青霉素G酰化酶在γ-氧化铝上的吸附交联固定化

以 γ-氧化铝为载体 ,利用戊二醛对巨大芽孢杆菌 (Bacillus megaterium) Bp931胞外青霉素 G酰化酶进行吸附交联固定化。酶活力回收为 31% ,在 37℃、p H8.0的条件下 ,固定化酶对青霉素 G的活力为30 4IU/ g;作用于青霉素 G的表观米氏常数 Km值为 3.2 3× 10 - 2 M。该固定化酶可耐高离子强度洗涤 ,连续合成头孢拉定 (cephradine) 10批次 ,酶活力剩余 82 %     

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。     

固定化青霉素酰化酶合成阿莫西林活性测定

固定化青霉素酰化酶的活性高低是影响酶催化合成阿莫西林速度的决定性因素,如何测定固定化青霉素酰化酶的合成活性起着至关重要的作用。目前,对青霉素酰化酶活性的测定主要集中在青霉素酰化酶的水解活性,而对其合成阿莫西林活性的研究较少,这导致不能快速的鉴别青霉素酰化酶合成阿莫西林的快慢,不能有效的指导生产。文章对固定化青霉素酰化酶合成阿莫西林的活性进行了定义,并研究了合成活性的测定方法,该测定方法准确可靠,操作简捷,便于比较不同厂家生产的青霉素酰化酶活性的高低,对工业生产起指导作用。     

青霉素G酰化酶在γ—氧化铝上的吸附交联固定化

以γ-氧化铝为载体,利用戊二醛对巨大芽孢杆菌(Bacillus megaterium)Bp931胞外青霉素G酰化酶进行吸附交联固定化.酶活力回收为31%,在37 C、pH8.0的条件下,固定化酶对青霉素G的活力为304IU/g;作用于青霉素G的表观米氏常数Km值为3.23×10-2M.该固定化酶可耐高离子强度洗涤,连续合成头孢拉定(cephradine)10批次,酶活力剩余82%.     

青霉素V酰化酶在生产6—APA中的潜力

目前发酵生产的青霉素G(Pen G)和青霉素V(Pen V)多半是用作生产β-内酰胺中间体6-氨基青霉烷酸(6-APA)和7-氨基脱乙酰氧头孢烷酸(7-ADCA)的原料.有效、稳定的固定化青霉素酰化酶的开发,为合成具有不同抗菌性能的6-APA和7-ADCA衍生物创造了有利条件.现6-APA年产量约7500t,为此需消耗10~30t固定化青霉素酰化酶.     

固定化青霉素酰化酶反应器的研制和应用(英文)

目的研制一个全新的 2 0 0L不锈钢固定化青霉素酰化酶反应器并在中试中应用。方法在罐内壁上安装三个盛酶柱 ,每根酶柱装入湿纤维状固定化酶 1 5kg,酶活力 76 2IU·g-1(以湿品为基础计算 )。以HBO3 作缓冲液 ,底物青霉素G钾的质量浓度为 80 g·L-1。每批投料 12kg,每批裂解反应 90min。结果 15批之后 ,剩余酶活力 6 85IU·g-1(以湿品为基础计算 ) ,6 APA收率平均达 89 8%。结论 6 APA生产中使用新的固定化青霉素酰化酶反应器将提高 6 APA的收率 ,减少固定化青霉素酰化酶的损耗。     

固定化青霉素G酰化酶的初步研究

本文研究固定化青霉素G酰化酶的不同制备方法,比较了各种固定化青霉素G酰化酶的载体。实验表明:聚丙烯腈纤维和无机载体制成的固定化酶具有较高的酶比活力,而且表面积大,易于装柱,抗污染能力大,又能裂解高浓度青霉素 G 溶液生成6-APA。     

环氧化磁性羟基微球的制备及在固定化青霉素G酰化酶中的应用

采用悬浮聚合法合成了一系列磁性羟基微球,以环氧氯丙烷活化后用于固定化青霉素G酰化酶.交联度为30%的微球固定化酶效果最优,并得到最适固定化条件为:给酶量2000u/g,反应时间24h,反应温度35℃,pH 8.0.制得固定化酶酶活力为782.2u/g.水解青霉素G钾盐的最适pH 8.5,最适温度45℃.     

