异丁香酚的生物转化及香兰素的合成

利用粘质沙雷氏菌菌株AB 90027产生的离体酶催化异丁香酚进行生物转化,结合TLC、GC、UV-VISI、R等测试手段对异丁香酚的转化途径进行了探讨,借助薄层扫描法对不同反应体系下产物香兰素的产率作了比较,并研究了可催化异丁香酚合成香兰素的酶在细胞的存在位置。结果表明:在酶的作用下,异丁香酚分别经过阿魏酸和香兰素两条途径开环降解成小分子,中间产物香兰素产率可达10.90%,催化合成香兰素的酶主要是胞外酶。     

香兰素生物合成的研究进展

香兰素是一种十分重要的香料,在较多行业中用途广泛。天然来源的香兰素受诸多因素的限制,不能满足市场需求,因此化学法合成的香兰素是主要原料来源。近年来,随着自然资源的不断枯竭以及人们对环境保护意识的增强,通过微生物转化适宜的底物生物合成香兰素逐渐变为研究热点。本文综述了以丁香酚、异丁香酚和阿魏酸为底物的细菌、真菌生产香兰素的相关研究进展,阐述丁香酚、异丁香酚、阿魏酸产香兰素代谢途径的研究,以及生物技术在这一领域的运用。香兰素的生物合成具有广阔的发展和市场应用前景。     

大豆异黄酮的微生物转化

大豆异黄酮是近年来发现的具有多种生物学活性的大豆营养成分。综述了微生物对大豆异黄酮的转化过程,对大豆异黄酮的生物利用和营养成分的影响,指出在富含大豆异黄酮类制品生产过程中合理利用微生物活动的重要意义和可能途径。     

微生物转化洛伐他汀条件的初步研究

以本实验室筛出的一株菌ST2710为出发菌,研究不同培养条件对转化率的影响。结果发现,菌株ST2710在种子培养基进行预培养,再转入转化培养基中培养2d后,加入洛伐他汀溶液1.0mL(10mg／mL)后再培养5d,产物的产率可达43．41％;就四种溶剂溶解底物条件进行了研究,结果发现用二甲基甲酰胺溶解洛伐他汀,产物产率为最高,并且转化作用不需要底物的诱导;以单独的菌丝体和上清液转化洛伐他汀时均低于培养液对洛伐他汀的转化作用。     

微生物转化法生产度洛西汀中间体手性醇菌株的鉴定

【目的】获得以DKTP为底物合成度洛西汀关键中间体手性醇(S)-DHTP的菌株。【方法】采用常规及改进的微生物转化法从土壤中进行筛选。【结果】筛选获得一株菌株,能够将底物DKTP对映选择性地还原为(S)-DHTP,且具有较高的转化率(>90%)和几乎绝对的对映体过量值(e.e.>99%),改进的筛选方法更为简便高效。形态学特征和26S rDNA序列分析综合判断,该菌株属于红酵母属,命名为红酵母Rhodotorula sp.507。【结论】供试菌株能够高效地、不对称地生物还原DKTP成度洛西汀前体物(S)-DHTP,使大量获得度洛西汀前体原料变得经济可行。     

大豆异黄酮微生物转化研究进展

大豆异黄酮是大豆在其生长过程中形成的一类次生代谢产物,具有抗氧化、抗癌、减少骨质流失、降低心脑血管发病率等多种生理功能。目前已知,被摄人机体的大豆异黄酮将被肠道微生物菌群转化为具有更高、更广生物学活性的不同产物。因此,大豆异黄酮对人体的有益调节作用强弱并不简单取决于摄人机体的净含量的多少,更在于被摄人机体的大豆异黄酮将如何被肠道菌群转化。本文从大豆异黄酮的组成与功能、大豆异黄酮体内吸收、代谢及微生物转化、转化产物的活性以及高效合成等方面进行了系统综述,对大豆异黄酮微生物生物转化研究现状和存在问题进行分析总结,并对今后发展趋势进行展望,旨在推动高活性大豆异黄酮微生物转化产物的研究与开发。     

微生物转化秸秆饲料

正秸秆作为农作物的主要副产品,是一种重要的可再生资源。利用微生物的降解能力,将秸秆转化为动物可利用的饲料,对于解决人畜争粮具有重大意义。微生物处理法具有提高营养价值、改善适口性、绿色无污染、能耗低等优点,是当前最具应用潜力和发展前景的秸秆饲料生产技术。本刊2013年第4期刊登了吴文韬、鞠美庭等发表的文章"一株纤维素降解菌的分离、鉴定及对玉米     

荧光假单胞菌香兰素脱氢酶基因功能的验证

验证了荧光假单胞菌(Pseudomonas fluorescensATCC13525)香兰素脱氢酶基因(vanillin dehydrogenasegene,vdh)的功能。基因vdh表达产物(Vdh)的活性测定结果显示Vdh具有很高的活性,而且不经IPTG诱导的Vdh也具有同样高的活性。经过4 h的体外酶促反应,重组蛋白Vdh能把95%以上的香兰素转化为香兰素酸,从而验证了vdh基因的表达产物具有香兰素脱氢酶的功能。同时发现NAD 是从香兰素到香兰素酸体外转化必不可少的因素。     

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10 g l⁻¹ isoeugenol was converted to 3.75 g l⁻¹ vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography–mass spectrometry. Based on the accurate masses obtained from gas chromatography–high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.     

