裂殖酵母生物合成L-苯基乙酰基甲醇的研究

用裂殖酵母作为生物催化剂，催化苯甲醛与丙酮酸合成L苯基乙酰基甲醇（L-PAC），后者为合成L-麻黄素等多种药物的前体，转化反应的最佳温度为28℃，pH6．0，时间16h，苯甲醛用量160-200mmol／L，丙酮酸120～180mmol／L，LPAC产量可达15g／L。     

辛醇/水两相体系中生物合成L-苯基乙酰基甲醇的研究

利用酿酒酵母作为生物催化剂合成L-苯基乙酰基甲醇(L-Phenylacetylcarbiol,L-PAC)的过程中,通过加入有机溶剂(辛醇)形成两相体系,可改善底物和产物的溶解度,以减少对细胞内丙酮酸脱羧酶(PDC)的强烈抑制和钝化作用,从而促进L-PAC产量的提高。实验结果表明,两相体系合成L-PAC的最佳条件为活化60 min,苯甲醛的起始浓度940 mmol/L,丙酮酸钠的起始浓度460 mmol/L,葡萄糖的含量8%,水醇两相体积比为3∶1,且加入一定量的甘油时,L-PAC产量可达在醇相中24.1 g/L和水相中3.7 g/L,比单一水相体系高出1.8倍。     

双酶法生物合成L-苯基乙酰基甲醇的研究

双酶偶联体系越来越受到人们重视,广泛应用于医药、食品等领域,是实现高效生物合成的重要方法。本文利用乳酸氧化酶(Lactate Oxidase,LOD)和丙酮酸脱羧酶(Pyruvatedecarboxylase,PDC)偶联构建双酶体系,催化乳酸钠和苯甲醛合成L-苯基乙酰基甲醇(L-Phenylacetylcarbinol,L-PAC),后者是合成L-麻黄素的重要中间体。转化反应的最佳温度为34℃,pH 6.8,转化时间6 h,苯甲醛用量160 mmol/L,乳酸钠用量330 mmol/L,葡萄糖用量3 g/100 mL,L-PAC产量达到8.79 g/L。在L-PAC的制备过程中,以廉价的乳酸钠代替丙酮酸大大降低了生产成本,缩短了反应时间,简化了操作步骤,有利于工业生产。双酶偶联体系为生物合成L-PAC提供了一个新的思路和新方法。     

响应面法优化微生物萃取转化制备L-苯基乙酰基甲醇的工艺

目的:优化微生物萃取转化制备L-苯基乙酰基甲醇(L-PAC)的条件。方法:以啤酒酵母萃取制备L-PAC。采用高效液相色谱法测定L-PAC浓度。以L-PAC浓度为响应值,通过Box-Behnken响应面法对苯甲醛、曲拉通(Triton)X-100和葡萄糖用量这3个主要因素进行考察,同时进行验证试验。结果:苯甲醛和Triton X-100之间的交互作用最为显著,最优因素组合为苯甲醛1.1%、Triton X-100 0.14 g/m L、葡萄糖0.028 g/m L;验证试验中L-PAC的平均浓度为28.04 mmol/L(RSD=1.35%,n=3),与预测值28.01mmol/L的相对误差为0.11%。结论:利用响应面法对微生物萃取转化制备L-PAC的条件进行了优化,得到了各因素的最优组合,可为大批量转化制备L-PAC提供有利参考。     

两相体系中固定化细胞生物合成(S)-3,5-双三氟甲基苯乙醇

建立了在有机/水两相体系中固定化酵母细胞牛物合成(S)-3,5-双三氟甲基苯乙醇的方法.考察了有机溶剂、缓冲液pH、底物浓度、温度、转化时间、辅助底物对转化率和对映体过量值(ee)的影响.结果显示,采用辛烷-缓冲液两相体系,缓冲液pH 8,初始底物浓度5.55 mmol/L,30℃转化36 h,转化率和产物的ee值分别为93.2%和96.3%.添加与底物等当量的葡萄糖或蔗糖作为辅助底物,固定化细胞可以反复使用6次,转化率维持在50%以上.     