用CHITOSAN固定化青霉素酰化酶

本文报道以Chitosan为载体制备固定化青霉素酰化酶的研究结果。1.4％ Chitosan醋酸溶液先用12.5％戊二醛交联,洗涤,研磨,得颗粒。再用1.5％pH5.5和1.5％pH8.5多聚磷酸处理,得机械强度良好的载体。此载体先后经0.5％戊二醛和对甲苯磺酰氯双活化后,再与青霉素酰化酶偶联。所得固定化酶的活力达20.8u/g。固定化青霉素酰化酶的反应最适温度略高于游离酶,最适pH略低于游离酶;其Km值基本接近游离酶。固定化酶经反复使用,活力基本不变。     

青霉素酰化酶制备6-APA的研究进展

6-氨基青霉烷酸（6-APA）是重要的抗生索药物中间体之一．目前均采用青霉素酰化酶酶促裂解青霉素获得。本文介绍近年来青霉索酰化酶催化青霉素水解的研究进展，青霉素酰化酶的性质及其催化机理，青霉素酰化酶的固定化方法．青霉素酰化酶反应器的设计，反应介质工程的研究进展。     

胰蛋白酶的固定化及其在玻璃酸制备中的应用

纸纤维为载体,对β－硫酸酯乙砜基苯胺为活化剂,以共价介联法固定化胰蛋白酶。在偶联反应ｐＨ８．０、酶量７．２６ｍｇ／ｍｌ的最佳固定化条件下,固定化酶活力达９３．３Ｕ／ｇ（湿纸纤维）,酶活回收率为５７．５％。固定化酶具有较高热稳定性和抗氯仿能力。采用固定化胰蛋白酶酶解工艺,所得玻璃酸成品符合质量标准,其酶解反应半衰期为４８０ｈ。     

Concanavalin A layered calcium alginate-starch beads immobilized beta galactosidase as a therapeutic agent for lactose intolerant patients

A novel therapeutic agent in the form of beta galactosidase immobilized on the surface of concanavalin A layered calcium alginate-starch beads has been developed. Immobilized beta galactosidase exhibited significantly very high stability against conditions of digestive system such as pH, salivary amylase, pepsin and trypsin. Soluble and immobilized beta galactosidase exhibited same pH-optima. However, the immobilized enzyme retained greater fraction of catalytic activity at higher and lower pH to pH-optima as compared to soluble enzyme. Immobilized enzyme preparation was quite stable under conditions present in mouth, stomach and intestine. Immobilized beta galactosidase retained 65% activity even after its sixth repeated use.     

Glucuronidation of the dietary fatty acids, phytanic acid and docosahexaenoic acid, by human UDP-glucuronosyltransferases.

Linoleic acid has recently been shown to be glucuronidated in vitro by human liver and intestinal microsomes and recombinant UGT2B7. In the present study, the dietary fatty acids (FA), phytanic acid (PA), and docosahexaenoic acid (DHA) have been used as substrates for human UDP-glucuronosyltransferases (UGTs). Both compounds were effectively glucuronidated by human liver microsomes (HLM; 1.25 +/- 0.36 and 1.12 +/- 0.32 nmol/mg x min for PA and DHA, respectively) and UGT2B7 (0.71 and 0.53 nmol/mg x min). Kinetic analysis produced relatively low K(m) values for PA with both HLM and UGT2B7 (149 and 108 microM, respectively). The K(m) for DHA glucuronidation by HLM (460 microM) was considerably higher than that for UGT2B7 (168 microM), suggesting the involvement in microsomes of other UGT isoforms in addition to UGT2B7. Glucuronidation of PA and DHA by gastrointestinal microsomes from 16 human subjects was determined. In general, both PA and DHA were glucuronidated by gastric and intestinal microsomes, and activity toward both substrates was lowest in the stomach, increased in the small intestine, and lower in the colon. However, there were large interindividual variations in UGT activity toward both substrates in all segments of the intestine, as has been seen with other substrates. Thus, PA and DHA are effective in vitro substrates for human liver, gastric and intestinal microsomes, and glucuronidation may play a role in modulating the availability of these FA as ligands for nuclear receptors.     

