A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.     

Comparative electrochemical study on monolignols and dimers relevant for the comprehension of the lignification process

The objective of the present study is to complete previous electrochemical studies on coniferyl alcohol analogs by focusing on its dimers to further elucidate the lignification process, in order to distinguish between steric and electronic factors that inhibit oxidation. The results demonstrated that monolignols exhibit lower oxidation potential values than dimers and the β-O-4 dimer exhibited a quite peculiar behavior allowing two-electron oxidation. Moreover, we noticed that the pH turned out to affect the redox potential values. Phenol/phenol cation radical couples had a significantly higher potential than the corresponding phenate/phenoxy radical couple. These results emphasize the importance of understanding the specificities of in planta lignin growth and structure.

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Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli

Two novel genes (tvms10a, tvmz2a) were identified in the metagenomic DNA of Rusa unicolor and Equus burchelli fecal samples. The amplified PCR product of tvms10a is composed of 917bp and the gene was found to encode a protein containing 165 amino acids, while the tvmz2a PCR product was 1053bp long encoding 298 amino acid proteins. The gene has 72% primary sequence identity with Clostridiales sp. These amplified PCR products which can encode FAE were cloned into pGEMT Easy TA cloning vector and then sub-cloned into the EcoRI site of pET32a expression vector to generate pET32-tvms10a and pET32-tvmz2a, which was then transformed into Escherichia coli BL21. The recombinants were grown in LB medium and gene expression was induced with IPTG for 6h. Purified recombinant Tvms10a and Tvmz2a proteins showed molecular masses of 18.6 and 31.2kDa respectively, and displayed hydrolytic activity towards substrate ethyl ferulate. The activities of Tvms10a and Tvmz2a produced in E. coli were 15 and 9U/min respectively, and their specific activities 16.6 and 10.4U/mg protein respectively. The optimal pH is between 5.0 and 8.0 and the optimal temperature is 37°C for enzyme reaction. Unusually, these proteins were found to be capable of releasing ferulic acid (FA) and diferulic acid (diFA) from untreated crude plant cell wall materials. The substrate utilization preferences and sequence similarity of these clones place it in the type-D sub-class of FAE.     

川芎晒干过程中阿魏酸和阿魏酸松柏酯含量的变化

为了解新鲜川芎采后干燥过程中阿魏酸和阿魏酸松柏酯含量的动态变化规律,采用高效液相色谱法测定了川芎晒干过程中总阿魏酸、游离阿魏酸和阿魏酸松柏酯的含量。结果显示,在整个晒干过程中(30 d),总阿魏酸、游离阿魏酸和阿魏酸松柏酯含量呈先升高后下降的变化趋势,晾晒第3 d时总阿魏酸含量最高(0.23%),因此在晾晒的第3 d利用快速干燥技术能较好地保留川芎药材中总阿魏酸含量,使其发挥更佳的药效。川芎药材中的阿魏酸松柏酯能水解产生阿魏酸,因此研究川芎干燥过程中的生理响应与含水量的关系对阿魏酸积累有重要意义。由于川芎在用药过程中是以总阿魏酸含量发挥药效的,所以以总阿魏酸含量作为川芎药材质量控制指标更加科学。     

苦荞FtF5H基因克隆及表达分析

阿魏酸-5-羟基化酶(Ferulate 5-hydroxylase)是调控S型木质素合成的关键酶,为研究其在苦荞木质素生物合成途径中的分子机制,该文从苦荞转录组数据中筛选获得一个F5H基因,命名为FtF5H(GenBank登录号:MW455111),采用生物信息学方法对苦荞F5H蛋白的理化性质、信号肽、跨膜结构、亚细胞定位、亲疏水性、蛋白质二级结构、蛋白质三级结构、氨基酸结构、系统进化树等进行分析和预测,并运用实时荧光定量PCR(qRT-PCR)技术分析FtF5H基因在厚果壳苦荞与薄果壳苦荞的叶、花、茎、果壳中的差异表达。结果表明:(1)FtF5H基因序列包含1395 bp的完整cDNA开放阅读框,编码464个氨基酸。(2)FtF5H蛋白具有P450超家族结构,为亲水性稳定酸性蛋白,不具有跨膜结构域,且为非分泌性蛋白。(3)FtF5H蛋白的二级结构主要由α-螺旋和无规则卷曲组成,三级结构预测显示FtF5H蛋白与5ylw.1.A的相似度较高。(4)系统进化分析显示FtF5H属于CYP84A亚家族。(5)qRT-PCR显示FtF5H基因在两种苦荞中的不同部位均有表达,且在厚果壳苦荞果壳中的表达量是薄果壳的5倍,表达具有极显著差异。该研究为进一步研究苦荞木质素合成的分子调控机制奠定了基础,对苦荞新品种的培育具有重要意义。     

