雄甾-4-烯-3,17-二酮(4-AD)转化菌种的筛选及鉴定

从土壤中筛选能将植物甾醇转化为雄甾-4-烯-3,17-二酮(4-AD)的菌种。采用富集培养基富集能降解植物甾醇的菌种、采用摇瓶进行发酵、采用薄层层析的方法对发酵产物进行检测、采用高效液相方法测定发酵液中4-AD的含量、采用PCR方法扩增菌种的16S rDNA序列。筛选出了一株转化能力最强的菌株,命名为:3-12。将这株菌的16S rDNA序列与GeneBank中收载的序列进行比对,3-12号菌株为分支杆菌。     

羊耳菊中咖啡酰基奎宁酸类化学成分研究

采用正相硅胶色谱柱、Sephadex LH-20以及制备HPLC等色谱方法对羊耳菊进行分离纯化,并通过理化常数和波谱数据鉴定其化学结构。深入研究羊耳菊干燥全草的咖啡酰基奎宁酸类化学成分。从羊耳菊中分离得到8个咖啡酰基奎宁酸类化合物,分别为1,5-O-二咖啡酰基奎宁酸(1)、1,3,5-O-三咖啡酰基奎宁酸(2)、3,5-O-二咖啡酰基奎宁酸甲酯(3)、3,4-O-二咖啡酰基奎宁酸甲酯(4)、3,4-O-二咖啡酰基奎宁酸乙酯(5)、4,5-O-二咖啡酰基奎宁酸乙酯(6)、3,5-O-二咖啡酰基奎宁酸(7)、3,4-O-二咖啡酰基奎宁酸(8)。化合物1~3为从该植物中首次分离得到,4~6从该属植物中首次分离得到。     

2-氧代-4-苯基丁酸乙酯还原酶产生菌筛选及产酶条件

研究了利用生物催化不对称还原的方法制备(R)-2-羟基-4-苯基丁酸乙酯[(R)-HPBE]。以2-氧代-4-苯基丁酸乙酯(OPBE)为底物,通过对实验室保藏菌株进行筛选,得到一株产物立体选择性较高的菌株G2ndida krusei SW2026,并对其发酵产酶条件进行研究。其最适的发酵培养基组成为4.5%葡萄糖,3%蛋白胨,1.5%牛肉膏,0.05%Mn~(2+);适宜的产酶发酵条件为初始pH 6.0,温度28℃,摇床转速180 r/min,发酵周期48 h。将此条件下发酵培养的菌体用于OPBE的不对称还原反应,产物(R)-HPBE的对映体过量值(e.e.)可达97.33%,产率最高达到72.54%。     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

奎宁的热解机理、动力学及贮存期研究

利用热重-红外联用技术测得奎宁在氮气气氛中的热分解曲线,协同使用Achar法和Coats-Redfern法两种方法同时进行动力学处理,根据热分解的表观活化能(E_a)和指前因子(A)计算推断奎宁的贮存期。奎宁晶体在219.93～389.93℃发生了第一步分解,先后释放了醇类、CO、CO_2、醚类、胺类等物质,反应机理为化学反应控制,对应的函数名称是反应级数方程,反应级数n=2;第二步分解(389.93～800℃)是由于残余分子骨架中苯并吡啶环的深度裂解碳化,过程较为缓慢,形成了CO_2、H_2O、NH_3等气体。经红外光谱解析和热重数据结合分析,晶体在第一步分解时又先后分为两个阶段,首先是苯并吡啶环上与两个氧原子相连的化学键断裂,其次是支链上的其它原子化学键相继发生断裂并裂解;根据第一步热分解的表观活化能(E_a)和指前因子(A)推断,在室温25℃下,奎宁的贮存期为4～5年。     