固定化细胞转化液中瓜氨酸的HPLC法测定

建立了HPLC法测定瓜氨酸固定化细胞转化液中底物、产物及杂质的浓度变化。采用Zorbax SB—Aq反相色谱柱，以6mmol／L硫酸为流动相，检测波长为210nm。瓜氨酸和精氨酸均在20～1000μg／ml浓度范围内线性关系良好。     

两相体系中固定化红酵母细胞催化3,5-双三氟甲基苯乙酮的不对称还原反应

采用海藻酸钠固定红酵母细胞为生物催化剂,对有机溶剂—水两相体系不对称还原3,5-双三氟甲基苯乙酮合成(S)-3,5-双三氟甲基苯乙醇的影响因素进行了考察,并与静息细胞转化法进行比较。结果表明,固定化红酵母细胞在5%辛烷中进行转化,于30℃p、H 8、1.42 mg/mL底物浓度并添加葡萄糖作为辅助底物的条件下,转化效果最佳,经6次重复使用后,仍可保留50%的转化活力。     

青霉素酰化酶基因重组大肠杆菌在两水相体系中的转化反应

以青霉素酰化酶基因重组大肠杆菌Ａ５６（ｐＰＡ２２），经戊二醛交联（防止细胞自溶）后，在ＰＥＧ２０００－ＤｅｘｔｒａｎＴ７０两水相体系中进行转化反应，生产６－氨基青霉烷酸（６－ＡＰＡ）。实现含酶菌体在两水相体系中的单侧分配。考察了菌体加量对青霉素转化率的影响。在两水相体系中进行５批半连续转化反应，底物浓度为７％，转化率在９０％以上，反应时间为４０分钟，比生产速率为３·６～４·６μｍｏｌ／ｍｌ·ｍｉｎ，下相酶活为５２ｕ／ｍｌ，并从上相中直接结晶６－ＡＰＡ，６－ＡＰＡ的纯度为９６·２％。实现了高底物浓度，高转化率，缩短反应时间和产物不需浓缩直接结晶之目的。在两水相体系中生产６－ＡＰＡ成为又一新的青霉素酰化酶应用形式。     

水/有机相两相体系中Saccharomyces cerevisiae B5不对称还原制备手性药物中间体R-2'-氯-1-苯乙醇的研究

The process of preparation of chiral pharmaceutical intermediate R-2'-chloro-1-phenyl-ethanol with the method of Saccharomyces cerevisiae B5 asymmetric reduction in water/organic diphasic system was investigated in detail. The result shows that the higher     

偶联法合成4-（2’，4’-二氟苯基）苯乙酮的研究*

目的合成4-(2',4'-二氟苯基)苯乙酮.方法以2,4-二氟苯硼酸和对溴苯乙酮为原料,四氢呋喃/水为溶剂,经新型Pd(Ⅱ)配合物催化交叉偶联反应合成目标化合物.结果和结论反应在空气中、室温下、水/有机两相体系中高效催化4-(2',4'-二氟苯基)苯乙酮的合成,产率达93%.     

Analysis of synthetic peptides by capillary zone electrophoresis in organic/aqueous buffers

Whereas synthetic peptides have been routinely analyzed for purity by reverse phase high performance liquid chromatography (RPHPLC) for a number of years, it is only in the last decade that the use of capillary zone electrophoresis (CZE) in aqueous buffers has been taken advantage of as an orthogonal method for the detection of impurities. However, we have found that hydrophobic amino acids and peptides often migrate as very broad, tailing absorbances or even precipitate in the aqueous buffers during CZE analysis. As a result, alternative buffer systems containing organic modifiers were sought. Varying concentrations of acetonitrile, methanol and isopropanol in sodium phosphate and triethylammonium phosphate buffers were used to study their effects on the electrophoretic migration of several synthetic peptides [gonadotropin releasing hormone (GnRH), corticotropin releasing factor (CRF) and analogs] and an enantiomeric synthetic amino acid. The organic/aqueous buffers used to obtain the best conditions for separation of porcine gonadotropin-releasing hormone (GnRH) and chicken II GnRH were then used to optimize a separation of nine native forms of GnRH decapeptides. Interestingly, several of these GnRHs have identical formal charges and yet could be separated. This suggests a mixed mechanism of separation that discriminates not only on the basis of peptide charge and structure but also of adsorptive properties (Van der Waals forces, dipole-dipole interactions and hydrogen bonding) of the capillaries.     

Pepsin-catalyzed peptide synthesis in organic media: studies with free and immobilized enzyme

