium chrysogenum) was purified to near homogeneity as judged by SDS/polyacrylamide-gel electrophoresis. The enzyme (Mr about 40,000) exhibited a pH optimum around 7.5. It required 2-oxoglutarate (Km 0.04 mM), Fe2+ and O2 as cofactors, and ascorbate and dithiothreitol were necessary for maximum activity. It was stable for over 4 weeks at -70 degrees C in the presence of 1 mM-dithiothreitol. Activity was inhibited by the thiol-quenching reagent N-ethylmaleimide, the metal-ion-chelating reagent bathophenanthroline, and NH4HCO3. The highly purified enzyme also showed deacetoxycephalosporin C hydroxylase (deacetylcephalosporin C synthetase) activity, indicating that both expandase and hydroxylase activities are properties of a single protein. These activities could not be separated by ion-exchange, dye-ligand, gel-filtration or hydrophobic chromatography. A beta-sulphoxide and a 3 beta-methylene hydroxy analogue of penicillin N were synthesized to test as potential intermediates in the ring-expansion reaction, Neither compound was a substrate for the enzyme. A synthetic analogue in which the 3 beta-methyl group and the 2-hydrogen atom of penicillin N were replaced by a cyclopropane ring was not a substrate but was a reversible inhibitor of the enzyme.     

Identification of rate-limiting steps in cephalosporin C biosynthesis in Cephalosporium acremonium: a theoretical analysis

Summary A kinetic model describing the biosynthesis of celphalosporin C in Cephalosporium acremonium has been developed to identify the rate-limiting step(s). Using this model and in-vitro kinetic data of the biosynthetic enzymes, the production kinetics of cephalosporin C were examined theoretically. The predicted time profile of the specific production rate during batch culture is in good agreement with that of experimental results published previously. Sensitivity analysis indicates that -(l--aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase is the rate-limiting enzyme. Our analysis also predicts that increasing ACV synthetase enhances the production rate initially until expandase/hydroxylase becomes rate-limiting. Furthermore, increasing expandase/hydroxylase reduces the accumulation of penicillin N, and thus, enhances the production of cephalosporin C. Based on our analysis, amplifying both ACV synthetase and expandase/hydroxylase concurrently should enhance the production rate to a great extent.Correspondence to: W. S. Hu     

Performance of a recombinant strain of<Emphasis Type="Italic"> Streptomyces lividans</Emphasis> for bioconversion of penicillin G to deacetoxycephalosporin G

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO₄ and α-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO₄ and 1.3 mM α-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO₄ and 1.9 mM α-ketoglutarate. Electronic Publication     

Construction of hybrid bacterial deacetoxycephalosporin C synthases (expandases) by in vivo homeologous recombination

Homeologous recombination (recombination between partially homeologous DNA sequences) was used to produce novel functional deacetoxycephalosporin C synthase (expandase) enzymes in vivo which are hybrids of the Streptomyces clavuligerus and Nocardia lactamdurans enzymes. DNA sequencing of hybrids obtained in E. coli showed that recombination had occurred at several locations within conserved sequences as short as 2 bp. Recombination events obtained in a Streptomyces background resulted in expandases with altered activity on penicillin G as determined by bioassay and HPLC.     

Cephamycin C biosynthesis in Streptomyces cattleya: nitrogen source regulation

Summary The production of cephamycin C by Streptomyces cattleya varies with the use of asparagine, glutamine or ammonium as nitrogen sources. hydroxylase and expandase activities were demonstrated for the first time with this species. A study of the biosynthetic regulation of these enzymes by two different nitrogen sources, glutamine and asparagine, was carried out. Asparagine proved to be a better nitrogen source, both for enzymatic biosynthesis and production of cephamycin C. Moreover, an excess of asparagine in the culture environment provokes, simultaneously, a reduction in cephamycin C production and a decrease in the biosynthesis of expandase and hydroxylase.Offprint requests to: A. Lebrihi     

Evolving enzyme technology for pharmaceutical applications: case studies

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent. Received 07 March 1997/ Accepted in revised form 15 June 1997     

Cloning, characterization, and expression in Escherichia coli of the Streptomyces clavuligerus gene encoding deacetoxycephalosporin C synthetase.

Biosynthesis of cephalosporin antibiotics involves an expansion of the five-membered thiazolidine ring of penicillin N to the six-membered dihydrothiazine ring of deacetoxycephalosporin C by a deacetoxycephalosporin C synthetase (DAOCS) enzyme activity. Hydroxylation of deacetoxycephalosporin C to form deacetylcephalosporin C by a deacetylcephalosporin C synthetase (DACS) activity is the next step in biosynthesis of cephalosporins. In Cephalosporium acremonium, both of these catalytic activities are exhibited by a bifunctional enzyme, DAOCS-DACS, encoded by a single gene, cefEF. In Streptomyces clavuligerus, separable enzymes, DAOCS (expandase) and DACS (hydroxylase), catalyze these respective reactions. We have cloned, sequenced, and expressed in E. coli an S. clavuligerus gene, designated cefE, which encodes DAOCS but not DACS. The deduced amino acid sequence of DAOCS from S. clavuligerus (calculated Mr of 34,519) shows marked similarity (approximately 57%) to the deduced sequence of DAOCS-DACS from C. acremonium; however, the latter sequence is longer by 21 amino acid residues.