天然辣椒素酯的酶促合成与生物活性

研究了天然辣椒素经化学．酶法转化为天然辣椒素酯类物质的方法。在30℃,香草醇、脂肪酸酯分别为50、75mmol／L的100mL脱水丙酮溶液,以1g固定化的脂肪酶Novozyme435为催化剂,摇床转速为200r／min条件下反应24h,目标化合物产率可达63％。经硅胶柱色谱分离纯化后,所得产物经质谱确证其含有天然辣椒素酯（capsiate）、二氢辣椒素酯（dihydrocapsiate）、降二氢辣椒素酯（nordihydrocapsiate）、高二氢辣椒素酯（homodi-hydrocapsiate）等。制备HPLC分离后得到辣椒素酯和二氢辣椒素酯,以^1HNMR及MS确定结构。活性测试表明辣椒素酯类物质具有激活PPARγ的生物活性及体外抑制乳腺癌细胞（MCF-7）和肝癌细胞（HepG2）的活性。     

有机介质中酶促合成壬酸香草醇酯的研究

壬酸香草醇酯是与天然辣椒素酯结构最接近的一个化合物,本文研究了有机相中脂肪酶催化合成这个化合物的方法,为天然辣椒素酯的酶促合成探索方法。考察了酶、溶剂、酶的用量、底物浓度、溶剂水含量以及温度等因素对反应的影响,结果表明脂肪酶Novozym e 435的活性最好,最适条件为:在1 mL脱水丙酮中,香草醇与壬酸甲酯的浓度分别为50、75 mmol/L,酶量为20 mg,30℃下反应24 h,产率可达到60%以上,产物经硅胶柱层析纯化,并以1H NMR及MS进行了表征。     

荔枝核化学成分的研究

利用硅胶柱色谱、聚酰胺柱色谱、制备薄层色谱等方法,从荔枝（Litchi chinensis Sonn．）核乙醇提取物中分离出9个化合物,根据理化性质和波谱分析鉴定了7个化合物,分别为胡萝卜苷（daucosterol,1）、原儿茶醛（protocatechuic aldehyde,2）、原儿茶酸（protoeatechuic acid,3）、表儿茶素（（-）-epicafichin,4）、procyanidin A2（5）、procyanidin A1（6）、芦丁（rulin,7）。化合物2,4-7为首次从荔枝核中分得,化合物2．4、5为首次从该属植物中分离得到。     

刺玫果果肉石油醚层化学成分及胰脂酶和α-糖苷酶抑制活性研究北大核心CSCD

采用硅胶柱色谱、葡聚糖凝胶柱色谱等方法进行分离纯化,通过波谱数据进行结构鉴定,对石油醚层通过荧光检测法测定胰脂肪酶抑制活性,通过96微孔板法测定不同来源α-葡萄糖苷酶的抑制活性。从刺玫果果肉石油醚层分离得到12个化合物,分别鉴定为二十九烷（1）、α-生育酚（2）、邻苯二甲酸二乙酯（3）、邻苯二甲酸二丁酯（4）、β-谷甾醇（5）、α-香树脂醇（6）、乌苏醇（7）、白桦脂醇（8）、白桦脂酸（9）、19α-羟基乌苏酸（10）、胡萝卜苷（11）和麦芽糖（12）。化合物1~3、6~7和12首次从该植物中分离得到,石油醚层对胰脂肪酶和酵母菌来源的α-葡萄糖苷酶具有一定的抑制活性。     

卡伍尔氏链霉菌NA4突变株〖WTHX〗ΔbafAI的次级代谢产物研究

链霉菌产生的次级代谢产物是农用和医药抗生素的重要来源。为了更深入挖掘卡伍尔氏链霉菌NA4潜在多样的次级代谢产物,对阻断NA4主产物bafilomycins的突变株ΔbafAI随机激活的代谢产物进行分离和鉴定。采用HP20固相萃取、硅胶柱色谱、凝胶柱色谱以及半制备HPLC等方法对ΔbafAI代谢产物进行分离纯化;使用NMR和MS确定化合物结构,并对单体化合物进行了生物活性测定。共分离鉴定6个化合物：环（L-异亮氨酸-L-脯氨酸）（1）、环（L-缬氨酸-L-脯氨酸）（2）、环（L-脯氨酸-L-亮氨酸）（3）、环（L-脯氨酸-L-苯丙氨酸）（4）、（2E, 4E）-5-（3-羟苯基）-戊-2, 4-二烯酰胺（5）和5′-脱氧-5′-甲硫肌苷（6）。6个化合物均是阻断NA4主产物后非特异激活的或产量显著提高的化合物,均为该菌中首次分离;化合物5和6是第二次从天然提取物中分离得到;化合物1和2显示出抗白色念珠菌活性,并首次报道了化合物5的抗氧化活性。进一步丰富了卡伍尔氏链霉菌NA4次级代谢产物研究,为激活沉默或低表达次级代谢产物合成基因簇提供了有益借鉴。     