Pepsin-catalyzed synthesis of protected peptides was studied in two-phase systems containing up to 50% (by volume) of aqueous phase. A methodological study was carried out to determine the optimum conditions for the synthesis of the model protected peptide Z-Phe-Phe-OMe. Several parameters such as concentrations of carboxylic and amino components, pH of the aqueous phase, ratio of organic to aqueous phase volumes and nature of the organic solvent were investigated. It was observed that the most hydrophobic solvents produced the best yields, despite the low solubility of substrates in these media. The log P of the solvent could be used to predict the solvent effect over the reaction yields. Pepsin immobilized by adsorption onto the solid supports Celite and Chromosorb was employed to perform a study of secondary specificity of the enzyme in organic media through the coupling between Z-X-Phe-OH (X = Ala, Asp, Glu, Gly, Phe, Ile, Val, Trp and Tyr) and Phe-OMe. This investigation was performed in two solvent systems: (A) ethyl acetate:citrate buffer pH 4.5 (98:02, v:v) and (B) acetonitrile:citrate buffer pH 4.5 (96:04, v:v). Reaction rate data showed that pepsin had a preference for more hydrophilic substituents in the P₂ position. These data are in contrast to the literature for a similar reaction performed in predominantly aqueous media. Thus, for mainly organic media, partition phenomena are very important and may cause an apparent modification of enzyme specificity.     

用固定化细胞生产D-氨基酸的研究

目的优化CPU 0 4 30 1菌株的发酵条件 ,提高酶活力 ,并建立D 氨基酰化酶酶活力的测定方法。方法对CPU 0 4 30 1菌株培养的碳源、氮源进行优化 ,同时观察温度、pH值、离子含量等对D 氨基酰化酶酶活力测定的影响。结果CPU 0 4 30 1经固定化后于 37℃反应 2 0h得产品 4 .78g ,纯度达 99.5 7%。结论CPU 0 4 30 1菌株经优化后 ,D 氨基酰化酶的产量和活力均明显提高 ,同时建立了准确、灵敏的酶活力测定方法。     

Relative hydrophobicity of amino acid residues in homooligopeptides as measured by aqueous two‐phase partitioning

Abstract: Partitioning of 17 amino acids and their homooligopeptides of different lengths in an aqueous dextran–PEG two‐phase system containing 0.15 m NaCl in 0.01 M sodium phosphate buffer, pH 7.4 and 0.11 m sodium phosphate buffer, pH 7.4 was examined. The relative hydrophobicity of the amino acid residues was estimated and expressed in equivalent numbers of methylene units. Analysis of the data shows that the additivity principle does hold for the hydrophobicity of homooligopeptides. The relative hydrophobicity of essentially all amino acid residues is noticeably affected by the ionic composition of aqueous media.     

Randomized phase II evaluation of aprinocarsen in combination with gemcitabine and cisplatin for patients with advanced/metastatic non-small cell lung cancer

Summary Aprinocarsen is a specific antisense oligonucleotide inhibitor of protein kinase C-. This study aimed to evaluate the response rate to combination therapy with aprinocarsen, gemcitabine and cisplatin, in chemonaive patients with advanced/metastatic NSCLC. Secondary objectives included comparison of response rate, time to event efficacy parameters, and toxicities on the 2 treatment arms. Patients with stage IV, or stage IIIB disease (N₃ and/or pleural/pericardial effusion), were randomized to either control or experimental arm. Patients on both arms received gemcitabine 1250 mg/m² on days 1 and 8, and cisplatin 80 mg/m² on day 1 of a 3-week cycle. Additionally, on the experimental arm, aprinocarsen was administered as 2 mg/kg continuous iv infusion on days 1–14, every 21 days. A total of 18 enrolled patients were randomized on the 2 arms. Further enrollment was terminated in March 2003 as a result of a phase III trial suggesting that aprinocarsen did not have an added survival benefit when combined with paclitaxel and carboplatin therapy in patients with NSCLC. Patients received a median of 4 cycles on control arm and 2.5 cycles on experimental arm. The response rate was 16.7% in the experimental arm and 44.4% in the control arm. Most frequent grade 3/4 toxicities were hematologic, with a higher incidence of thrombocytopenia in the experimental arm (87.5% vs. 33.3%). Despite the 14-day continuous infusion schedule, infection rate was not increased in the experimental arm. The present study did not show any advantage, in response rate or secondary endpoints, with aprinocarsen; however, the toxicity was not unduly increased, and aprinocarsen regimen was safely administered.     

Survivin基因在鼻型NK／T细胞淋巴瘤的表达及其与肿瘤细胞增殖的关系

目的探讨Survivin基因在鼻型NK／T细胞淋巴瘤中的表达,并初步分析肿瘤组织中Survivin基因表达与肿瘤细胞增殖的关系及其在鼻型NK／T细胞淋巴瘤发生发展中的意义。方法应用免疫组化Envision System检测鼻型NK／T细胞淋巴瘤和正常淋巴结组织中Survivin和ki-67的表达情况,计算肿瘤组织ki-67增殖指数,并对Survivin表达阳性组和阴性组ki-67增殖指数的差异进行分析。结果50例肿瘤组织中Survivin阳性表达率为60％（30／50）,12例正常淋巴结组织中Survivin阳性表达率为16．7％（2／12）,且主要局限于少数生发中心细胞（χ^2=7．28,P〈0．05）。Survivin基因阳性组ki-67增殖指数明显高于Survivin基因阴性组（t=2．80,P〈0．05）。结论Survivin高表达在鼻型NK／T细胞淋巴瘤的发生发展中可能有一定的作用。Survivin与增殖指数呈正相关,Survivin可能促进肿瘤细胞的增殖。     

