發(fā)布時間：2021-05-12 13:06

　　酶在合成有機化學和生物技術(shù)的應(yīng)用上通常受到以下限制:（1）、由于酶作用的底物范圍比較窄,許多我們需要的化合物不能被酶催化;（2）、酶的活性高,但是立體選擇性較差;（3）、酶在催化條件下不穩(wěn)定;（4）、產(chǎn)物抑制使得轉(zhuǎn)化率低。在近10-15年間,定向進化技術(shù)發(fā)展起來。定向進化,有時又被稱為實驗室進化（試管中的進化）,試圖通過反復的基因突變、表達、篩選循環(huán)重復自然進化過程,直到篩選（或選擇）到符合要求的生物催化劑。這一概念在一定程度上改變了傳統(tǒng)采用“合理設(shè)計”構(gòu)建定點突變的蛋白質(zhì)工程方法。對來自魚腥藻（Anabaena variabilis）中的苯丙氨酸解氨酶（AvPAL）進行合理設(shè)計,以低成本并且市場上可購買的反式肉桂酸（t-CA）為原料,大規(guī)模生產(chǎn)L-苯丙氨酸以及β-苯丙氨酸。通過計算機模擬（對接）的方法,得到兩個突變體可以提高L-苯丙氨酸的產(chǎn)量,對突變體的氫鍵分析揭示了催化活性提高的原因。突變體所催化反應(yīng)的反應(yīng)速率,底物抑制以及動力學數(shù)據(jù)與野生型基本一致。根據(jù)我們的結(jié)果,Arg103突變體的酶活性相對于野生型AvPAL提高了 11倍。我們通過酶法,以低價的肉桂酸為原料,一鍋法得到大量光... 

