微生物转化方法生产香草酸与香草醛的初步研究

从实验室保藏的菌种中筛选到一株黑曲霉（Aspergillus niger）SW-33,能够将1g/L的阿魏酸底物转化为0.23g/L的香草酸,相应的摩尔转化率为29.35％;流加四次底物阿魏酸后,产物浓度达到1.11g/L,相应的摩尔转化率为44.9％。为了提高产物浓度,对培养基和发酵条件进行优化,使得该菌株能够将1g/l的阿魏酸底物转化为0.46g/L的香草酸,相应的摩尔转化率为57.81％。提取得到的香草酸,经HPLC测定,纯度为85.9％;提取收率为75.2％。用含香草酸的转化液,或者用提取的结晶香草酸,加入朱红密孔菌（Prcnporus cinnabarnus）SW-0203发酵培养液,可得到转化产物香草醛。     

两相体系中微生物法转化异丁香酚生成香草醛的研究

从土壤中筛选获得一株能耐受高浓度异丁香酚并高效转化生成香草醛的纺锤芽孢杆菌Bacillus fusiformis菌株CGMCC1347,研究了微生物细胞在异丁香酚-水两相体系中转化异丁香酚制备香草醛的过程。在异丁香酚体积分数60％,初始pH 4．0,温度37℃,转速180 r/min的条件下,转化72h,湿细胞质量浓度迭60 g/L时,香草醛质量浓度高达46.10 g/L。     

转化异丁香酚生成香草醛纺锤芽孢杆菌的筛选

以底物异丁香酚为唯一碳源从七壤中筛选获得了一株能高效转化异丁香酚生成香草醛的芽孢杆菌。根据生理生化特性及16SrRNA序列分析鉴定其属于纺锤芽孢杆菌（Bacillus fusiformis）,初步试验表明该菌能转化2％异丁香酚生成4．20g/L香草醚。     

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。     

转化异丁香酚生成香草醛纺锤芽孢杆菌的筛选*

以底物异丁香酚为唯一碳源从土壤中筛选获得了一株能高效转化异丁香酚生成香草醛的芽孢杆菌。根据生理生化特性及16S rRNA序列分析鉴定其属于纺锤芽孢杆菌(Bacil-lus fusiformis),初步试验表明该菌能转化2%异丁香酚生成4.20 g/L香草醛。     

酶法转化异丁香酚制备香草醛的研究

研究了由大豆粗酶转化异丁香酚制备香草醛的方法,对转化条件进行了优化,并研究了H2O2、和吸附剂的影响,在优化后的条件下转化36h产物浓度达2．46g/L,摩尔转化率为13．27％。     

纺锤芽孢杆菌CGMCC1347发酵生产异丁香酚单加氧酶的条件优化

对从土壤中筛选获得的纺锤芽孢杆菌CGMCCl347生产异丁香酚单加氧酶的发酵条件进行了单因素考察及正交实验优化,确定了最适的发酵摇瓶培养基组成和培养条件。在发酵培养基组成为尿素1g／L,玉米浆55g/L,K2HP042g／L,MgSO4·7H2O1g／L,初始pH7．5,发酵温度37℃,摇床转速180r／min的条件下培养16h获得的细胞,能转化2％的异丁香酚生成2．49g／L香兰素,异丁香酚单加氧酶酶活达3．79U／L。     

Optimization of fermentation conditions for isoeugenol monooxygenase from Bacillus fusiformis CGMCC1347

对从土壤中筛选获得的纺锤芽孢杆菌CGMCC1347生产异丁香酚单加氧酶的发酵条件进行了单因素考察及正交实验优化,确定了最适的发酵摇瓶培养基组成和培养条件.在发酵培养基组成为尿素1 g/L,玉米浆55 g/L,K2HPO4 2g/L,MgSO4·7H2O 1 g/L,初始pH 7.5,发酵温度37℃,摇床转速180 r/min的条件下培养16h获得的细胞,能转化2％的异丁香酚生成2.49 g/L香兰素,异丁香酚单加氧酶酶活达3.79 U/L.     