黑曲霉催化雄甾-4-烯-3,17-二酮16β-羟基化

为进一步确定黑曲霉菌株TCCC41650的生物转化能力,以雄甾-4-烯-3,17-二酮(Androstenedione)为底物,利用黑曲霉菌株TCCC41650进行催化,产物经纯化、重结晶后,通过单晶衍射鉴定为16β-羟基雄甾-4-烯-3,17-二酮。转化条件为:培养液pH 6.0,乙醇添加量为2%,投料浓度为1‰时,72 h转化率为85.8%。目前甾体研究领域对于C16β-羟基化的微生物转化未见报道,研究结果为C16β-羟基甾体药物的研发奠定了基础。     

不对称还原前手性芳香酮微生物的筛选及反应特性

含芳香基手性醇是许多手性药物合成的关键手性砌块,生物催化不对称还原前手性酮是合成该类醇的重要方法之一.以4'-氯-苯乙酮为模型底物,从土壤中筛选得到一株能高效催化前手性芳香酮不对称还原合成相应手性醇的菌株,鉴定表明该菌株为白地霉( Geotrichum candid ).进一步考察了其催化4'-氯-苯乙酮不对称还原的反应特性,发现还原4'-氯-苯乙酮的产物主要为 S-4'-氯苯乙醇.在合适的反应条件下,其产率达到35%,对映选择性高于97%.     

来源于西梅的(R)-醇腈酶促立体选择性转氰合成(R)-酮醇腈

在水/有机溶剂双相反应体系中,研究了来源于西梅的(R)-醇腈酶催化酮与丙酮醇腈合成(R)-酮醇腈的立体选择性转氰反应．系统探讨了不同酶源、酶粉颗粒大小、底物浓度、两底物配比、酶浓度和底物结构对转氰反应的影响．结果发现西梅醇腈酶能高效催化三甲基硅酮与丙酮醇腈的立体选择性转氰．酶粉颗粒大小以直径0.3～0.45 mm为优,底物浓度以21 mmol/L左右为佳,底物丙酮醇腈与三甲基硅酮摩尔浓度比以2∶1为宜,酶浓度以60.9 g/L左右为好．西梅醇腈酶对3, 3-二甲基-2-丁酮几乎没有催化活性,而对其硅结构类似物三甲基硅酮却具有非常高的立体选择性和催化活性,在上述优化反应条件下反应24 h的底物转化率和产物光学纯度均高达99%以上,表明底物中的硅原子对西梅醇腈酶的催化活性有非常显著的促进作用．     

微生物转化雄甾-4-烯-3，17-双酮的菌种筛选和转化条件的初步研究

雄甾-4-烯-3,17-双酮(简称4AD)是甾体药物的重要中间产物,其11α羟化产物可制成治疗心血管疾病的药物。通过对30株不同种属真菌转化4AD能力的筛选,获得球孢白僵菌(Beauveria bassiana)QY2A对4AD有高效C11α羟化能力,得到目标产物C11α-羟基雄甾-3,17-双酮(简称11α-OH-4AD)。另对该菌株的转化条件进行优化,结果表明:初始pH值6.0,温度28℃,转速180r/min,转化时间60h,助溶剂甲醇终浓度和底物浓度分别为2.5%和2.5g/L时,11α-OH-4AD的转化率为65%,比未优化的转化率提高了51.2%。     

代谢工程改造Gluconobacter suboxydans生产2-酮基-D-葡萄糖酸

2-酮基-D-葡萄糖酸是重要的抗氧化剂和食品添加剂——D-异抗坏血酸的重要前体。弱氧化葡糖酸杆菌(Gluconobacter suboxydans)具有丰富的周质空间氧化还原酶类,可将葡萄糖氧化为葡萄糖酸再氧化为2-酮基-D-葡萄糖酸。以提高2-酮基-D-葡萄糖酸的产量和减少副产物为目标,采用同源重组染色体修饰策略,将编码甘油脱氢酶的基因gldh置换为编码葡萄糖脱氢酶的基因gdh,将编码山梨醇脱氢酶的基因sdh置换为编码2-酮-D-葡萄糖酸脱氢酶的基因ga-2-dh。经PCR、酶活性显色及发酵产物HPLC检测验证表明:构建的工程菌株gdh和ga-2-dh基因被强化而gldh和sdh被敲除;使用10%的葡萄糖复合培养基,摇瓶发酵72h,工程菌2KGA3发酵液中没有副产物5-酮基-葡萄糖酸,2-酮基-D-葡萄糖酸的含量终浓度达到72.3 g/L,比野生菌株提高42.2g/L,工程菌和野生菌的2-D-KGA质量转化率分别为72.3%和30.1%,工程菌比野生菌提高1.4倍。构建获得的工程菌,不需要外加抗生素,可以保持稳定遗传,对于工业化规模生产具有一定优势,为获得可产业化显示的优势遗传资源打下了基础。     

<Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis>, a carotenoid producing yeast strain from a patagonian high-altitude lake

The red yeast Rhodotorula mucilaginosa strain CRUB 0138 (previously identified as R. lactosa) was isolated from a high-altitude Patagonian Lake Toncek (1700 m a.s.l.), and assigned with mucilaginosa species. Its biochemical, physiological and molecular features were assessed and compared to R. mucilaginosa PYCC 5166 type strain using a polyphasic approach; in addition, biomass and carotenoid pigment production at different C/N ratios were determined in an incubator shaker. Phenetic characterization by means of 70 current physiological tests including assimilation of aldaric acids and aromatic compounds, and also the ability to grow with amino acids as sole carbon sources, was carried out. According to numerical taxonomy calculations, similarity indexes between R. mucilaginosa CRUB 0138 and PYCC 5166 type strain were 0.86 and 0.77, corresponding to a complete set of physiological tests and MSP-PCR (Mini/Micro Satellite Primed PCR; (GTG)5, M13 and (GAC)5 primers were employed) fingerprinting. Killer activity against 2 native strains, Rhodosporidium kratochvilovae and R. mucilaginosa was detected. Maximum biomass-glucose conversion efficiency (87%) and maximum carotenoid yield (2.32 mg/L) were obtained at C/N = 5 in culture medium containing 10 and 40 g/L glucose, respectively. Different C/N ratios did not influence carotenoid pigment production but low C/N enhanced biomass yield.     

胶红酵母JB401降解脱色三苯甲烷类染料

从烟梗中分离筛选得到1株能够对三苯甲烷类染料高效脱色的微生物,经ITS-5.8S rDNA分析鉴定为胶红酵母,命名为Rhodotorula mucilaginosa JB401。全波长扫描实验结果证实染料的脱色由胶红酵母降解结晶紫引起。为了提高R.mucilaginosa JB401脱色结晶紫的能力,通过单因素试验对R.mucilaginosa JB401的培养条件进行了优化,得出菌体生长24 h后以2%接种量接入初始pH为5的脱色培养基并在37℃摇床培养,可以取得最优脱色效果,此时脱色50、100和200 mg/L的结晶紫达到90%去除率分别需要3、6和14 h。此外,胶红酵母对温度和pH良好的适应性使其具有应用于工业废水处理的潜力。     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

不对称还原苯乙酮的酵母菌株筛选与分子鉴定

利用苯乙酮作为模式底物,对145株菌株进行初筛和复筛,获得一株具有高效立体选择性的酵母菌株YS6-2,能够不对称还原苯乙酮生成(S)-1-苯基乙醇.在苯乙酮浓度为70mmol/L时,底物的初始转化率达26.8%,产物(S)-1-苯基乙醇的对映体过量值为98.8%.基于形态学、生理生化特征、18S rDNA和26S rDNA D1/D2区域的分析表明,YS6-2为胶红酵母(Rhodotorula muci-laginosa).     

Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. III. Reaction rates

The absolute rate for the repair reaction of radiation-induced, oxygen-dependent lesions in bacterial transforming DNA with the sulphydryl (SH)-containing compound dithiothreitol (DTT) has been determined using a fast response method, the gas explosion technique, to be 1.6 X 10(6) mol-1 s-1. Glutathione reacts ten times slower than DTT with the irradiated transforming DNA. It can also be calculated that transforming DNA radicals react with O2 in a damage-fixing reaction with a rate of about 3 X 10(8) dm3 mol-1 s-1. These rates are compared with values in the literature for reaction rates of SH, compounds and O2 with irradiated DNA constituents and with bacterial cells.     