【文章來源】：北京化工大學北京市 211工程院校 教育部直屬院校

【文章頁數(shù)】：89 頁

【學位級別】：碩士

【文章目錄】：
Abstract
摘要
Abbreviations list
Chapter 1 Introduction
    1.1.Phenylalanine Ammonia Lyase
    1.2.Discovery of phenylalanine ammonia-lyase enzyme
    1.3.Sources of Phenylalanine Ammonia Lyase (PAL)
    1.4.Structure of Phenylalanine Ammonia Lyase and Phenylalanine Aminomutases Enzymes
        1.4.1.Determination the structure of PAM
            1.4.1.1.PAM Monomer Structure
            1.4.1.2.PAM Tetramer Structure
    1.5.Application of Phenylalanine ammonia-lyase
    1.6.Rational Designing of PAL
    1.7.Our Aim and Objective
Chapter 2 The Production of L-Phenylalanine
    2.1 Introduction of L-Phenylalanine and its importance
    2.2 Methods used for the synthesis of L-phenylalanine
    2.3 Materials and Method used for the Production of L-Phenylalanine
        2.3.1.Media Used
            2.3.1.1.LB (Luria-Bertani) Liquid Medium
            2.3.1.2.LB (Luria-Bertani) Solid Medium
        2.3.2.Chemicals
    2.4.Strains and Plasmids
    2.5.Induced Expression of Recombinant Protein AvPAL
        2.5.1.The Pet-28A-Avpal Recombinant Plasmid Was Transferred Into Competent Cells
        2.5.2.Inducing Expression of the Target Protein
        2.5.3.Ultrasonic Disruption and SDS-PAGE Electrophoresis Protein Detection
    2.6.Polyacrylamide Gel Electrophoresis
        2.6.1.Stock Solutions
            2.6.1.1.Acrylamide/Methylene-bis-acrylamide (30% Acr, 0.8% Bis)
            2.6.1.2.Determining Gel Buffer Stock(1.50 M Tris-HCl pH 8.8):
            2.6.1.3.Stacking Gel Buffer Stock (0.5 M Tris-HCl pH 6.8)
            2.6.1.4.50%(v/v) Glycerol
            2.6.1.5.10%(W/V) Sodium Dodecyl Sulfate
            2.6.1.6.10% (w/v) Ammonium per Sulfate: (AP)
            2.6.1.7.5x Protein Loading Buffer
            2.6.1.8.Coomessive Brilliant Blue solution
            2.6.1.9.Coomessive Brilliant Blue Decolonization Solution 1L
        2.6.2.Separating and Stacking Gel Making Method
        2.6.3.Staining and De staining
    2.7.Expanding Culture and Induction
    2.8.Ultrasonic Crushing
    2.9.His-Tag Purification of Target Protein
    2.10.Anabaena Variabilis PAL (AvPAL) Optimized DNA Sequence
    2.11.Site-Directed Mutagenesis
    2.12.PCR (Polymerase Chain Reaction) Magnification
        2.12.1 Quick Change Pcr Setup;
        2.12.2 Quick Change Pcr Cycling
    2.13.Agarose Gel Electrophoresis
    2.14.Digestion
    2.15.Transformation
    2.16.Phe79 and Arg103 Expression and Purification
    2.17.L-Phenylalanine Production Reaction Condition
    2.18.Enzyme Activity Assay
    2.19.Methodology of Molecular Docking Studies
    2.20.Results and Discussion of L-Phenylalanine
        2.20.1.His-Tag Purification of the Target Protein
        2.20.2.Study of Substrates As Well As Yields
        2.20.3.Optimization of Bio-Reaction Conditions
            2.20.3.1.The Influence of pH on ee and Conversion
            2.20.3.2.Effect of Temperature over ee and Conversion
            2.20.3.3.Influence of the Incubation Period on ee and Conversion
        2.20.4.The Comparison of the Hydromanition Activity of AvPAL, Mutant Arg103 and79 Phe towards Cinnamic Acid for the Production of L-Phenylalanine
        2.20.5.Docking Study
    2.21.Conclusion
Chapter 3 Engineer the Phenylalanine Ammonia Lyase of AnabaenaVariabilis (AvPAL) for the Production of β-Phenylalanine from LowCost Trans-Cinnamic Acid
    3.1.Introduction and Brief History of β-phenylalanine
    3.2.Application of β-Phenylalanine
    3.3.Synthetic Ways of β-Phenylalanine
    3.4.Materials and Method used for the Production of b-Phenylalanine
        3.4.1.Materials
        3.4.2.Extraction of Plasmids and Genomes
            3.4.2.1.Extraction of Plasmid
            3.4.2.2 Extraction of Genomic DNA
        3.4.3.Strains and Plasmids
        3.4.4.Induced Expression and Purification of Recombinant Protein AvPAL
        3.4.5.Bioinformatics Tools
        3.4.6.Site-Directed Mutagenesis
        3.4.7.PCR (Polymerase Chain Reaction) Amplification
            3.4.7.1.PCR System
            3.4.7.2.Setting PCR Machine Programmed
        3.4.8.Agarose Gel electrophoresis
        3.4.9.Val165Ser, Leu358 and Ile105 Expression and Purification
        3.4.10.β-Phenylalanine Production Reaction Condition
        3.4.11.Enzyme Assay
    3.5.Result and Discussion of b-Phenylalanine
        3.5.1.Sequence and Structure Alignment
            3.5.1.1.Aligning of Sequence
            3.5.1.2.Structure Alignment
        3.5.2.His-Tag Purification of the Target Protein
        3.5.3.Investigation of Substrates and Products
        3.5.4.Optimizations of Bio Reaction Conditions
            3.5.4.1.Effect of pH over ee and Conversion
            3.5.4.2.Effect of Temperature over ee and Conversion
            3.5.4.3.Effect of Incubation Period over ee and Conversion
        3.5.5.Comparison of the Hydromanition Activity of Aminomutases and Ammonia Lyasetowards Cinnamic Acid for the Production of β-Phenylalanine
    3.6 Conclusions
Chapter 4 Summary
    4.1.Conclusion
    4.2.Rational designing of AvPAL mutant for L-phenylalanine and β-PhenylalanineProduction
        4.2.1.L-Phenylalanine Production
        4.2.2.β-Phenylalanine Production
    4.3.Expression and Protein Purification of AvPAL and their Mutant
    4.4.Innovation Points
    4.5.Outlook
Chapter 5 Reference
ACKNOWLEDGEMENTS
About the author and mentor
附件



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