Hepatic steatosis is one of the most important causes of liver disease worldwide. Heat shock protein 90 (HSP90) is essential for numerous client proteins. Recently, more attention was focused on increased HSP90 levels in hepatic steatosis, especially HSP90β. Thus, great efforts have been made to develop HSP90β inhibitors, and most natural inhibitors are derived from microorganisms. In this study, using microarray chips and surface pasmon resonance (SPR) technology, we screened 189 antibiotics in order to obtain an inhibitor directly binding to the non-N-terminal domain of HSP90β. Finally, we discovered an antibiotic, 7-aminocephalosporanic acid (7ACA), with a KD value of 6.201 μM between 7ACA and non-N-terminal domain of HSP90β. Besides, 7ACA was predicted to interact with the middle domain (MD) of HSP90β. In HepG2 cells, we found that 7ACA reduced cellular total cholesterol (TC) and triglyceride (TG) by decreasing sterol regulatory element-binding proteins (SREBPs). In HFD fed mice, administration of 7ACA (5, 10, and 25 mg kg-1 d-1, ig, for 12 weeks) dose-dependently decreased serum TC and TG and played an important role in protecting liver and adipose tissue from lipid accumulation. In conclusion, our study demonstrated that antibiotic 7ACA, as an HSP90β middle domain inhibitor, was promising for the development of lipid-lowering drugs.

The pharmaceutical industry has developed various highly effective semi-synthetic cephalosporins, which are generated by modifying the side chains of the core molecule 7-aminocephalosporanic acid (7-ACA). In industrial productions, the 7-ACA nucleus is obtained in vitro from cephalosporin C (CPC) by chemical or enzymatic processes, which are waste intensive and associated with high production costs. Here, we used a transgenic in vivo approach to express bacterial genes for cephalosporin C acylase (CCA) in the CPC producer Acremonium chrysogenum. Western blot and mass spectrometry analyses verified that the heterologous enzymes are processed into α- and β-subunits in the fungal cell. Extensive HPLC analysis detected substrates and products of CCAs in both fungal mycelia and culture supernatants, with the highest amount of 7-ACA found in the latter. Using different incubation times, temperatures, and pH values, we explored the optimal conditions for the active bacterial acylase to convert CPC into 7-ACA in the culture supernatant. We calculated that the best transgenic fungal strains exhibit a one-step conversion rate of the bacterial acylase of 30%. Our findings can be considered a remarkable contribution to supporting future pharmaceutical manufacturing processes with reduced production costs.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic β-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC2 to p-NPC8. Maximum enzyme activity was recorded at pH 9.0 and 50 °C, where Km and Vmax were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 μM min-1, respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3\' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.

OBJECTIVE: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation.
RESULTS: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the α/β hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 °C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the Kcat/Km value was 2.04 s-1 mM-1.
CONCLUSIONS: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.

BACKGROUND: Cephalosporins are the most widely used semisynthetic antibiotics, which acted on bacterial cell wall (peptidoglycan) synthesis. The key intermediate for fabricating about twothirds of cephalosporins in clinical use is 7-aminocephalosporanic acid (7-ACA), which is derived from chemical or enzymatic deacylation of the natural antibiotic cephalosporin C (CPC). The chemical deacylation process has been replaced by the enzymatic deacylation process because the chemical process required harsh conditions and released toxic waste.
METHODS: A two-step enzymatic process that utilized D-amino acid oxidase (DAAO) and 7-β-(4carboxybutanamido)-cephalosporanic acid acylase (GLA) for two successive reactions has been applied for the conversion of CPC to 7-ACA in an industrial scale.
RESULTS: To simplify the process and lower costs, the one-pot enzymatic processes were developed by the application of the mono-enzymatic process (application of cephalosporin C acylase or the variants of GLA), di-enzymatic process (simultaneous action of DAAO and GLA) or the tri-enzymatic process (simultaneous action of DAAO, GLA and catalase) for direct conversion of CPC to 7-ACA.
CONCLUSIONS: Here, we mainly focused on the description of these one-pot enzymatic processes and emphasized on the preparation of the involved biocatalysts.