利拉鲁肽和门冬胰岛素30对肥胖2型糖尿病患者疗效和β细胞功能影响的比较

目的比较利拉鲁肽和门冬胰岛素30分别与二甲双胍联合对肥胖2型糖尿病患者的疗效和β细胞功能的影响。方法将65例新诊断以及口服降糖药物效果欠佳的肥胖2型糖尿病患者,分为利拉鲁肽组[利拉鲁肽联合二甲双胍（n=30）]和门冬胰岛素30组[门冬胰岛素30联合二甲双胍治疗（n=35）],均治疗12周。分别测定所有对象治疗前后的体重（BW）、体重指数（BMI）、空腹血糖（FPG）、餐后2 h血糖（PPG）、糖化血红蛋白（Hb A1c）、糖化白蛋白（GA）、血清总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白-胆固醇（LDL-C）、高密度脂蛋白-胆固醇（HDL-C）、血清空腹C肽（FCP）、餐后2 h C肽（PCP）等指标,并计算β细胞功能指数（HOMA-β）和胰岛素抵抗指数（HOMA-IR）。比较各组治疗前后及两组间各项指标变化情况。结果利拉鲁肽组治疗12周后BW及BMI均较治疗前下降（P〈0.01）,与门冬胰岛素30组相比差异具有统计学意义（P〈0.01）。两组治疗后FPG、PPG、Hb A1c和GA均较治疗前下降（P〈0.01）。利拉鲁肽组治疗后FCP、PCP和HOMA-IR较治疗前下降（P〈0.05）,HOMA-β上升（P〈0.01）;而门冬胰岛素30组治疗后FCP和PCP较治疗前上升（P〈0.05）,HOMA-β上升（P〈0.01）。两组治疗后HOMA-IR下降差异具有统计学意义（P〈0.05）。利拉鲁肽组治疗后TC、TG及LDL-C较治疗前下降（P〈0.05）;而门冬胰岛素30组治疗后TC和LDL-C较治疗前下降（P〈0.05）。结论利拉鲁肽或门冬胰岛素30联合二甲双胍治疗对肥胖2型糖尿病患者的血糖指标控制均有效,并均可改善其β细胞功能。但在控制体重及改善胰岛素抵抗方面,利拉鲁肽更优于门冬胰岛素30。     

SH-SY5Y细胞膜固相色谱法的建立及其对远志皂苷碱水解产物效应物质的研究

目的建立SH-SY5Y细胞膜固相色谱法,并运用该方法分析远志皂苷碱水解产物中的活性成分。方法应用SH-SY5Y细胞膜作为固定相选择性地结合远志皂苷碱水解产物中的活性成分,排除未与细胞膜靶点结合的非活性成分后,使细胞碎裂,被结合的活性成分释放出来,样品经SPE固相萃取处理后用HPLC对这些成分进行分析。结果远志皂苷碱水解产物中的活性成分初步确定为4个,分别为细叶远志皂苷、黄花倒水连皂苷A、3,4,5-三甲氧基肉桂酸和对甲氧基肉桂酸。结论细胞膜固相色谱法基本可以反映化合物与细胞膜生物靶点的相互作用,该体系中的保留成分和其药理作用有显著的相关性。     

高效液相色谱-串联质谱法测定白内障患者应用加替沙星滴眼液后房水药物浓度

目的 定量分析白内障患者术前不同时间应用加替沙星(抗生素)滴眼液后房水中的药物浓度.方法 随机选取白内障患者36例,分为4组.各组患者均在术前3天内,于手术的眼局部应用加替沙星滴眼液,每日4次,每次1滴.手术当日,各组分别于术前15、30、60、120 min用加替沙星滴眼液1次;于术中取房水0.1 mL,用高效液相色谱-串联质谱法测量房水中的药物浓度.结果 4组房水中药物浓度分别为(0.15±0.29)、(0.33±0.22)、(0.31±0.19)、(0.74±0.32)μg·mL~(-1).结论 加替沙星滴眼液具有良好的角膜穿透性,局部应用能够在房水中获得有效的药物浓度;于术前120 min用药时,房水药物浓度最高.