人参皂苷Rb2促进牛主动脉内皮细胞纤溶活性

AIM:The effect of ginsenoside Rb2 purified from Panax ginseng on fibrinolytic activity of bovine aortic endothelial cells (BAEC) was in vestigated.METHODS:Cellular plasminogen activator(PA) level of the lysates was measured by the chromogenic substrate S-2403.Fibrin underlay technique was carried out to observe fibrinolysis by growing endothelial cells in the culture medium.Cella viability was the determined by measurement of the activity of mitochondrial dehydrogenase.The ability of Rb2 of potentiating cellular PA activity was investigated by measuring the amounts of PA and PA inhibitor-1(PAI-1) in the culture medium using zymography and reverse zymography.Changes in the expression of urokinase-type PA (uPA),uPA receptor,and PAI-1 mRNA in BAEC atfer treatment with Rb2 were analyzed by Northern blot,RESULTS:Rb2 enhanced cellular PA activity in a concentration-and time-dependent manner.Treatment of BAEC with Rb2 10 mg/L for 9 h resulted in a 3.5-fold increase of PA activity without a marked cytotoxic effect,as shown by LDH levels in culture.Increased PA levels caused the increase in surface plasmin levels as observed by fibrin underlay tecnique Rb2 greatly or moderately increased the amount of urokinase-type PA(uPA) or its inhibitor (PAI-1),present in the culture medium,whereas saponin did not influencce mRNA levels of uPA,its surface receptor,and PAI-1,suggesting that Rb2 may stimulate the secretion of uPA without enhancing its gene expression.The enhancement of PA levels by retinoic acid alone,a stimulator of PA synthesis,was potentiated by the simultaneous addition of ginsenoside Rb2 1 mg/L.Therefore, Rb2 might exert a strong synergism in the synthesis of cellular PA in BAEC.CONCLUSION:Ginsenoside Rb2 enhanced the PA activity levels in BAEC as well as the surface plasmin actvity of BAEC.Rb2 may stimulate the secretion of uPA without enhancing the gene expression of uPA,uPA receptor (uPAR),and PAI-1.     

Separation of three exotoxic factors of Bacillus anthracis by sequential immunosorbent chromatography

Protective antigen and lethal factor components were isolated directly from crude culture supernatant of Bacillus anthracis by sequential immunosorbent chromatography using immobilized monoclonal antibodies (MAB) against the respective toxins. The immunological activity of protective antigen, lethal factor and edema factor were purified by 1.2-, 6.3- and 2.3-fold, respectively, with recoveries of 63, 70 and 46%, respectively. All three components retained biological activity when combined to form lethal toxin or edema toxin, PA + LF and PA + EF, respectively, after the purification process, and were not contaminated with any of the other components. The order of immunosorbent columns during the purification process was found to be important. The best results were obtained when the protective antigen was removed initially from the crude culture supernatant.     

Nanostructured Lipid Carriers for Oral Delivery of a Corticosteroid: Role of Formulation on Biopharmaceutical Performance

Corticosteroids are potent anti-inflammatory and immunosuppressive drugs widely used world-wide for treatment of diverse conditions. However, their use is restricted by their poor bioavailability and high risk-benefit ratio. Therefore, the aim of this study was to develop nanostructred lipid carriers (NLC) of prednisolone acetate (PA) to improve the drug's therapeutic outcome by altering its pharmacokinetic profile and/or allow preferential targeting to inflammatory tissues. PA-loaded NLCs were formulated by solvent injection method using Compritol (solid lipid), oleic acid (liquid lipid) and Tween 80 or Pluronic F68 (surfactant). Formulation conditions, such as liquid lipid concentration, total lipids, drug:lipid ratio and surfactant type were optimized based on particle size (PS), polydispersity index (PDI), and encapsulation efficiency (EE%) results. Optimized formulation was further characterized for its surface morphology, thermal properties, storage stability and anti-inflammatory activity in an animal acute inflammation model. Selected NLCs displayed PS of 170.7 nm, EE% of 67.4%, sustained release over 72 h and good stability for 30 days at refrigeration conditions. PA NLCs displayed superior anti-inflammatory activity of 83.9 ± 4.46% compared to PA suspension (40.5 ± 7.03%) and drug-free NLCs (54.7 ± 6.12%). The current work delineates the potential of NLCs for distinctly improved biopharmaceutical performance of PA.     