离子液体中固定化脂肪酶催化拆分（±）-薄荷醇

以自制的平均粒径为4．5um磁性高分子微球为载体,采用离子交换法固定化Candida rugosa脂肪酶,催化（±）-薄荷醇的酯化反应,以考察反应时间、pH、反应温度、水活度等因素对酶的固定化以及酯化反应的影响。在固定化反应150min、pH5．0、酯化反应温度30℃、固定化酶的水活度为0．78的条件下,所制备的固定化脂肪酶在离子液体[bmim]PF6中催化拆分（±）-薄荷醇的效果最佳,与游离酶相比固定化脂肪酶的立体选择性有很大的提高,对映体过量率可达93％,对映体选择值为35。     

乌桕桕脂中甾醇和其他成分分离鉴定

通过柱层析、薄层层析等化学方法,从乌桕桕脂中分离得到甾醇及脂肪酸酯类化合物。经气相色谱和气-质连用分析,鉴定出下列化合物：β—谷甾醇、菜油甾醇、r7—豆甾烯醇,肉豆蔻酸甲酯、肉豆蔻酸乙酯、棕榈酸甲酯、硬酯酸甲酯和硬酯酸乙酯。     

高分子载体固定化酵母脂肪酶的研究

合成了一系列丙烯酸甲酯－双甲基丙烯酸乙二醇酯、甲基丙烯酸羟乙酯－双甲基丙烯酸乙二醇酯、苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物作为固定化酵母脂肪酶的载体,比较了这些载体固定化酵母脂肪酶催化橄榄油水解反应的效果,结果发现疏水性较强的苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物固定化酵母脂肪酶的效果最好．比较了酵母脂肪酶溶液和苯乙烯－双甲基丙烯酸乙二醇酯文联共聚物固定化酶催化橄榄油水解反应的最适ｐＨ值、最适离子强度、最适温度和Ｋ＿ｍ值．同时考察了苯乙烯－双甲基丙烯酸乙二醇酯交联共聚物固定化酶在有机溶剂中催化十六酸戊酯合成反应的活力．     

海洋放线菌Streptomyces variabilisstrain 6-1次级代谢产物的研究

目的：对来自海洋软珊瑚的链霉菌6-1（Streptomyces variabilisstrain6-1）进行次级代谢产物的分离和鉴定,寻找具有生物活性的化合物,为人类健康服务。方法：采用液体培养基对分自海洋软珊瑚Scleronephthya sp中的链霉菌6．1（Streptomyces vafiabilisstrain6-1）进行发酵培养,用乙酸乙酯对发酵液进行萃取;采用半制备高效液相色谱（semi-preparative HPLC）分离方法对乙酸乙酯萃取物进行分离纯化,得到单体化合物;运用电喷雾质谱（ESI—MS）、核磁共氢振（1HNMR）、核磁共振碳谱（13C NMR）和物理性质对所得单体化合物进行结构鉴定。结果：从海洋链霉菌6-1（strain6-1）发酵液的乙酸乙酯萃取物中分离得到3个单体化合物,分别鉴定为：7,4’-二羟基异黄酮（1）、5,7,4’-三羟基异黄酮（2）和丁烯酸内酯-I（3）。结论：丁烯酸内酯．I是从链霉菌属首次分离得到,化合物1和2均是从Streptomyces variabilis中首次分离得到;变异链霉菌6-1（Stmptomyces variabilis strain6-1）可以作为活性化合物3（丁烯酸内酯-I）的重要来源。     

