生物法催化水解外消旋安息香酸薄荷酯制备左旋薄荷醇的研究

论文摘要

l-薄荷醇是一个很重要的香料化学品。由于它独特香气和清凉效果被广泛用于医药、食品、烟草、化妆品及口腔保健等产品。由于生物催化反应具有高效、高选择性、副产物少、安全和环境友好等优点,所以近年来利用生物催化对映选择性拆分技术制备l-薄荷醇的研究引起了学术界和工业界的广泛关注。安息香酸薄荷酯作为一个重要的拆分底物已被用于千吨级左旋薄荷醇的工业生产。本文的目的是以外消旋安息香酸薄荷酯作为底物,建立一个酶促对映选择性水解制备左旋薄荷醇的生物催化过程。本论文的主要工作包括以下两个部分：第一,高效生物催化剂的定向筛选。以待拆分的底物（dl-安息香酸薄荷酯）作为微生物富集培养的唯一碳源,以目标产物（l-薄荷醇）的产量和纯度作为筛选的指针。从土样分离得到的341个微生物菌落中,通过薄板层析（TLC）初筛得到129株具有目标反应活性的菌株,通过对其中活性较高的酯酶产生菌的复筛,获得13株对目标底物转化较好且所得产物光学纯度在80%ee以上的菌株。对13株候选菌株的底物耐受性进一步考察,最终筛选到一株具有高选择性和高水解活性的生物催化剂。该菌株经16S rDNA鉴定,确定属于不动杆菌类,命名为Acinetobacter sp.ECU2040。第二,Acinetobacter sp.ECU2040的催化特性与过程优化及放大研究。经过优化,确定最佳反应温度为37℃,最佳反应pH为7.5,最佳乳化剂是吐温-80,其最适添加浓度为0.5%（w/v）。在最适反应条件下,它能够较好地催化外消旋混合物中l-薄荷酯的对映选择性水解生成左旋的l-薄荷醇。此外,还考查了酶的底物耐受性,确定不动杆菌酯酶最适的反应底物浓度可达10mM。最后将该反应扩大到1升规模,底物转化率仍可达到48%,表明该酶催化的反应过程具有进一步开发和较好的潜在应用前景。

论文目录

• Abstract
• 摘要
• Chapter 1 Introduction
• 1.1. Chirality
• 1.2. Chirality in bio-industry
• 1.3. l-Menthol
• 1.4. Application of menthol
• 1.5. The research on production of menthol in the world
• 1.5.1. Extraction from natural plants
• 1.5.2. Chemical synthesis
• 1.5.3. Biosynthesis
• 1.5.3.1. Enzymatic enatioselective esterification or transesterification
• 1.5.3.2. Enzymatic enantioselective hydrolysis
• 1.6. The main content and objective of this study
• Chapter 2 Screening of biocatalysts for enantioselective hydrolysis of dl-menthyl benzoate to l-menthol
• 2.1. Forewords
• 2.2. Main process for strain screening
• 2.3. Materials and methods
• 2.3.1. Main materials and apparatuses
• 2.3.2. Soil samples
• 2.3.3. Culture medium
• 2.3.4. Analytic methods
• 2.3.4.1. Thin layer chromatoraphy （TLC） analysis
• 2.3.4.2. Gas chromatography （GC） analysis
• 2.3.4.3. High-performance liquid chromatography（HPLC）
• 2.3.5. Experimental methods
• 2.3.5.1. Chemical synthesis of racemic menthyl benzoate
• 2.3.5.2. Soil sample collection
• 2.3.5.3. Enrichment culture
• 2.3.5.4. Isolated strains
• 2.3.5.5. Preliminary screening
• 2.3.5.6. Reselection of target strains
• 2.3.5.7. Crude enzyme preparation
• 2.3.5.8. Strain identification
• 2.4. Results and discussion
• 2.4.1. Chemical synthesis of racemic menthyl benzoate
• 2.4.2. Calibration curve of menthol concentration
• 2.4.3. The results of isolated microbial strains from soil samples
• 2.4.4. Isolated strains
• 2.4.5. Preliminary screening
• 2.4.6. Reselection and confirmation
• 2.4.7. Strain identification
• 2.4.8. Fermentation process of Acinetobacter sp.ECU2040
• 2.5. Summary
• Chapter 3 The process study of enzymatic hydrolysis for l-menthol production
• 3.1. Forewords
• 3.2. Materials and apparatuses
• 3.2.1. Main materials and apparatuses
• 3.2.2. Analytic methods
• 3.2.2.1. Thin layer liquid chromatography（TLC）analysis
• 3.2.2.2. Gas chromatography analysis（GC）
• 3.2.2.3. High-performance liquid chromatography（HPLC）
• 3.2.2.4. Enzyme activity assays
• 3.2.3. Experimental methods
• 3.2.3.1. Cultivation
• 3.2.3.2. Effect of pH
• 3.2.3.3. Effect of temperature
• 3.2.3.4. Effect of surfactants
• 3.2.3.5. Effect of substrate concentration on the enzyme tolerance
• 3.2.3.6. Enzymatic hydrolysis of different dl-menthyl esters
• 3.2.3.7. The scale-up of l-menthol production
• 3.3. Results and discussion
• 3.3.1. Effect of pH
• 3.3.2. Effect of temperature
• 3.3.3. Effect of surfactants
• 3.3.4. Effect of substrate concentration on enzyme tolerance
• 3.3.5. Enzymatic hydrolysis of different dl-menthyl esters
• 3.3.6. Scale up of bioreaction and preparation of l-menthol product
• 3.4. Summary
• References
• Acknowledgements