三角酵母整细胞酶促转化头孢菌素C为戊二酰-7-氨基头孢烷酸

研究了利用含D-氨基酸氧化酶(Damino acid oxidase, DAO EC1.4.3.3)的透性化三角酵母多倍体FA10(Trigonopsis variabilis FA10)细胞酶促转化头孢菌素(Ccephalosporin> C, CPC)为戊二酰-7-氨基头孢烷酸(Glutaryl-7-ACA,GL-7-ACA)的反应过程和细胞中同时存在的过氧化氢酶(Catalase, CAT)通过水解H₂O₂而对转化反应产生的干扰作用及其对策。实验证明适量添加外源H₂O₂(6%)或在反应体系中加入过氧化氢酶抑制剂NaN₃(0.13mg/mL)可使GL-7-ACA生成率分别为73.0%和70.1%。如果将透性化的FA10细胞在pH10.5～11.0,20℃条件下保温30min,CAT被不可逆性完全钝化,以无过氧化氢酶的FA10细胞进行CPC的酶促转化反应,GL-7ACA的生成率可达84%。     

提高三角酵母细胞DAO表观活力的透性化研究

三角酵母(Trigonopsis variabilis)的D-氨基酸氧化酶(D-amino acid oxidase,DAO,EC1.4.3.3)是一种胞内酶,完整细胞并不呈现酶促活性。为了获得三角酵母细胞的较高表观活力,需对细胞进行处理以改变壁和膜对反应底物和产物的通透性。研究中比较了冻融、丙酮、丁醇和十六烷基三甲基溴化铵(Cetyltrimethylammonium bromide,CTAB)等理化因子的透性化效率,并对丙酮的透性化条件进行了优化。证明丙酮的透性化作用与溶剂浓度、保温时间和温度有关。30%～35%丙酮浓度,4～28℃保温,可得到最大酶活力。透性化所需时间极短,在5min内即可完成。同时,透性化细胞中的DAO比无细胞抽提液中的酶对热更为稳定。用丙酮透性化细胞作为生物催化剂可有效地转化头孢菌素C(Cephalosporin C, CPC)为戊二酰-7ACA(Glutaryl-7-ACA,GL-7ACA)。     

GL-7ACA酰化酶表达检测系统的建立

戊二酰－7－氨基头孢烷酸（GL－7ACA）酰化酶能够催化GL－7ACA分解生成7－ACA,后者是工业半合成生产头孢类抗菌素所需的重要前体。为了准确地检测GL－7ACA酰化酶及其突变体的表达,本研究通过构建一系列质粒载体,建立了两个简便有效地测定GL－7ACA酰化酶基因acy表达量的系统,从而可对酶的比活力进行定量。我们将两个报告基因,即儿茶酚双加氧酶基因（xylE)和β－半乳糖苷酶基因（lacZ)分别置于acy基因的下游,使之与acy基因共用一个启动子,进行串联表达,各自构成一个多顺反子系统。实验证明,基因融合后的儿茶酚双加氧酶或β－半乳糖苷酶的活力可以间接反映acy的表达量。     

假单胞菌sp.130 GL-7-ACA酰化酶的核苷酸和蛋白质序列分析

对来源于假单胞菌sp.130的戊二酰-7-氨基头孢烷酸（GL－7－ACA）酰化酶结构基因的全序列及所编码蛋白质的α,β亚基的N末端和C末端的氨基酸序列进行了测定。将蛋白质序列与其他同类的GL－7－ACA酰化酶进行了同源性比较,结果显示该酶与来源于假单胞菌GK16和C427的酰化酶的序列有较高同源性,而与其它同类酰化酶的同源性较低。这些酶的α亚基N－末端差别较大,但是β－亚基的N－末端有较高的保守性。     