Objective: To observe serum levels of periostin, ECP, IgE in the antibiotic enterprise workers, and study the role of periostin, ECP, IgE in the development of allergic inflammation. Methods: 90 cases with asthma or rhinitis were enrolled as disease group, another 117 workers exposed to 7-ACA、6-APA dust without suffering from allergic illness, are chosen as group of dust exposed, and 192 healthy workers who didn\'t contact dust were chosen as control group. Questionnaires were used to learn their basic information.Lung function was determined with a portable spirometer.The expression levels of periostin、ECP and IgE in serum were measured by enzyme-linked immuno sorbent assay. Results: The exposure group and disease group had significantly lower forced vital capacity (FVC) , forced expiratory volume in 1 second (FEV(l.0)) , and FEV(l.0)/FVC ratio than the control group (P<0.05) . The disease group had significantly higher eosinophil than the control group (P<0.05) . Compared with the control group, the exposure group, the disease group, asthma subgroup, rhinitis subgroup of serum periostin and IgE increased, the differences are statistically significant (P<0.05) . Serum levels of ECP in the workers of asthma subgroup were significantly higher than that in control group (P<0.05) . Serum expression levels of periostin were positively correlated with IgE, ECP in workers (P<0.001) , serum levels of periostin were negatively correlated with FEV(1.0) in workers (P<0.05) . Multiple logistics regression analysis found that exposure to 7-ACA or 6-APA (OR=3.09, 95%CI: 1.83-5.21) , age>47years (OR=2.53, 95%CI: 1.22-5.26) , higher ECP (OR=1.04, 95%CI: 1.01-1.06) were risk factors for increased serum periostin level. Conclusion: Occupational exposure to 7-ACA or 6-APA can result in higher serum periostin level, exposure to 7-ACA or 6-APA, age>47 years, higher ECP are risk factors for increased serum periostin level.
目的： 观察β-内酰胺类抗生素接触工人血清骨膜蛋白（periostin）、嗜酸粒细胞阳离子蛋白（ECP）、免疫球蛋白E（IgE）水平，探讨其在过敏性炎症发生发展中的作用。 方法： 选取90名接触7-氨基头孢烷酸（7-ACA）、6-氨基青霉烷酸（6-APA）粉尘哮喘或过敏性鼻炎患者为病例组（哮喘亚组32人，鼻炎亚组58人），选取117名接触7-ACA、6-APA粉尘无哮喘或鼻炎等过敏性疾病工人为接触组，选择192名不接触粉尘体检健康者作为对照组。通过问卷调查基本情况，便携式肺功能仪测定肺功能，ELISA法测定血清骨膜蛋白、ECP和IgE水平。 结果： 接触组和病例组工人用力肺活量（FVC）、第1秒用力呼气容积（FEV(1.0)）及FEV(1.0)/FVC均低于对照组，差异有统计学意义（P<0.05）。病例组嗜酸粒细胞计数明显高于对照组，差异有统计学意义（P<0.05）。接触组、病例组、哮喘亚组、鼻炎亚组血清骨膜蛋白、IgE水平均高于对照组，哮喘亚组血清ECP水平高于对照组，差异有统计学意义（P<0.05）。血清骨膜蛋白与IgE、ECP均呈正相关（P<0.01），血清骨膜蛋白与FEV(1.0)呈负相关（P<0.05）。多元logistic回归分析发现，接触7-ACA或6-APA（OR=3.09，95%CI：1.83~5.21）、年龄>47岁（OR=2.53，95%CI：1.22~5.26）、ECP增高（OR=1.04，95%CI：1.01~1.06）为血清骨膜蛋白升高的危险因素。 结论： 职业接触7-ACA、6-APA可导致血清骨膜蛋白表达水平升高，接触7-ACA或6-APA、年龄>47岁、ECP增高可作为骨膜蛋白升高的危险因素。.