小花清风藤的化学成分研究

利用Sephadex LH-20,硅胶柱色谱和硅胶制备薄层色谱等分离方法反复分离纯化,从小花清风藤（Sabia parviflora Wall．ex Roxb）中分离得到9个化合物,通过波谱数据分析分别鉴定它们为二十五烷酸（1）、木栓酮（2）、5-氧阿朴菲碱（3）、3-氧化齐墩果酸甲酯（4）、齐墩果酸（5）、羽扇豆-20（29）-烯-3-酮（6）、羽扇豆醇（7）、β-谷甾醇（8）、脱镁叶绿甲酯酸（9）。化合物2,6、7、9首次从该植物中分离得到。     

Enzymatic synthesis of a capsinoid by the acylation of vanillyl alcohol with fatty acid derivatives catalyzed by lipases

Capsinoids are a novel group of compounds produced by the Capsicum plant. We synthesized a capsinoid by the lipase-catalyzed esterification of vanillyl alcohol with fatty acid derivatives in an organic solvent. The use of seven out of 17 commercially available lipases, especially Novozym 435, was applicable to the synthesis of vanillyl nonanoate, a model compound of capsinoids. The yield of vanillyl nonanoate under the optimum conditions of 50 mM vanillyl alcohol and 50 mM methyl nonanoate in 500 microl of dioxane, using 20 mg of Novozym 435 and 50 mg of 4 A molecular sieves at 25 degrees C, was 86% in 20 h. Several capsinoid homologues having various acyl chain lengths (C6-C18) were synthesized at 64-86% yields from the corresponding fatty acid methyl ester. The natural capsinoids, capsiate and dihydrocapsiate, were obtained by a 400-fold-scale reaction at these optimum conditions in 60% and 59% isolated yields, respectively.     

Synthesis of stable isotope-labeled precursors for the biosyntheses of capsaicinoids, capsinoids, and capsiconinoids

Stable isotope-labeled precursors were synthesized for an analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the biosynthetic flow of capsaicinoids, capsinoids, and capsiconinoids. [1'-(13)C][5-(2)H]-Vanillin was prepared by the condensation of guaiacol with [(13)C]-chloroform and a D(2)O treatment. Labeled vanillylamine, vanillyl alcohol, ferulic acid, and coniferyl alcohol were prepared from the labeled vanillin. The labeled vanillylamine was converted to labeled capsaicinoid in a crude enzyme solution extracted from pungent Capsicum fruits.     

Characterization of newly developed pepper cultivars (Capsicum chinense) ‘Dieta0011-0301’, ‘Dieta0011-0602’, ‘Dieta0041-0401’, and ‘Dieta0041-0601’ containing high capsinoid concentrations and a strong fruity aroma

ABSTRACT

Capsaicinoids are responsible for the pungent flavor of peppers (Capsicum sp.). The cultivar CH-19 Sweet is a non-pungent pepper mutant that biosynthesizes the low-pungent capsaicinoid analogs, capsinoids. Capsinoids possess important pharmaceutical properties. However, capsinoid concentrations are very low in CH-19 Sweet, and Capsicum cultivars with high content capsinoids are desirable for industrial applications of capsinoids.

Habanero, Bhut Jolokia, and Infinity are species of Capsicum chinense, and have strong pungency and intense fruity flavors. In the present study, we report new cultivars with high concentrations of capsinoids (more than ten-fold higher than in CH-19 Sweet), and showed that these cultivars (Dieta0011-0301 and Dieta0011-0602 from Bhut Jolokia, Dieta0041-0401 and Dieta0041-0601 from Infinity) are of nutritional and medicinal value and have fruity aromas. We also obtained a vanilla bean flavor, vanillyl alcohol, and vanillyl ethyl ether from capsinoids in the fruit of these cultivars following the addition of ethanol at room temperature.     