Enhanced vanillin production from ferulic acid using adsorbent resin

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l⁻¹ ferulic acid could be added continually and 19.2 g l⁻¹ vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.     

Synthesis of two new hydroxylated derivatives of spironolactone by microbial transformation

Spironolactone is a medicinally important molecule that is clinically used in the treatment and management of many diseases such as oedema and ascites in cirrhosis of the liver, malignant ascites, nephrotic syndrome, chronic lung disease, resistant hypertension, congestive heart failure, and primary hyperaldosteronism. Microbial transformations of spironolactone by Cunninghamella elegans ATCC 9245 was carried out. Two new hydroxylated derivatives, 12β-hydroxy-spironolactone and 2α-hydroxy-spironolactone, were synthesized. Their structures were characterized on the basis of the spectroscopic data. The substrate can be efficiently converted into the products within 72 h after its addition to the fermentation broth of C. elegans ATCC 9245.     

微生物转化技术在中药开发中的应用进展

中药现代化是目前中药研究最迫切的需要,微生物转化技术可以利用微生物的特性解决中药现代化研究中难以解决的诸多问题,我们总结了微生物转化中药的特点,转化酶系统及反应类型,分析了微生物生物转化对中药的影响,认为微生物转化技术酶系统广泛,选择性强,反应条件温和可控,反应类型广泛,适用于所有类型的中药有效成分的生物转化,经过转化后,可以提高中药药效,降低毒性,去除杂质,帮助有效成分的体内代谢及产生新的药物成分。中药微生物转化技术必定成为中药学与微生物学完美契合的典范,推进中药现代化的进程。     

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of bacillus coagulans

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and ¹H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.     

Calculated ionisation potentials determine the oxidation of vanillin precursors by lignin peroxidase

In view of the biocatalytic production of vanillin, this research focused on the lignin peroxidase (LiP) catalysed oxidation of naturally occurring phenolic derivatives: O-methyl ethers, O-acetyl esters, and O-glucosyl ethers. The ionisation potential (IP) of a series of model compounds was calculated and compared to their experimental conversion by LiP, defining a relative IP threshold of approximately 9.0 eV. Based on this threshold value only the O-acetyl esters and glucosides of isoeugenol and coniferyl alcohol would be potential LiP substrates. Both O-acetyl esters were tested and were shown to be converted to O-acetyl vanillin in molar yields of 51.8 and 2.3%, respectively.     

High-performance liquid chromatographic method for determination of vanillin and vanillic acid in human plasma, red blood cells and urine

A simple high-performance liquid chromatographic method was developed for the determination of vanillin and its vanillic acid metabolite in human plasma, red blood cells and urine. The mobile phase consisted of aqueous acetic acid (1%, v/v)–acetonitrile (85:15, v/v), pH 2.9 and was used with an octadecylsilane analytical column and ultraviolet absorbance detection. The plasma method demonstrated linearity from 2 to 100 μg/ml and the urine method was linear from 2 to 40 μg/ml. The method had a detection limit of 1 μg/ml for vanillin and vanillic acid using 5 μl of prepared plasma, red blood cells or urine. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of vanillin in patients undergoing treatment for sickle cell anemia.     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

Quantitative isotopic C nuclear magnetic resonance at natural abundance to probe enzyme reaction mechanisms via site-specific isotope fractionation: The case of the chain-shortening reaction for the bioconversion of ferulic acid to vanillin

Isotope fractionation is a powerful technique by which to probe the reaction mechanism of enzymes. The effect of a heavy isotope on the reaction energetics can be used to predict transition state architecture and reaction mechanism. In order to examine simultaneously the isotope fractionation in ¹³C at multiple sites within the substrate and product molecules without any need for site-selective isotope enrichment, a technique exploiting quantitative isotopic nuclear magnetic resonance (NMR) spectrometry at natural abundance (NAQ–NMR) has been developed. Here we report the first application of this technique to the study of an enzyme-catalyzed reaction, the bioconversion of ferulic acid to vanillin in cultures of Streptomyces setonii. We were able to show that the NAQ–NMR methodology is sufficiently precise and robust to measure the isotope shifts in the ¹³C/¹²C ratios in both substrate and product of this biotransformation, thereby permitting meaningful data to be obtained even at carbon positions that take part only indirectly in the reaction and show only secondary isotope fractionation. The results obtained provide direct evidence in support of the current hypothesis for the reaction mechanism of the enzyme hydroxycinnamoyl–CoA hydratase/lyase, notably the proposed involvement of the quinone methide enolate of feruloyl–CoA as intermediate in the catalytic pathway.