Studies on the biosynthesis of carbapenem antibiotics. II. Isolation and functions of a specific acylase involved in the depantothenylation of the OA-6129 compounds

A specific acylase designated A933 acylase was isolated and purified to 90% protein homogeneity from Streptomyces fulvoviridis A933 17M9 which produces PS-5, epithienamycins A and C and MM 17880 together with minor carbapenem analogs, penicillin N and cephamycin C. This enzyme was found to catalyze the depantothenylation of OA-6129 carbapenems; the acyl exchange of OA-6129 carbapenems with acyl CoA's; the deacetylation of N-acetyl-L-amino acids; and the acylation of NS-5 and 6-aminopenicillanate with acyl CoA's, whereas the deacetylation of PS-5 and N-acetyl-D-amino acids; and the deacylation of benzylpenicillin and cephalosporin C were not observed. Similar enzyme activities were also detected in Streptomyces cattleya, Streptomyces cremeus subsp. auratilis and Streptomyces argenteolus which are all carbapenem producers.     

胞外青霉素酰化酶产生菌的筛选及产酶条件

采用青霉素梯度琼脂平皿法，从土壤中快速筛选出３５株产胞外青霉素酰化酶的菌株，经复筛有５株酶活力较高，经鉴定均为芽胞杆菌，Ｂ３５－１７菌株酶活力最高．研究了Ｂ３５－１７菌株的胞外青霉素酰化酶产生条件，该菌株在最适产酶条件下，酶活力达２．６７ｕ／ｍＬ．在发酵培养基中添加０．２％的玉米浆能降低Ｆｅ２＋对酶合成的抑制作用．     

3-硝基-5-(6-溴己酰胺)苯甲酸的合成及其作为头孢菌素酰化酶产生菌筛选指示剂的研究

为筛选头孢菌素Ｃ酰化酶产生菌,由环己酮和３－硝基－５－乙酰胺基苯甲酸出发,合成了一种新的生色物质３－硝基－５－（６－溴己酰胺）苯甲酸（３－Ｎｉｔｒｏ－５－（６－ｂｒｏｍｏｈｅｘａｎｏｙｌａｍｉｎｏ）ｂｅｎｚｏｉｃａｃｉｄ,ＮＢＨＡＢ）。通过红外光谱法、核磁共振法、质谱法鉴定了其结构。ＧＬ－７ＡＣＡ酰化酶和青霉素Ｇ酰化酶（ｐｅｎｉｃｉｌｉｎＧＡｃｙｌａｓｅ,ＰＧＡ）及其产生菌对该化合物的作用表明,该物质能够检测头孢菌素酰化酶活力,用来筛选头孢菌素Ｃ酰化酶产生菌,其灵敏性有待进一步提高。     

Direct evaluation of phenylacetyl-CoA: 6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum by bioassay

The enzyme phenylacetyl-CoA: 6-Aminopenicillanic acid acyltransferase of Penicillium chrysogenum was evaluated by direct bioassay against Micrococcus luteus ATCC 9341. The enzyme required dithiothreitol, was inactivated by 0.2 mM Hg2+ (100%), Zn2+ (80%), Cu2+ (60%), 1 mM N-ethylmaleimide (80%), and showed maximal catalytic activity at pH 8.4 and 20 degrees C. The V50 values for phenylacetyl-CoA and 6-aminopenicillanic acid were 0.55 mM and 1 microM, respectively. When octanoyl-CoA was employed as substrate similar results were obtained. In both cases the product generated showed strong antibacterial activity which was quickly lost when incubation was carried out with beta-lactamase. Reactions performed in the presence of Escherichia coli penicillin acylase did not generated active products when phenylacetyl-CoA was the substrate; they did with octanoyl-CoA. Time-course experiments revealed that the highest enzyme levels are found in 36 hours mycelium and remained almost constant from 48 to 96 hours; thereafter the level of the enzyme slowly decreased.     