Proanthocyanidin from grape seeds potentiates anti-tumor activity of doxorubicin via immunomodulatory mechanism

The purpose of this study was to investigate the anti-tumor activities of proanthocyanidin (PA) from grape seeds and doxorubicin (DOX) in vitro as well as in vivo, either alone or in combination and to explore the immunomodulatory mechanism in tumor-bearing mice. PA (12.5 approximately 200 mg/l) or DOX (0.01 approximately 1 mg/l) for 24 h significantly inhibited YAC-1 cell proliferation (IC(50) 57.53 or 0.198 mg/l, respectively) in a concentration-dependent manner using microculture tetrazolium (MTT) assay. Meanwhile, a combination of PA (12.5, 25 mg/l) with DOX strongly inhibited cell proliferation with IC(50) values of DOX decreasing by 0.09 and 0.045 mg/l, respectively. In mouse tumor xenograft models, intraperitoneal administrations of PA (10 mg/kg) daily or DOX (2 mg/kg) every other day for 9 days significantly inhibited the growth of sarcoma 180, whereas a combination of the two strongly inhibited tumor growth as compared with PA or DOX alone (p<0.01). In contrast to PA treatment, DOX inhibited Con A-stimulated lymphocyte proliferation, IL-2 and IFN-gamma productions, NK cell cytotoxicity and CD4+/CD8+ ratio, while the administration of PA combined with DOX significantly enhanced the above immune responses as compared with the tumor-bearing control (p<0.01). Taken together, these results suggest that PA has anti-tumor activity and increases the anti-tumor activity of DOX, and the mechanism might be related partially to immunopotentiating activities through the enhancements of lymphocyte proliferation, NK cell cytotoxicity, CD4+/CD8+ ratio, IL-2 and IFN-gamma productions.     

The anti-tumor effect of pachymic acid on osteosarcoma cells by inducing PTEN and Caspase 3/7-dependent apoptosis

Pachymic acid (PA) is a lanostane type triterpenoid isolated from Poria cocos, which possesses an anti-tumor effect in breast cancer, prostate cancer, lung cancer, and bladder cancer cells. In this study, we investigated the effect of PA on the growth and apoptosis of human immortalized cell line (HOS) and primary osteosarcoma cells by a Cell Counting Kit-8 (CCK-8) and Annexin V and propidium iodide (PI) staining, respectively. Western blot was used to measure the expression of cleaved Caspase 3, PTEN, and AKT, as well as the AKT phosphorylation. The Caspase 3 activity was determined using the Caspase-3 Colorimetric Assay Kit. From the results, PA significantly reduced cell proliferation in a concentration- and time-dependent manner. PA also induced cell apoptosis in a dose-dependent fashion. PA treatment led to increased Caspase 3 activation and PTEN expression, as well as reduced AKT phosphorylation. Moreover, Ac-DEVD-CHO (a Caspase 3/7 inhibitor) pre-treatment or PTEN knockdown partially blocked the effects of PA on cell proliferation and apoptosis. Caspase 3/7 inhibitor had an additive effect with PTEN knockdown. Collectively, our results suggested that induction of apoptosis by PA was mediated in part by PTEN/AKT signaling and Caspase 3/7 activity. This study provides evidence that PA might be useful in the treatment of human osteosarcoma.     

Improved liquid chromatographic determination of procainamide and N-acetylprocainamide in serum

A liquid chromatographic method for the determination of procainamide (PA) and N-acetylprocainamide (NAPA) in serum has been developed. This method utilizes isocratic conditions, ambient temperature, and a conventional fixed-wavelength 280-nm detector. Sample pretreatment involves extraction of PA and NAPA, along with p-amino-N-(2-dipropylaminoethyl)-benzamide hydrochloride as internal standard, into an organic phase and reextraction into an aqueous acidic phase. Using this sample pretreatment, interferences due to commonly used drugs are eliminated. The method accurately measures PA and NAPA to levels as low as 1 mg/L.     

On-line chemiluminescence determination protocatechuic aldehyde and protocatechuic acid in pharmaceutical preparations by capillary electrophoresis

Capillary electrophoresis (CE) with on-line direct chemiluminescence (CL) detection was first used in detecting protocatechuic aldehyde (PAH) and protocatechuic acid (PA) in their pharmaceutical preparations. It was found that the weak CL produced from the reaction of luminol with ferricyanide in an alkaline solution was strongly increased by PAH and PA which was separated by CE. Parameters affecting separation process and CL detection have been examined in detail. Under the optimum conditions, the baseline separation of PAH and PA was obtained within 6 min. The relative standard deviation (R.S.D.) for the analysis of PAH and PA was less than 1.1% for the migration time and 1.6% for the peak height. The detection limits (S/N=3) of PAH and PA were 7.0 x 10(-8)M and 5.0 x10(-8)M, respectively. The proposed method has been satisfactorily applied to the determination of PAH and PA in Salivia miltorrhrza pharmaceutical preparations.