High-level expression of extracellular lipase Lip2 from Yarrowia lipolytica in Pichia pastoris and its purification and characterization

The extracellular lipase gene from Yarrowia lipolytica (YlLip2) was cloned into the pPICZalphaA and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The lipase was successfully expressed and secreted with an apparent molecular weight of 39kDa using Saccharomyces cerevisiae secretion signal peptide (alpha-factor) under the control of the methanol inducible promoter of the alcohol oxidase 1 gene (AOX1). The lipase activity of 12,500,000U/l (2.10g total protein and 0.63g lipase per liter) was obtained in a fed-batch cultivation, where methanol feeding was linked to the dissolved oxygen content after initial glycerol culture. After fermentation, the supernatant was concentrated by ultrafiltration with a 10kDa cut off membrane and purified with ion exchange chromatography using Q Sepharose FF. Deglycosylation showed that the recombinant lipase is a glycoprotein which contains the same content of sugar (about 12%) as the native lipase from Y. lipolytica. The optimum temperature and pH of the recombinant lipase was 40 degrees C and 8.0, respectively. The lipase showed high activity toward long-chain fatty acid methyl esters (C12-C16).     

产脂肪酶菌株C7828-5的筛选、鉴定以及产酶条件的优化

以花生油为唯一碳源,从海口市各地被油脂污染土样中分离筛选出1株中温碱性脂肪酶菌株C7828-5。形态学、生理生化特征和分子生物学鉴定结果表明,该菌株为铜绿假单胞菌(Pseudomonas aeruginosa)。该菌所产脂肪酶的最适温度为37℃,最适pH为8.0。优化了菌株的产酶条件,最适产酶培养基(g/L)为:蔗糖5、牛肉膏20、(NH_4)_2SO_41、MgSO_4·7H_2O 0.5、CaCl_20.5,聚乙烯醇花生油乳化液120 mL,发酵72 h,获得高达8.08 U/mL的脂肪酶表达量。     

Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167

To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram-positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected.     

固定化脂肪酶合成二元酸酯

The syntheses of dicarboxylic esters by immobilized lipase from Candida sp. -1619 were investigated. The reaction system was composed of 1 mmol dicarboxylic acid, 2 mmol alcohol, 3 mL hexane and 15 mg celite-adsorbed im mobilized lipase(300 u), in a closed 100 mL Erlenmeyer flask, shaken at 40°C for 5h. Sebacic acid was the best substrate among nine dicarboxylic acids selected. Among the 18 saturated fatty n-alcohols, the alcohols with carbon chain length rangin from C4～C18 had good reactivity. The primary alcohols had much better reactivities than corresponding secondary alcohols and multihydroxy-alcohols. Tertiary alcohols showed no reactivity. Hydrocarbons, benzene, toluene, xylene and te trachloride were favorite reactants among 15 organic solvents selected, in none-solvent stationary system, (5 mmol sebacic acid, 10 mmol dndecanol, 150 mg immobilized lipase(3000 u))reacted without plug for 3.5h, the optimum temperature was 60°C. The conversion degree was over 92% when reaction carried out at 50～90°C for 17h. The suitable reaction pH ranged from 6～8. The reactant was developed on GF254 plate(hexane ethyl ether acetic acid = 30201 ( V V V).There were three spots with different Rf value at 0.96, 0.55 and 0 corresponding to product, oleyl alcohol and sebacic acids, respectively.     

低温脂肪酶产生菌的筛选、产酶发酵及粗酶性质研究

利用含有Tween 80的琼脂平板和摇瓶发酵法,从若尔盖高原土壤中筛选产脂肪酶菌株.通过菌落形态和菌体特征观察初步对菌种进行鉴定,得到一株产低温脂肪酶的适冷菌Pseudomonassp.DL-B,并设计正交试验对该菌株的产酶发酵培养条件进行了优化.摇瓶实验表明,该菌株最适产酶发酵培养基为:蔗糖10 g/L,蛋白胨20 ...     

Ordered mesoporous materials containing Mucor Miehei Lipase as biocatalyst for transesterification reaction

Herein, we report the design of a biocatalyst by the immobilization of Mucor Miehei Lipase (Mm-L) onto mesoporous silica materials. Supports with different pore diameters have been considered. Infrared spectroscopy was used to determine the adsorption isotherms in different pH conditions.Then, the biocatalyst was tested for the methanolysis of colza oil. The production of methyl esters was monitored over time by gas chromatography coupled to a mass spectrometer. The results show that to reach maximum performance of the biocatalyst, a certain amount of water is required (5 wt.%). By using ratio lower than the stoichiometry (1:1), the methanol conversion is completed and high transesterification yields could be obtained even in the absence of non polar solvents (i.e. hexane). Herein, the lipase uses the fatty substrate as lipophilic interface required for the opening of the active site of the enzyme.