药物中间体7-氨基头孢烷酸的制备

药物中间体7-氨基头孢烷酸（7-ACA）的工业化生产国内多局限于化学裂解法，由于该法有某些缺点，因此，本文介绍了环保型的酶法催化生产7-ACA的制备方法。该制备方法以头孢菌素C为起始原料，经固定化D-AOD氧化酶和GL-脱酰酶的作用，酶法裂解制得7-ACA，得到7-ACA的总收率达到74％。实验结果说明该方法是可行的，并且具有重要的现实意义。本文还对影响反应的因素做了进一步的讨论。     

Thermal stability of penicillin G acylase : effect of stabilizing agents

The role of sugars, polyhydroxy compounds, phenylacetic acid and 6-aminopenicillanic acid in stabilization of immobilized penicillin G acylase (IMPGA) was studied. The loss in the activity of IMPGA at 50 degrees C, 2 h, after incorporation of sucrose and mannitol at 0.1 M concentration was 16 and 18% respectively; the loss in the activity of the enzyme under these conditions in the absence of stabilizing agents was 40%.     

Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C

Cephalosporin C biosynthesis by Cephalosporium acremonium was delayed until most glucose in the medium was used. Addition of increasing concentrations of glucose up to 55 g/liter decreased cephalosporin C biosynthesis but stimulated growth. Sequential formation of penicillin N (an intermediate in the cephalosporin C biosynthetic pathway) and cephalosporin C was found when the culture was developed synchronously. Little cephalosporin C formation was observed until most penicillin N had already been formed. The sequential formation of penicillin N and cephalosporin C was due to the sequential formation of the "penicillin N synthetase system" and the "cephalosporin C synthetase system". Cells grown in the presence of glucose showed an increased accumulation of penicillin N and clear reduction of the conversion of penicillin N to cephalosporin C. Resting cell studies indicated that the glucose effect was due to the repression of one or more of the enzymes converting penicillin N into cephalosporin C. Little inhibition by glucose of the activity of these enzymes, once formed, was observed. Glucose did not effect significantly the pool sizes of either precursor amino acids of cephalosporin (alpha-aminoadipic acid and valine) or methionine (an inducer of penicillin N and cephalosporin C biosynthesis). On the basis of these data it is suggested that glucose catabolism specifically represses the enzyme system converting penicillin N into cephalosporin C.     

吸附法纯化青霉素酰化酶

目的纯化发酵液中的青霉素酰化酶。方法采用吸附纯化法。结果按 1 2 % (W /V)的比例将皂土加到含青霉素酰化酶的发酵上清液中 ,可将 98%的酶吸附 ,而同时吸附的杂蛋白质仅占发酵上清液总蛋白质的 14 5 %左右。吸附过程受pH影响较大 ,在 pH 6 5～ 7 5时 ,吸附效果最佳。使用不同种类的缓冲液洗涤酶 -皂土复合物 ,发现缓冲液的种类对酶的洗涤影响不大。使用含5 0 %以上的PEG和 8 0 %的NaCl的磷酸缓冲液可以将酶全部洗脱。结论此法可在常温下操作 ,酶活力收率较高 ,具有潜在的工业应用价值     

Biosynthesis of β-lactam antibiotics by cell-free extract fromLysobacter lactamgenus

Using cell-free extract ofLysobacter lactamgenus, enzymatic conversion of δ-l-(α-aminoadipyl)-l-cysteinyl-d-valine (ACV), the first substrate of β-lactam biosynthesis, into antibiotic compounds was attempted. In high performance liquid chromatographic (HPLC) analysis, the biosynthetic intermediates for cephalosporin antibiotics including isopenicillin N, deacetoxycephalosporin C, deacetylcephalosporin C and unknown cephem compound were detected in reaction mixtures. It implies that cephabacin compounds fromL. lactamgenus could be produced by biosynthetic routes through penicillin ring formation and its expansion to cephalosporin ring, likely as cephalosporin C fromCephalosporium or cephamycin C fromStreptomyces. Among biosynthetic enzyme in cell-free extract, the ring formation activity (isopenicillin N synthetase activity) was separated in 50–60% of ammonium sulfate fraction, and ring expansion activity (deacetoxycephalosporin C synthetase activity) was found to be in 40–50% fraction. The partially purified isopenicillin N synthetase could convert as much as 90% ACV to isopenicillin N during 6-hour reaction.     