NO和H2O2在介导热激诱发金丝桃细胞合成金丝桃素中的信号互作

热激处理（40℃,10min）可以诱发金丝桃细胞中金丝桃素的生物合成并诱导细胞产生一氧化氮（NO）和过氧化氢（H2O2）．过氧化氢酶（CAT）和NO专一性淬灭剂（cPTIO）不仅可以分别抑制由热激诱发的H2O2积累和NO合成,而且还可以阻断热激处理对金丝桃素生物合成的促进作用．H2O2单独处理虽然不能提高细胞的金丝桃素产量,但是H2O2和NO共同处理对金丝桃素产量的促进作用显著高于NO单独处理,表明NO和H2O2对金丝桃素的生物合成具有协同诱导效应．NO处理可以提高细胞的H2O2水平,而外源H2O2对金丝桃细胞的NO合成积累也具有促进作用,说明NO和H2O2对彼此的合成反应具有促进作用．CAT在抑制热激诱发H2O2合成的同时还能够部分抑制热激细胞中NO的合成,而cPITO也可以同时降低热激细胞的H2O2水平．上述实验结果提示,在热激处理下金丝桃细胞中的NO和H2O2可能通过互作反应提高各自的信号水平．质膜NAD（P）H氧化酶抑制剂DPI和NO合酶抑制剂PBITU可以抑制NO和H2O2之间的互作反应,并且解除NO和H2O2对金丝桃素合成的协同诱导作用,说明NO和H2O2对金丝桃素合成积累的协同效应依赖于两种信号分子之间的互作反应．本文实验结果不仅证实了NO和H2O2是参与热激诱发金丝桃细胞中金丝桃素合成所必需的两种信号分子,而且揭示了NO和H2O2在介导热激诱发金丝桃素生物合成过程中特殊的信号互作现象．     

家蚕胚胎发育中过氧化氢的代谢

过氧化氢（H2O2）是生物体内主要的活性氧来源之一。在超氧化物歧化酶（SOD）、过氧化氢酶（CAT）等的催化作用下,H2O2被降解,释放出活性氧。所以,生物个体发育过程中体内H2O2、SOD和CAT含量的变化反映着H2O2的代谢水平。另外,家蚕是蚕卵滞育昆虫,实验设计考虑到了滞育前后可能会有的差别。取产后10分钟内的卵为供试材料。采用即时浸酸法解除卵滞育。采用比色法和氧电极法测定并比较家蚕胚胎滞育形成与解除过程中过氧化氢的代谢。结果表明：（1）受精初期最低水平（Fig.2);（2）胚胎发育过程中（即时浸酸除滞有）,H2O2量除168－216h处于低水平外均显著高于滞育卵（Fig.3),SOD活性分别在72h、168h,形成大小两峰,后期显著高于滞育卵（Fig.4),而CAT活性72－192h保持平衡,随后急剧上升,前期显著低于滞育卵,后于滞育卵（Fig.4),而CAT活性72－192h保持平衡,随后急剧上升,前期显著低于滞育卵,后期相反（Fig.5);(3)滞育形成过程中H2O2水平变化平缓（Fig.6),SOD活性前期剧烈活动,但后期保持平稳（Fig.7),CAT活性逐步升高（Fig.8),而浸酸解除滞育过程中H2O2水平显著高于滞育卵（Fig.6),SOD活性前期出现新峰,后期显著升高（Fig.7),CAT活性显著低于滞育卵（Fig.8)。结合他人的研究结果,可以推测：家蚕卵H2O2代谢状况可能在其滞育形成和解除中具有重要意义,或许酯酶A4计器假说与卵孔堵塞说可以通过H2O2而联系起来。     

过氧化氢刺激RAW264.7巨噬细胞促炎细胞因子和趋化因子基因表达

免疫反应细胞经呼吸瀑布作用产生的活性氧是巨噬细胞促炎细胞因子和趋化因子表达的信号分子,但目前缺乏过氧化氢(H2O2)刺激巨噬细胞表达促炎细胞因子和趋化因子的直接证据．本研究以离体培养的小鼠RAW264.7巨噬细胞为研究体系,探讨外源H2O2对RAW264.7巨噬细胞促炎因子和趋化因子基因表达和生成的影响．MTT法结合实时荧光定量PCR(qRT-PCR)、酶联免疫吸附试验(ELISA)结果显示,RAW264.7细胞在H2O2浓度低于40 μmol/L时不影响RAW264.7细胞的增殖活力．20 μmol/L和40 μmol/L H2O2显著增强RAW264.7细胞TNF-α、IL-1β、MCP-1和MIP-2基因转录和蛋白质生成,并存在剂量依赖效应;而200 U/mL过氧化氢酶预处理则可减弱由H2O2刺激的TNF-α、IL-1β、MCP-1和MIP-2基因表达和蛋白生成．这些结果提示,H2O2是刺激巨噬细胞促炎因子和趋化因子表达或生成的重要因子,对机体炎症反应的发生具有重要作用．     