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-glucosidase 1 for efficient biomass conversion

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus β-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher β-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous β-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and β-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.     

The role of carotenoids in preventing oxidative damage in the pigmented yeast, Rhodotorula mucilaginosa

Rhodotorula mucilaginosa is an obligate aerobic yeast which contains a high concentration of carotenoid pigment. To test whether carotenoids are able to protect R. mucilaginosa against oxidative injury, yeast cells in liquid culture were incubated with duroquinone (DQ) (100 microM), a redox-cycling quinone known to generate intracellular O2-. or were grown in a hyperoxic atmosphere (80% O2) under conditions where carotenoid concentrations were altered either intracellularly or extracellularly. Neither of these oxidative challenges affected cell growth unless carotenogenesis was blocked by the addition of diphenylamine (50 microM). In the diphenylamine-treated nonpigmented cells, growth was completely inhibited by DQ and by hyperoxia. In normoxia, however, diphenylamine alone reduced growth by only 30%. The growth inhibition observed in diphenylamine-treated cells exposed to hyperoxia was primarily mycocidal rather than mycostatic since plating of these cells onto solid media revealed that only 25% of the cells were viable after 50 h of incubation when compared to plated control cells. Addition of 10 microM beta-carotene to diphenylamine-treated cells completely prevented the growth inhibition caused by either hyperoxia or DQ. Carotenoids, therefore, are able to prevent oxidant-induced cytotoxicity in R. mucilaginosa. Analysis of the absorption spectra of chloroform extracts of beta-carotene-supplemented cells showed that beta-carotene, not the endogenous carotenoid, torularhodin, was the major carotenoid present in these cells. Superoxide dismutase (SOD) activity in R. mucilaginosa was compared with that of another yeast, Saccharomyces cerevisiae by two methods: (i) activity staining of proteins separated by gel electrophoresis and (ii) measurement of inhibition of ferricytochrome c reduction. By these techniques, the R. mucilaginosa SOD activity had the characteristics of Mn-SOD. No Cu/ZnSOD activity was detected. Thus, the apparent absence of Cu/ZnSOD may make the antioxidant capability of endogenous carotenoids even more critical in preventing oxidative damage in R. mucilaginosa.     

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Action of restriction endonucleases on transforming DNA of Haemophilus influenzae.

Cleavage of DNA from Haemophilus influenzae with restriction endonucleases caused inactivation of transforming ability to an extent that depended on the genetic marker and the enzyme. The rate of inactivation, but not the final level of survival, depended on the concentration of enzyme in the restriction digest. In general, the greatest extent of inactivation of transforming activity was obtained with endonucleases that are known to produce the shortest fragments. We electrophoresed restriction digests of H. influenzae DNA in agarose gels and assayed transforming activity of DNA extracted from gel slices. In this way, we determined the lengths of restriction fragments that contain genetic markers of H. influenzae. For the marker that we studied most thoroughly (nov), the shortest restriction fragment that possessed detectable transforming activity was a 0.9-kilobase pair fragment produced by endonuclease R . PstI. The shortest marker-bearing restriction fragment that retained substantial transforming activity (50% of value for undigested DNA) was a 2.1-kilobase pair EcoRI fragment bearing the kan marker. Among marker-bearing restriction fragments 1 to 4 kilobase pairs in length, survival of transforming activity varied 10,000-fold. We relate these observations to the recent findings by Sisco and Smith (Proc. Natl. Acad. Sci. U.S.A. 76:972-976, 1979) that efficient entry of DNA into competent H. influenzae cells appears to require the presence of a recognition sequence that is scattered throughout the Haemophilus genome in many more copies than in unrelated genomes.