Acyl-CoA: 6-APA acyltransferase from Penicillium chrysogenum studies on its hydrolytic activity.

Acyl-CoA: 6-APA acyltransferase (AT) from Penicillium chrysogenum Wis 54-1255 catalyzes the hydrolysis of different acyl-CoA derivatives generating, in the absence of 6-APA, free acid and CoA. The hydrolytic efficiency of AT is highest for acyl-CoA variants in which the acyl-moiety is higher than six carbon atoms. The maximal rate of catalysis was achieved in 50 mM Tris-HCl buffer, pH 8.5 at 35 degrees C. Unlike the AT activity, the acylase activity has a different optimum temperature and substrate specificity and dithiothreitol is not required for the reaction.     

穿龙薯蓣内生真菌分离鉴定及其抑菌活性研究

目的 分离纯化穿龙薯蓣内生真菌,并对分离得到的内生真菌进行抑菌活性筛选及分类鉴定。方法 采用组织分离法纯化穿龙薯蓣内生真菌,采用载片培养法、挑片培养法、插片培养法,对照《真菌鉴定手册》和《半知菌纲手册》进行初步鉴定;以金黄色葡萄球菌Staphylococcus aureus、白色葡萄球菌Staphylococcus albus、柠檬色葡萄球菌Staphylococcus citreu、四联球菌Micrococcus tetragenus、大肠杆菌Escherichia coli、伤寒杆菌Salmonella typhi、铜绿色假单胞菌Pseudomonas aeruginosa、变形杆菌Bacterium termo、肺炎杆菌Bacillus endocarditidis capsulatus、福氏痢疾杆菌Shigella flexneri、乙型副伤寒杆菌Bacterium paratyphosum B作为受试对象,检测内生真菌菌株发酵液以及菌丝提取物的抑菌活性,应用分子生物学方法对筛选出的活性菌株进行分类鉴定。结果 从穿龙薯蓣叶片中分离得到52株内生真菌,根据形态学特征将10株产孢子的内生真菌初步鉴定为2个目、3个科、9个属;其中有3株内生真菌的菌丝提取物具有广谱抗菌活性,结合形态学和分子生物学特征鉴定C37菌为赤霉属Gibberella真菌,C30和C50菌为链格孢属Alternaria真菌。结论 穿龙薯蓣叶片中存在具有抑菌活性的内生真菌,具有开发抑菌生物制剂的潜力。     

游离态和固定态青霉素酰化酶对环境的敏感性

在优化的固定化条件下，通过戊二醛交联直接将青霉素酰化酶固定化。比较了环境因素对游离酶和两种化酶活性的影响，结果表明青霉固定化后，抗环境变化的能力明显提高。现任中固定化酶活化的比较说明由游离酶直接交联得到的固定化酶更适合工业应用。     

Industrial landmarks in the development of sustainable production processes for the β-lactam antibiotic key intermediate 7-aminocephalosporanic acid (7-ACA)

Reflecting its importance as key intermediate for the large scale manufacture of β-lactam-type antibiotic drugs being widely used in antibacterial therapy, tremendous achievements have been made over the past decades in developing both, economically and ecologically attractive production routes for 7-aminocephalosporanic acid (7-ACA). Representative achievements and industrial landmarks thereof are described in this review. With cephalosporin C a starting material being accessible through fermentation is readily available in bulk quantities. Initially a chemical production process in organic medium based on various reagents for protection group chemistry and acyclic amide activation served as the large scale production technology for transforming cephalosporin C into 7-ACA. Subsequently, a bi-enzymatic production in aqueous reaction medium based on the use of a D-amino acid oxidase for side chain modification and glutaryl acylase for side chain cleavage was developed. This biocatalytic approach also turned out as an industrial scale solution and dramatically reduced the amount of waste from 31 kg (for the "original chemical process") to less than 1 kg. The latest innovation has been the extension of the two-step bi-enzymatic process towards a one-step monoenzymatic process based on a direct cleavage of the acyclic amide bond of cephalosporin C by means of a cephalosporin C acylase. This alternative, economically efficient and sustainable biotechnological process has already been established on industrial scale in recent years. These biotechnological achievements represent industrial landmarks in biocatalysis and examples that modern biocatalysis can contribute to the development and implementation of highly economical as well as sustainable industrial manufacturing processes.     