NADH-细胞色素 b5 还原酶在甲状腺过氧化氢生物合成中的作用

应用高香草酸荧光分析技术及NADH－高铁氰化钾还原酶法,对正常和Graves病甲状腺过氧化氢（H2O2）和NADH－细胞色素b5还原酶（b5R)进行测定,发现Graves病甲状腺b5R活性和H2O2水平均明显高于正常,而H2O2酶活性在Graves病和正常甲状腺间无显著差异。加b5R抑制剂对氯汞苯甲酸抑制b5R活性,Graves病和正常甲状腺b5R活性降低近85％,同时H2O2降低近50％,蛋白结合碘形成减少近52％。b5R活性和H2O2水平两者呈显著正相关关系。以上结果表明,b5R参与甲状腺内H2O2的生物合成,是甲状腺内产生H2O2的重要酶系。     

外源NO供体对小麦离体叶片过氧化氢代谢的影响

分析了外源一氧化氮（nitric oxide ,NO）供体硝普钠（sodium nitroprusside,SNP）对离体小麦（Triticum aestivumL.）叶片过氧化氢（H2O2）含量及其清除酶活力的调节作用。不同浓度的SNP (1 mmol/L和5 mmol/L) 处理30 min内, 离体小麦叶片H2O2含量均有一个显著上升的过程, 同时过氧化物酶（POD） 活力受到显著抑制,而过氧化氢酶（CAT）活力则轻微下降;处理30 min到240 min时,POD活力的抑制状态基本维持不变,而CAT 活力开始恢复上升, H2O2含量也相应地开始下降。粗酶液的体外实验也表明,SNP对POD和CAT的抑制类型不同,前者可能是不可逆抑制,后者则可能是可逆抑制。因此NO可通过对POD和CAT的不同抑制作用来调节小麦叶片内源H2O2含量。     

B7家族的新成员

T细胞的活化需要共刺激分子和其受体及特异性抗原与T细胞受体的双信号作用。B7家族成员是重要的共刺激分子,除B7－1和B7－2,新近又发现了一些新成员：B7RP－1（又名B7h,GL50或ICOSL）为鼠ICOS（inducible costimulator,CD28家族的第三位成员）的配体,其人的同源物命名为B7－2（也称为GL50或ICOSL）,它对TG细胞生长及细胞因子产生的共刺激作用已明显,B7－H1（也称PD－1L）,B7H3是一类不与ICOS结合的B7家族新成员。现已证实。现已证实,这些分子与其受体之间的作用在T细胞增殖及发挥效应中扮演重要角色,它们在许多疾病中的作用也引起学者的普遍关注。     

Two-step immobilized enzyme conversion of cephalosporin C to 7-aminocephalosporanic acid

The first large-scale production of 7-aminocephalosporanic acid (7ACA) from cephalosporin C (CPC) using a wholly enzymatic synthesis method is reported here. We produced 7ACA from CPC in as high a molar yield as 85% using the immobilized enzymes D-amino acid oxidase (D-AOD) and glutaryl-7-ACA acylase (GL-acylase). In the first reactor, CPC is converted to keto-adipyl-7-aminocephalosporanic acid (keto-7ACA) using an immobilized D-AOD isolated from a yeast, Trigonopsis variabilis. The keto-7ACA is then spontaneously converted to glutaryl-7-aminocephalosporanic acid (GL-7ACA) via a chemical reaction with hydrogen peroxide. The hydrogen peroxide is also a product of the D-AOD reaction. Near quantitative conversion of the keto-7ACA to GL-7ACA was observed. The second reactor converts GL-7ACA to 7ACA using an immobilized GL-acylase, which was isolated from a reconbinant Escherichia coli. The final 7ACA crystalline product is a high quality product. The reactions are conducted under very mild aqueous conditions: pH 8.0 and 20 degrees to 25 degrees C. The production of desacetyl side products is minimal. This process is currently being implemented on an industrial scale to produce 7ACA. (c) 1995 John Wiley & Sons, Inc.     

Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells

The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme.     

头孢菌素酰化酶

7-氨基头孢烷酸(7-amino cephalosporanic acid, 7-ACA)是医药工业合成大多数头孢菌素的重要原料.头孢菌素酰化酶(cephalosporin acylase, CA)催化头孢菌素C(CPC)和戊二酰-7-氨基头孢烷酸(GL-7ACA)的水解反应, 生成7-ACA.根据CA催化底物的不同, 可将其划分为两类：CPC酰化酶和GL-7ACA酰化酶.由CA的同源性、分子质量大小和基因结构, 可以把头孢菌素酰化酶划分为五种;讨论了酶的基本性质.通过CA与N端亲核水解酶(Ntn水解酶)的比较, 推测CA属于Ntn水解酶, 并由此可以进一步理解它们的生理功能.     

In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130.

Cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C (CPC) and/or glutaryl 7-amino cephalosporanic acid (GL-7ACA) to produce 7-amino cephalosporanic acid (7-ACA). The acylase from Pseudomonas sp. 130 (CA-130) is highly active on GL-7ACA and glutaryl 7-aminodesacetoxycephalosporanic acid (GL-7ADCA), but much less active on CPC and penicillin G. The gene encoding the enzyme is expressed as a precursor polypeptide consisting of a signal peptide followed by alpha- and beta-subunits, which are separated by a spacer peptide. Removing the signal peptide has little effect on precursor processing or enzyme activity. Substitution of the first residue of the beta-subunit, Ser, results in a complete loss of enzyme activity, and substitution of the last residue of the spacer, Gly, leads to an inactive and unprocessed precursor. The precursor is supposed to be processed autocatalytically, probably intramolecularly. The two subunits of the acylase, which separately are inactive, can generate enzyme activity when coexpressed in Escherichia coli. Data on this and other related acylases indicate that the cephalosporin acylases may belong to a novel class of enzymes (N-terminal nucleophile hydrolases) described recently.     

Two novel engineered bacteria for secretory expression of glutaryl 7-amino-cephalosporanic acid acylase

Two novel engineered bacteria, BL21(DE3)/pETCA1S and TG1/pSuperCA1S, were obtained which can secretory express the gene encoding glutaryl 7-amino-cephalosporanic acid acylase (GL-7ACA acylase) from Pseudomonas sp. 130 with high activity. The growth conditions of transformants for overproduction of GL-7ACA acylase were optimized: in intact cells of BL21(DE3)/pETCA1S and TG1/pSuperCA1S the activity of GL-7ACA acylase was 415 and 600 units g^–1 dry cells, respectively. The highest specific activity of GL-7-ACA acylase is in the intact cell as compared with that of transformants constructed in our laboratory. In fiftieth generation of mutants transferred on agar plates the specific activity of GL-7ACA acylase remained constant.     

Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide

In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min⁻¹, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h⁻¹ to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

透性化嗜酸乳杆菌细胞转化亚油酸为共轭亚油酸的反应动力学

本实验旨在研究透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的反应动力学。探讨了细胞浓度、底物浓度、反应体系pH值和温度等因素对生物转化共轭亚油酸反应速度的影响;建立了透性化嗜酸乳杆菌细胞生物转化共轭亚油酸的动力学模型。结果表明,透性化嗜酸乳杆菌细胞有利于共轭亚油酸的生物转化,最适细胞浓度、pH值和反应温度分别为10×1010ufc/mL、4.5和45℃;生物转化共轭亚油酸存在底物抑制现象,当亚油酸的浓度为0.6mg/mL时,反应速度达到最大值17.8μg/(mL·min)。在低亚油酸浓度下,反应初始阶段的反应规律与经典米氏方程相符,而在高亚油酸浓度下,存在底物抑制现象。在最适反应条件下建立了动力学模型,模型基本反映了共轭亚油酸的生物转化特性。