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。     

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。     

最新专利文摘

CN1712518:微生物转化异丁香酚制备香草醛的菌种和方法本发明涉及一株从土壤中筛选获得的纺锤芽孢杆菌CGMCC1347(SW-B9)及其培养发酵,并用于转化异丁香酚制备香草醛的方法。在优化条件下发酵培养,发酵液或游离细胞或其固定化细胞用于异丁香酚转化24~96 h,转化液中含香草酸2~4 g     

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.     

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。     

芽孢杆菌异源转化和转导的比较研究

以枯草杆菌168和Ki-2-148作转化受体,8个种和1个变种的17株芽孢杆菌DNA作给体进行转化,证实大多数异源DNA能将给体的遗传标记传递给受体,但种间转化的频率与同源转化的频率相比,往往降低。借助转导噬菌体PBS1,在枯草杆菌Ki-2,浸麻芽孢杆菌AS1·64,短小芽孢杆菌AS1·386和枯草杆菌168之间能进行异源转导。抗链霉素标记的转化频率比色氨酸标记和尿嘧啶标记的转化频率高,表明芽孢杆菌的抗链霉素标记具有较大的同源性。插     

维生素C二步发酵中巨大芽孢杆菌对氧化葡萄糖酸杆菌生长和产酸的影响

通过测定氧化葡萄糖酸杆菌转化L－山梨糖中成ZKGA的细胞酶活性、摇瓶发酵及中长变化,研究了Vc:步发酵中巨大茅孢杆菌对氧化葡萄糖酸杆菌生长和产酸作用的影响。结果显示：巨大芽孢杆菌胞外液和胞内液均可促进氧化葡萄糖酸杆菌的增殖,主要表现为缩短其中长周期中的延迟期;巨大芽孢杆菌通过所产生的部分生物活性物质增强氧化葡萄糖酸杆菌产酸的细胞酶活性,促进氧化葡萄糖酸杆菌转化L一山梨糖生成2KGA.     

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。     

芽孢杆菌原生质体的形成和质粒转化的研究

测定了芽孢杆菌属中21个种、68个菌株的原生质体形成率。在高渗缓冲液(SMMP 或SMN)中,原生质体形成率在90％以上的有37株。降低高渗缓冲液中钠离子浓度,有利于原生质体的形成。用质粒(pUB 110或pC194) DNA对16株芽孢杆菌的原生质体进行了转化试验。转化成功的共8株：纳豆芽孢杆菌AS 1.107、AS 1.921、幼虫芽孢杆菌AS 1．430、球芽孢杆菌AS 1.1362、迟缓芽孢杆菌#50、苏云金芽孢杆菌松蠋亚种AS 1．294、地衣芽孢杆菌# 18和坚强芽孢杆菌#28。原生质体在DM3再生培养基上的再生率分别为0．1％一19．2％,转化效率分别为1．4×102一1．0×105转化子／μgDNA。转化效率低或未转化成功的菌株,其原生质体的再生率一般都很低或不能再生,有的菌株在形成原生质体后发生自溶。     

枯草芽孢杆菌感受态细胞的制备及质粒转化方法研究

为便于枯草芽孢杆菌工业化生产应用,对Spizizen创立的枯草芽孢杆菌DNA转化方法进行改进.用GMI和GMII溶液处理枯草芽孢杆菌野生型菌株BS501a、营养缺陷型突变株DBl342和非营养缺陷型突变株WB800,用改进的方法制备感受态细胞,用7.5kb质粒pSBPTQ进行转化,并研究RNA、酵母粉、水解酪蛋白、培养方法对枯草芽孢杆菌质粒转化的影响.结果表明,该方法适用于不同基因型枯草芽孢杆菌的质粒转化,营养缺陷型突变株DBl342的转化率为750 CFU/μg/DNA,非营养缺陷型突变株WB800转化率为1 070 CFU/xg DNA,野生型菌株BS501a转化率为270 CFU/μg/DNA.根据影响转化效率的因素,推测在该方法中,枯草芽孢杆菌质粒转化原理:一定生物量的枯草芽孢杆菌在外界营养条件和钙、镁离子作用下,细胞壁和细胞膜形成缺陷,使外源DNA转入枯草芽孢杆菌细胞内.     

Biotransformation of Isoeugenol to Vanillin by a Novel Strain of <Emphasis Type="Italic">Bacillus fusiformis</Emphasis>

A novel strain of Bacillus fusiformis, producing high amounts of vanillin from isoeugenol, was isolated from soil. Using 60% (v/v) isoeugenol as substrate and solvent and at pH 4.0, 37 °C and 180 rpm, vanillin was produced at 32.5 g l⁻¹ over 72 h. The unused isoeugenol was reusable.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.     