Tissue distribution of and species differences in deacetylation of N-acetyl-L-cysteine and immunohistochemical localization of acylase I in the primate kidney

Species differences in the biotransformation of N-acetyl-L-cysteine (NAC) have been investigated to evaluate the usefulness of NAC as a constituent in parenteral nutrition solutions in place of cysteine. The activity of NAC-deacetylating enzyme (acylase) was measured in various tissues of different species (rat, rabbit, dog, monkey, and man). Acylase activity was highest in the kidney in all species studied. Enzyme activity in the liver was 10 %-22 % of that in the kidney in the rat, rabbit, monkey, and man, but almost no hepatic activity was seen in the dog. NAC-deacetylating activity was very low in other organs. The tissue distribution of acylase I was determined by Western blotting and an immunohistochemical method employing specific antibody against porcine acylase I (EC 3.5.1.14). The immunoblotting study showed a 46-kDa protein band corresponding to porcine acylase I in the kidney of all species. In liver cytosol, 46 kDa and/or 29 kDa bands were observed in the rat, rabbit, monkey, and man, but not in the dog. In the immunohistochemical study, positive staining with anti-acylase I antibody was observed clearly in the renal proximal tubules in the monkey and man. These results suggested that the kidney and liver were the main organs responsible for the biotransformation of NAC to cysteine in mammals other than the dog.     

Synthesis of benzylpenicillin by cell-free extracts from Streptomyces clavuligerus

Cell-free enzyme concentrates from Streptomyces clavuligerus were found to convert phenylacetyl-L-cysteinyl-D-valine (PCV) directly into benzylpenicillin when incubated under reaction conditions which support the activity of isopenicillin N synthetase. The formation of benzylpenicillin was detected both by biological assay and by high performance liquid chromatography. Supplementation of PCV-containing reaction mixtures with cofactors required for ring expansion activity did not result in the production of cephalosporins. Incubation of phenoxyacetyl-L-cysteinyl-D-valine (PoCV) under the same reaction conditions did not result in the formation of penicillin V or any cephalosporin product.     

我院生化药物和酶诊断试剂的研制

概述近２０年我院在药用酶（胶原酶，链激酶，尿激酶，弹性酶，溶菌酶）、工业用酶（青霉素酰化酶，头孢菌素酰化酶，乙内酰脲酶）、诊断用酶（胆固醇，甘油三酯，肌酸激酶，乳酸脱氢酶，葡萄糖和尿素氮的测定）及透明质酸方面的研究开发     

Studies on the biosynthesis of carbapenem antibiotics. III. Enzymological characterization of the L-amino acid acylase activity of A933 acylase

A933 acylase, which is involved in exchange of the pantothenyl substituent of OA-6129 carbapenems with acetyl CoA, was characterized as an L-amino acid acylase with a molecular weight of 100,000 (+/- 8,000) and a pI value of 5.1. The highest L-amino acid acylase activity of A933 acylase was observed at 37 degrees C and pH 7 approximately 7.5 for N-chloroacetyl-L-phenylalanine. Unlike other amino acid acylases, A933 acylase was severely inhibited by cobalt ions and p-chloromercuribenzoate. The acylase also showed peptidase activity with some di- and tripeptides. A protein fraction with A933 L-amino acid acylase activity from blocked mutant 1501 lacked OA-6129A-depantothenylating activity.     