1,3-Diferuloyl-<Emphasis Type="Italic">sn</Emphasis>-glycerol from the biocatalytic transesterification of ethyl 4-hydroxy-3-methoxy cinnamic acid (ethyl ferulate) and soybean oil

1,3-Diferuloyl-sn-glycerol is found ubiquitously throughout the plant kingdom, possessing ultraviolet adsorbing and antioxidant properties. Diferuloyl glycerol was synthesized and isolated as a byproduct in up to 5% yield from a pilot plant scale packed-bed, biocatalytic transesterification of ethyl ferulate with soybean oil or mono- and diacylglycerols from soybean oil. The yield of the diferuloyl glycerol byproduct was directly proportional to the overall water concentration of the bioreactor. The isolated diferuloyl glycerol exhibited good ultraviolet adsorbing properties, 280–360 nm with a λ_max 322 nm, and compared well to the efficacy of commercial sunscreen active ingredients. The antioxidant capacity of diferuloyl glycerol (0.25–2.5 mM) was determined by its ability to scavenge 2,2-diphenyl-1-picrylhydrazyl radicals and was comparable to that of ferulic acid. At current pilot plant scale production capacity, 120 kg diferuloyl glycerol byproduct could be isolated per year. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: capillary zone electrophoresis study of pH effect

Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.     

Continuous synthesis of alkyl ferulate by immobilized Candida antarctica lipase at high temperature

1-Pentyl, 1-hexyl and 1-heptyl ferulates were continuously synthesized at 60–90°C using a reactor system in which a column packed with ferulic acid powders and another column packed with immobilized Candida antarctica lipase particles were connected in series. Conversions greater than 0.9 were achieved for the synthesis of the 1-hexyl and 1-heptyl ferulates at 90°C. The system could be stably operated for the 1-heptyl ferulate synthesis at 90°C for at least two weeks.     

阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液的抗抑郁样效果

阿魏酸(ferulic acid,FA)是一种广泛存在的低毒酚酸,阿魏酸钠(sodium ferulate,SF)则是其钠盐。先前的研究已经证实,阿魏酸钠具有显著的神经保护和神经发生增强作用及抗抑郁效果。该研究的目的在于探讨阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液可能的抗抑郁效果。PC12细胞在含80μmol/L阿魏酸钠的DMEM培养基中孵育6d,无菌条件下制备阿魏酸钠诱导分化的PC12细胞液的无细胞滤液,测定PC12细胞裂解液无细胞滤液中残留的阿魏酸钠量。以慢性不可预期的多种刺激制造大鼠抑郁模型,用行为学、形态学、免疫组织化学和BrdU掺入等方法观察并检测阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液对慢性应激大鼠抑郁模型行为学、海马的组织病理学、海马和大脑皮质的神经生长因子(nerve growth factor,NGF)及脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)的表达及神经发生的影响。实验证实,阿魏酸钠诱导分化的PC12细胞裂解液的无细胞滤液能改善抑郁症样模型大鼠的行为学障碍,上调其海马和大脑皮质NGF和BNDF的表达,增加海马神经干细...     

Regulation of Syringyl to Guaiacyl Ratio in Lignin Biosynthesis

p -hydroxyphenyl (H)-, guaiacyl (G)- and syringyl (S) propane, in situ is described. New pathways that regulate the ratio of S to G moieties operating at the stages of cinnamoyl CoA, cinnamyl aldehyde and cinnamyl alcohol are introduced. The roles of monolignol glucoside in the lignification of tree xylem are discussed. The results of gene manupulations that alter the lignin structures are also introduced. Received 15 September 2001/ Accepted in revised form 16 October 2001