Isolation of a Bacillus sp. capable of transforming isoeugenol to vanillin

Natural aroma compounds are of major interest to the flavor and fragrance industry. Due to the limited sources for natural aromas, there is a growing interest in developing alternative sources for natural aroma compounds, and in particular aromatic aldehydes. In several microbial species aromatic aldehydes are detected as intermediates in the degradation pathway of phenylpropanoids. Thus, bioconversion of phenylpropanoids is one possible route for the production of these aroma compounds. The present work describes the isolation of microbial strains, capable of producing vanillin from isoeugenol. Bacterial strains isolated from soil, were screened for their ability to transform isoeugenol to vanillin. One of these strains, strain B2, was found to produce high amounts of vanillin when grown in the presence of isoeugenol, and was also capable of growing on isoeugenol as the sole carbon source. Based on its fatty acids profile, strain B2 was identified as a Bacillus subtilis sp. The bioconversion capabilities of strain B2 were tested in growing cultures and cell free extracts. In the presence of isoeugenol, a growing cultures of B. subtilis B2 produced 0.61 g l-1 vanillin (molar yield of 12.4%), whereas cell free extracts resulted in 0.9 g l-1 vanillin (molar yield of 14%).     

Candida galli Strain PGO6: A Novel Isolated Yeast Strain Capable of Transformation of Isoeugenol into Vanillin and Vanillic Acid

Candida galli strain PGO6 isolated from oil-contaminated water is the first isolated yeast strain which is capable to form vanillin and vanillic acid during isoeugenol biotransformation. The products were confirmed by thin-layer chromatography (TLC), changes in the UV absorption pattern and high-performance liquid chromatography (HPLC). The phenotypic and physiochemical characteristics as well as molecular phylogenetic analysis based on amplification the ITS1-5.8S-ITS2 rDNA regions indicated the isolated strain PGO6 was identified as C. galli (GenBank accession number HM641231). Resting cells of C. galli PGO6 from the late-exponential of growth phase were used as biocatalysts for the biotransformation of isoeugenol. The optimal molar conversion of vanillin (48%) and vanillic acid (19%) was obtained after a 30 h incubation using 0.1% (v/v) of isoeugenol and 6 mg of dry weight of cells per ml without further optimization. Under these conditions, the total amount of vanillin and vanillic acid was 583 mg l(-1). Further biotransformation was carried out using 0.5% (v/v) of isoeugenol under the resting cells conditions, yielding a vanillin concentration of 1.12 g l(-1) (molar yield 25.7%) after 60 h incubation. This study brings the first evidence for biotransformation of isoeugenol to vanillin and vanillic acid by a yeast strain.     

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.     

Optimization of media composition for improving conversion of isoeugenol into vanillin with Pseudomonas sp. strain KOB10 using the Taguchi method

Abstract

The popular demand for natural food additives has resulted in a number of processes for producing natural vanillin. Although there are chemical procedures and plant sources for vanillin production, microbial bioconversions are being sought as a suitable ‘natural’ alternative. The present paper describes the conversion of isoeugenol to vanillin by a novel bacterial strain isolated from soil. The strain was identified as Pseudomonas sp. strain KOB10 based on morphological and physiochemical characteristics and its 16S rDNA gene sequence. We optimized medium composition for vanillin production using a Taguchi experimental design. Eight factors, i.e. isoeugenol, glycerol, tryptone, K₂HPO₄, KH₂PO₄, Cu²⁺, Mg²⁺ and Ca²⁺ concentrations, were selected and experiments based on an orthogonal array layout of L₁₈ (2² × 3⁶) were performed. Analysis of the experimental data using the Taguchi method indicated that Cu²⁺ and glycerol concentrations had the highest impact on isoeugenol conversion into vanillin at a substrate concentration of 0.9 g L^?1. Under the optimized conditions, growing cells of Pseudomonas sp. strain KOB10 produced 0.153 g vanillin L^?1 from 0.9 g isoeugenol L^?1, with a molar yield of 18.3% after incubation for 48 h. To improve the vanillin yield, the effect of other bioconversion parameters including time of isoeugenol addition, initial isoeugenol concentration and conversion time was studied; the results showed a maximum concentration of 3.14 g vanillin L^?1 after a total incubation time of 88 h with 15 g isoeugenol L^?1, which corresponded to a molar yield of 22.5%. Further standardization and optimization for vanillin production was challenging.     

Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.