Sulfur metabolism of a mutant of Cephalosporium acremonium with enhanced potential to utilize sulfate for cephalosporin C production

Characteristics of a mutant of Cephalosporium acremonium with enhanced potential to utilize sulfate for cephalosporin C production were investigated with sulfur-starved cells. DL-Norleucine showed an inhibitory effect on cephalosporin C and penicillin N production by the mutant in the presence of a sulfur source such as sulfate, sulfite, thiosulfate, and L-cystine, but it exhibited no effect when it was added after a certain period of incubation. On the contrary, antibiotic production by the parent was stimulated by norleucine regardless of the addition time. An increase in the intracellular cysteine pool was found when the cells were incubated with L-methionine or norleucine and sulfate. Enzymatic studies revealed that methionine and norleucine stimulated the cysteine desulfhydrase formation, and this effect was significant in the mutant. Finally the mutant was found to have an enhanced L-serine sulfhydrylase activity. The increase in this enzyme activity in the mutant seems responsible for the increase in the sulfate-utilizing ability and the methionine sensitivity by maintaining a high level of the cysteine pool. Accordingly, the effect of methionine and norleucine is assumed to be exerted through cysteine.     

头孢氨苄酶法合成影响因素概述

头孢氨苄作为第一代口服的头孢菌素类抗生素,对多种革兰阳性菌和革兰阴性菌均有很强的抗菌活性。目前我国制药工业中生产头孢氨苄主要是化学合成法,合成过程步骤繁琐,对环境污染严重。与传统化学合成法相比,生物酶法合成头孢氨苄具有反应条件温和、工艺简便、绿色环保、洁净安全等优点。本文概述了酶法合成头孢氨苄固定化酶的制备,合成过程中各个反应物浓度、温度、pH、投酶量、非水介质对反应转化率的影响以及酶法合成头孢氨苄存在的问题。     

The influence of cefpirome on intestinal bacterial flora

Cefpirome (CPR, HR810), a new parenteral cephalosporin antibiotic, was studied for its effect on the intestinal bacterial flora in pediatric patients. The subjects were children admitted for infections (6 males and 3 females, 1 month to 5 years 1 month old, weighted 3.94 to 21.0 kg). CPR was intravenously administered at a dose between 19.0 to 40.0 mg/kg, 3 to 4 doses daily over 6 to 12 days. The feces from these children were collected before, during, and after administration, and bacteria were identified and counted. CPR concentration, beta-lactamase activity, and Clostridium difficile D-1 antigen were also assayed. Bacterial flora changes in feces during CPR administration showed some variance, but generally 5 cases out of the 9 showed a significant decrease in Enterobacteriaceae and Enterococcus faecalis among aerobic bacteria. The other 4 cases showed some transient decrease, but no significant change was observed. No significant changes were recognized for Enterococcus avium and Enterococcus faecium, and the total aerobic bacterial count decreased in a transient manner in only one patient. Regarding anaerobic bacteria, Bifidobacterium and Eubactrium revealed a significant decrease, a transient decrease or no change from case to case. Bacteroides showed little change in count. Consequently, the total anaerobic bacteria count did not reveal a large change aside from 1 case in which Bacteroides was not detected before administration and a significant decrease of other bacteria was noted. In no case, glucose nonfermentative Gram-negative bacilli or fungi were found dominant. Although C. difficile and C. difficile D-1 antigen were detected in 3 and 4 cases, respectively, there was no exact relationship between the number of C. difficile and the characteristics of the feces. CPR was detected in fecal samples from 6 cases during administration with concentrations ranging between 1.20 to 22.4 micrograms/g. High values of CPR tended to be found in specimens with low beta-lactamase activity in the feces. When drug sensitivities of the bacteria isolated from feces before and after administration were compared, higher levels of resistance were found in some bacteria such as Enterococci and Bacteroides during or after administration than before administration. The above results suggest that CPR is a drug with a relatively small influence on the intestinal bacterial flora in children, but a particular attention is required for diarrhea and microbial replacement during a continuous, long-term administration of the drug.