BACKGROUND: Enzymatic approaches have become promising alternatives to chemical methods for the production of semi-synthetic β-lactam antibiotics. In this work, enzymatic synthesis of N-bromoacetyl-7-aminocephalosporanic acid (N-bromoacetyl-7-ACA), the key intermediate for the production of cefathiamidine, was reported for the first time.
RESULTS: Of the immobilized penicillin acylases (PAs) tested, PGA-750 was the best biocatalyst. Optimization of the biocatalytic process was conducted. The optimal acyl donor, molar ratio of acyl donor to 7-ACA, pH, temperature, 7-ACA concentration, and enzyme dosage were methyl bromoacetate, 3, 7.5, 20 °C, 50 mmol/L and 4 U/mL, respectively. Under the optimal conditions, enzymatic N-acylation of 7-ACA with methyl bromoacetate afforded the desired product with the yield of 85% in 2 h, where the synthesis/hydrolysis (S/H) ratio was approximately 1.5. The immobilized enzyme PGA-750 exhibited good operational stability, and the relative yields of approximately 90% and 63% were achieved, respectively, when it was reused in 7th and 11th batch.
CONCLUSIONS: An enzymatic approach to N-bromoacetyl-7-ACA, the key intermediate for the industrial production of cefathiamidine, has been developed successfully in a fully aqueous medium. The present work may open up a novel opportunity for the production of cefathiamidine through a simple and green process.Graphical abstractEnzymatic synthesis of N-bromoacetyl-7-ACA, the key intermediate for the production of cefathiamidine, was reported for the first time.

Ethyl 4-amino-2-fluorophenylpiperazin-1-carboxylates containing a 1,3-oxazol(idin)e, 5-thioxo-1,2,4-triazole, 1,3,4-thiadiazole, 5-thioxo-1,3,4-oxadiazole, or 1,3-thiazole nucleus were obtained starting from ethyl piperazine-1-carboxylate (1) by several steps. The treatment of amine, 3 or hydrazide, 9 with several aromatic aldehydes generated the corresponding arylmethyleneamino (3a-f) or arylidenehydrazino (12a-c) compounds. The Mannich reaction between the 1,2,4-triazole or 1,3,4-oxadiazole compounds and 7-aca produced cephalosporanic acid derivatives. Penicillanic acid derivatives were obtained when 6-apa was used in the Mannich reactions. The synthesized compounds were screened for their antimicrobial, antilipase, and antiurease activities. Some of them were found to possess good-moderate antimicrobial activity against the test microorganisms. Two compounds exhibited antiurease activity, and four of them displayed antilipase activity.

The treatment of 7-ACA with 4-substituted benzensulfonyl chlorides afforded the compounds containing 4-nitro/aminophenyl sulfonylamino moiety in the cephalosporanic acid skeleton (2, 4). The synthesis of the cephalosporanic acid derivatives containing 1,3-thiazole or 5-oxo-1,3-thiazolidine nucleus and sulfonamide function (8a, 8b, 10) was performed starting from 7-ACA by several steps. The reaction of 7-ACA with [4-(2-fluoro-4-nitrophenyl)piperazin-1-yl]acetyl chloride afforded the corresponding 7-{[4-(2-fluoro-4-nitrophenyl)piperazin-1-yl]acetyl}amino derivative (13). The synthesized compounds were screened for their antimicrobial and antiurease activities. Some of them were found to possess good-moderate antimicrobial activity against the test microorganisms. Compound 5d was observed to have moderate anti-urease activity.

In light of unrestricted use of first-generation penicillins, these antibiotics are now superseded by their semisynthetic counterparts for augmented antibiosis. Traditional penicillin chemistry involves the use of hazardous chemicals and harsh reaction conditions for the production of semisynthetic derivatives and, therefore, is being displaced by the biosynthetic platform using enzymatic transformations. Penicillin G acylase (PGA) is one of the most relevant and widely used biocatalysts for the industrial production of β-lactam semisynthetic antibiotics. Accordingly, considerable genetic and biochemical engineering strategies have been devoted towards PGA applications. This article provides a state-of-the-art review in recent biotechnological advances associated with PGA, particularly in the production technologies with an emphasis on using the Escherichia coli expression platform.