香草醛对杉木幼苗生长的影响

为了解杉木连栽土壤中有毒化感物质对杉木幼苗毒害作用 ,采用水培杉木幼苗方法 ,通过投加不同浓度香草醛 ,发现 1mg·kg- 1 香草醛显著抑制杉木种子胚根的伸长 (P<0 .0 5) ,只为对照的 70 % ;香草醛浓度达 1 0mg·kg- 1 时 ,叶绿素总量明显下降到对照的80 % ;超过 2 0mg·kg- 1 对杉木幼苗地径与高度生长产生明显抑制作用 ;50mg·kg- 1 以上将明显影响地上部分枝叶的正常生长发育 ,及至植株冠层的生长 ;超过 1 0 0mg·kg- 1 ,整个植株的生长受到显著抑制 .香草醛是连栽土壤中毒性较大的一种有毒化感物质 ,是杉木存活率低的重要原因之一 .     

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。     

Recent advances in biotechnological production of 2-phenylethanol

2-Phenylethanol (2-PE) is an important aromatic alcohol with a rose-like fragrance. It has been widely applied in the cosmetic, perfume, and food industries and is mainly produced by chemical synthesis. An alternative method for the production of natural flavors and fragrances is the microbial transformation process, which is attracting increasing attention because it is an environmentally friendly process and the products are considered “natural”. The production of 2-PE from L-phenylalanine by biotransformation is possible through the Ehrlich pathway and considerable progress has been made in the development of this process. The present report reviews recent advances in biotechnological production of 2-PE, with emphasis on the strategies used to increase production and the applications of in situ product removal techniques. Future research should focus on product scale-up and product recovery processes for the industrialization of microbial processes.     

Transformation of ferulic acid to vanillin using a fed‐batch solid–liquid two‐phase partitioning bioreactor

Amycolatopsis sp. ATCC 39116 (formerly Streptomyces setonii) has shown promising results in converting ferulic acid (trans‐4‐hydroxy‐3‐methoxycinnamic acid; substrate), which can be derived from natural plant wastes, to vanillin (4‐hydroxy‐3‐methoxybenzaldehyde). After exploring the influence of adding vanillin at different times during the growth cycle on cell growth and transformation performance of this strain and demonstrating the inhibitory effect of vanillin, a solid–liquid two‐phase partitioning bioreactor (TPPB) system was used as an in situ product removal technique to enhance transformation productivity by this strain. The thermoplastic polymer Hytrel^® G4078W was found to have superior partitioning capacity for vanillin with a partition coefficient of 12 and a low affinity for the substrate. A 3‐L working volume solid–liquid fed‐batch TPPB mode, using 300 g Hytrel G4078W as the sequestering phase, produced a final vanillin concentration of 19.5 g/L. The overall productivity of this reactor system was 450 mg/L. h, among the highest reported in literature. Vanillin was easily and quantitatively recovered from the polymers mostly by single stage extraction into methanol or other organic solvents used in food industry, simultaneously regenerating polymer beads for reuse. A polymer–liquid two phase bioreactor was again confirmed to easily outperform single phase systems that feature inhibitory or easily further degraded substrates/products. This enhancement strategy might reasonably be expected in the production of other flavor and fragrance compounds obtained by biotransformations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:207–214, 2014     

Recent advances on applications and biotechnological production of d-psicose

D-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of D-psicose. In addition, the biochemical properties of D-tagatose 3-epimerase family enzymes and the D-psicose-producing enzyme are compared, and the biotechnological production of D-psicose from D-fructose is reviewed.     

Phenylpropanoid 2,3-dioxygenase involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia

Enzyme catalyzing the cleavage of the phenylpropanoid side chain was partially purified by ion exchange and gel filtration column chromatography after (NH₄)₂SO₄ precipitation. Enzyme activities were dependent on the concentration of dithiothreitol (DTT) or glutathione (GSH) and activated by addition of 0.5 mM Fe²⁺. Enzyme activity for ferulic acid was as high as for 4-coumaric acid in the presence of GSH, suggesting that GSH acts as an endogenous reductant in vanillin biosynthesis. Analyses of the enzymatic reaction products with quantitative NMR (qNMR) indicated that an amount of glyoxylic acid (GA) proportional to vanillin was released from ferulic acid by the enzymatic reaction. These results suggest that phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and GA in Vanilla planifolia.     

Production of vanillin from ferulic acid using recombinant strains ofEscherichia coli

Vanillin is one of the world's principal flavoring compounds, and is used extensively in the food industry. The potential vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with P_BAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L obtained before the optimization of vanillin production.     

Recent advances on physiological functions and biotechnological production of epilactose

Epilactose (4-O-β-d-galactopyranosyl-d-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed.