Applied Biocatalysis E-Book
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- Herausgeber: Wiley-VCH Verlag GmbH & Co. KGaA
- Kategorie: Wissenschaft und neue Technologien
- Sprache: Englisch
This reference book originates from the interdisciplinary research cooperation between academia and industry. In three distinct parts, latest results from basic research on stable enzymes are explained and brought into context with possible industrial applications. Downstream processing technology as well as biocatalytic and biotechnological production processes from global players display the enormous potential of biocatalysts. Application of "extreme" reaction conditions (i.e. unconventional, such as high temperature, pressure, and pH value) - biocatalysts are normally used within a well defined process window - leads to novel synthetic effects. Both novel enzyme systems and the synthetic routes in which they can be applied are made accessible to the reader. In addition, the complementary innovative process technology under unconventional conditions is highlighted by latest examples from biotech industry.
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Veröffentlichungsjahr: 2016
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Table of Contents
Cover
Related Titles
Title Page
Copyright
List of Contributors
Preface
Part A: Molecular Biology, Enzyme Screening and Bioinformatics
Chapter 1: Engineering Lipases with an Expanded Genetic Code
1.1 Introduction
1.2 Enzyme Activity of Lipases from Different Sources and Engineering Approaches
1.3 Noncanonical Amino Acids in Lipase Design and Engineering
1.4 Case Study: Manipulating Proline, Phenylalanine, and Methionine Residues in Lipase
1.5 “Unnatural” Lipases Are Able to Catalyze Reactions under Different Hostile Environments
1.6 Lipase Engineering via Bioorthogonal Chemistries: Activity and Immobilization
1.7 Conclusions and Perspectives
References
Chapter 2: Screening of Enzymes: Novel Screening Technologies to Exploit Noncultivated Microbes for Biotechnology
2.1 Introduction
2.2 Sequence- versus Function-Based Metagenomic Approach to Find Novel Biocatalysts
2.3 Alternative Hosts, Metatranscriptomics, and Metaproteomics
2.4 Future Perspectives
References
Chapter 3: Robust Biocatalysts – Routes to New Diversity
3.1 Introduction
3.2 Metagenomics to Retrieve New Genes from Extremophilic Microorganisms
3.3 Microbial Expression Hosts for the Production of Extremozymes
3.4 Molecular Biology Approaches for Enzyme Improvement
3.5 Conclusions and Future Perspectives
References
Chapter 4: Application of High-Throughput Screening in Biocatalysis
4.1 Introduction
4.2 Discussions
4.3 Summary
References
Chapter 5: Supporting Biocatalysis Research with Structural Bioinformatics
5.1 Introduction
5.2 Computational Tools to Assist Biocatalysis Research
5.3 From Active Site Analysis to Protein Stability Considerations
5.4 Applying DoGSiteScorer and HYDE to Biocatalytical Questions
5.5 Conclusion and Future Directions
Acknowledgments
References
Chapter 6: Engineering Proteases for Industrial Applications
6.1 Proteases in Industry
6.2 Serine Proteases and Subtilisins
6.3 Proteases as Additives in Laundry Detergents
6.4 Engineering
B. lentus
Alkaline Protease toward Increased Inhibition by Benzylmalonic Acid
6.5 Engineering Subtilisin Protease toward Increased Oxidative Resistance
6.6 Increasing Protease Tolerance against Chaotropic Agents
6.7 Directed Evolution of Subtilisin E toward High Activity in the Presence of Guanidinium Chloride and Sodium Dodecylsulfate
6.8 Summary
Acknowledgment
References
Part B: Biocatalytic Synthesis
Chapter 7: Biocatalytic Synthesis of Natural Products by O-Methyltransferases
7.1 Introduction
7.2 Classification and Mechanistic Aspects of
O
-Methyltransferases
7.3 Cofactor Dependence and Regeneration
7.4 Natural OMT Products in Industrial Applications
7.5 OMTs in Biocatalytic Synthesis
7.6 Challenges and Perspectives
7.7 Conclusions
Abbreviations
Acknowledgments
References
Chapter 8: Biocatalytic Phosphorylation of Metabolites
8.1 Introduction
8.2 Synthetic Aspects of Biocatalytic Phosphorylations
8.3 Development of Analytical Methods
8.4 Stability of Phosphorylated Metabolites
8.5 Phosphate Donors
8.6 Emerging Biocatalytic Phosphorylation Reactions
8.7 Reaction Engineering for Biocatalytic Phosphorylation Processes
8.8 Summary and Outlook
References
Chapter 9: Flavonoid Biotechnology – New Ways to High-Added-Value Compounds
9.1 Flavonoids
9.2 Metabolic Pathways of Flavonoids
9.3 Biotechnological Processes for the Production of High-Added-Value Flavonoids
9.4 Future Prospects
Acknowledgments
References
Chapter 10: Transaminases – A Biosynthetic Route for Chiral Amines
10.1 Introduction
10.2 Biocatalysts as Attractive Alternatives to Access Enantiopure Chiral Amines
10.3 Transaminases as a Biosynthetic Route for Chiral Amines
10.4 Amine Transaminases (ATAs) for the Production of Chiral Amines
10.5 Kinetic Resolution and Asymmetric Reductive Amination Using ATAs
10.6 Outlook
Acknowledgment
References
Chapter 11: Biocatalytic Processes for the Synthesis of Chiral Alcohols
11.1 Introduction
11.2 Statin Side Chain
11.3
o
-Chloromandelic Acid and Its Derivatives
11.4 Ethyl 2-Hydroxy-4-phenylbutyrate
11.5 Ethyl 4-Chloro-3-hydroxybutanoate
11.6 3-Quinuclidinol
11.7 3-Hydroxy-3-phenylpropanenitrile
11.8 Menthol
11.9 Halogen-Substituted 1-Phenylethanol
11.10 Summary and Outlook
References
Part C: Reaction and Process Engineering
Chapter 12: Inorganic Adsorbents in Enzymatic Processes
12.1 Introduction
12.2 Porous Inorganic Adsorbents for Enzyme Purification Processes (Alumina, Aluminosilicates, Precipitated Silica)
12.3 Immobilization of Phospholipase A1 and A2 for the Degumming of Edible Oils
12.4 Immobilization of
Alcohol Dehydrogenase
‘A’ and
Candida antarctica
Lipase B on Precipitated Silica by Layer-by-Layer-Technology
12.5 Molecular Modeling Calculations of the ADH-‘A’ Immobilization onto Polyelectrolyte Surfaces
12.6 Application of Clays and Zeolites for Adsorption of Educts and Products of Reactions with Alcohol Dehydrogenase in Aqueous Reaction Media
12.7 Product Separation from Complex Mixtures of Biocatalytic Transformations
12.8 Continuous Production and Discontinuous Selective Adsorption of Short-Chain Alcohols in a Fixed-Bed Reactor with Alumina Oxides
12.9 Summary and Outlook
Acknowledgment
References
Chapter 13: Industrial Application of Membrane Chromatography for the Purification of Enzymes
13.1 Introduction
13.2 Membrane Adsorber
13.3 Case Studies and Used Model Enzymes
13.4 Experimental
13.5 Case Study 1: Purification of Penicillin G Amidase
13.6 Case Study 2: Purification of Cellulase Cel5A
13.7 Case Study 3: Purification of Lipase aGTL
13.8 Conclusion and Outlook
Acknowledgment
References
Chapter 14: Fermentation of Lactic Acid Bacteria: State of the Art and New Perspectives
14.1 Introduction
14.2 Factors Effecting Growth and Productivity of Lactic Acid Bacteria
14.3 Fermentation Techniques for Growth and Production
14.4 Case Study: Fixed-Bed Reactor with Immobilized Cells
14.5 Conclusions
Acknowledgment
References
Chapter 15: The Bubble Column Reactor: A Novel Reactor Type for Cosmetic Esters
15.1 Introduction
15.2 Bubble Column Reactor in Comparison to Other Reactor Types
15.3 Case Study: Enzymatic Production of Cosmetic Esters
15.4
In situ
Online Measurements in a Bubble Column Reactor by Means of Fourier Transformed Mid-Infrared Spectroscopy
15.5 Summary and Outlook
References
Chapter 16: Pharmaceutical Intermediates by Biocatalysis: From Fundamental Science to Industrial Applications
16.1 Introduction
16.2 Boceprevir: Oxidation of 6,6-Dimethyl-3-azabicyclo[3.1.0]hexane by Monoamine Oxidase
16.3 Pregabalin: Enzymatic Preparation of (
S
)-3-Cyano-5-methylhexanoic Acid Ethyl Ester
16.4 Glucagon-Like Peptide-1 (GLP-1): Enzymatic Synthesis of (
S
)-Amino-3-[3-{6-(2-methylphenyl)} pyridyl]-propionic Acid
16.5 Rhinovirus Protease Inhibitor: Enzymatic Preparation of (
R
)-3-(4-Fluorophenyl)-2-hydroxy Propionic Acid
16.6 Saxagliptin: Enzymatic Synthesis of (
S
)-
N
-boc-3-Hydroxyadamantylglycine
16.7 Sitagliptin: Enzymatic Synthesis of Chiral Amine
16.8 Montelukast: Enzymatic Reduction for the Synthesis of Leukotriene D (LTD) 4 Antagonists
16.9 Clopidogrel: Enzymatic Preparation of (
S
)-2-Chloromandelic Acid Esters
16.10 Calcitonin Gene-Related Peptide Receptors Antagonist: Enzymatic Preparation of (
R
)-2-Amino-3-(7-methyl-1 H-indazol-5-yl)propanoic Acid
16.11 Chemokine Receptor Modulators: Enzymatic Desymmetrization of Dimethyl Ester
16.12 Regioselective Enzymatic Acylation of Ribavirin
16.13 Atorvastatin: Enzymatic Preparation of (
R
)-4-Cyano-3-hydroxybutyrate
16.14 Atazanavir, Telaprevir, Boceprevir: Enzymatic Synthesis of (
S
)-Tertiary-leucine
16.15 Relenza (Zanamivir): Enzymatic Synthesis of
N
-Acetylneuraminic Acid
16.16 Atorvastatin, Rosuvastatin: Aldolase-Catalyzed Synthesis of Chiral Lactol Intermediates
16.17 Anticancer Drugs: Epothilone B and Microbial Hydroxylation of Epothiolone B
16.18 Corticotropin-Releasing Factor-1 (CRF-1) Receptor Antagonist: Enzymatic Synthesis of (
S
)-1-Cyclopropyl-2-methoxyethanamine
16.19 Conclusion
Acknowledgment
References
Chapter 17: Biocatalysis toward New Biobased Building Blocks for Polymeric Materials
17.1 Introduction
17.2 Questions and Answers that Lead Us toward Sustainability in Plastic Materials
17.3 Criteria and Qualifiers for New Biobased Building Blocks for Plastics Applications
17.4 Criteria and Qualifiers for Launching New Biobased Building Blocks for Plastics Applications in New Value Chains
17.5 Position of Biobased Building Blocks Innovation in the Plastics Pyramid
17.6 Biocatalysis Conversions and Challenges toward newBBBB
17.7 Biocatalytic Cascade Reactions to Functional Building Blocks for Materials
17.8 Conclusion
References
Index
End User License Agreement
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Guide
Cover
Table of Contents
Begin Reading
List of Illustrations
Chapter 1: Engineering Lipases with an Expanded Genetic Code
Figure 1.1 3D structure of TTL obtained by homology modeling. The TTL structure contains the α/β hydrolase fold typical of hydrolytic enzymes. The active site with the conserved catalytic triad is shown in yellow. The lid is a mobile loop that modulates the access of the substrate to the active site.
Figure 1.2 Protein engineering by classic mutagenesis versus genetic code engineering. The common site-directed mutagenesis and directed evolution techniques make use of the 20 canonical amino acids or building blocks. Genetic code engineering, instead, allows the insertion of noncanonical amino acids, which bring new chemical functionalities into the proteins.
Figure 1.3 Lipase engineering with noncanonical amino acids. Methionine (M), tryptophan (W), proline (P), tyrosine (Y), and phenylalanine (F) side chains were subjected to global replacements with related analogs as follows: Met → Aha (azidohomoalanine) and Nle (norleucine); Trp → 4NW (4-aminotryptophan), 4FW (4-fluorotryptophan) and 7AW (7-azatryptophan); Pro → cFP (
cis
-4-fluoroproline), tFP (
trans
-4-fluoroproline), cHP (
cis
-4-hydroxyproline) and tHP (
trans
-4-hydroxyproline), Tyr → m-FY (meta-fluorotyrosine) and
o
-FY (
ortho
-fluorotyrosine), Phe →
m
-FF (
meta
-fluorophenylalanine) and
p
-FF (
para
-fluorophenylalanine). Side chains of Met142, Pro143, Met147, Tyr154, Met158, Phe163, and Phe175 belong to the “lid” domain of TTL (in purple) [20]. Note that buried or surface exposed residues are showed un-bold/-underlined or bold/underlined, respectively.
Figure 1.4 Lipase variants generated by the substitution of 16 Phe residues with
p
-FF and
m
-FF. The TTL[
m
-FF] congener showed a 25% increase in activity whereas TTL[
p
-FF] displayed only 40% of the enzymatic activity when compared with the parent TTL. Lipase activity was determined by measuring the hydrolysis of
p
-nitrophenyl palmitate (
p
NPP) according to Winkler and Stuckmann [23].
Figure 1.5 Improved lipase congeners generated by the substitution of Pro residues with cFP and tFP. The global replacement of six Pro residues in TTL with the fluorinated counterparts cFP and tFP conferred activity enhancement upon treatment with several organic solvents used in industries. Residual activity was measured using the
p
-nitrophenyl palmitate assay after 1 h incubation at 25 °C with 90% of organic solvent.
Chapter 2: Screening of Enzymes: Novel Screening Technologies to Exploit Noncultivated Microbes for Biotechnology
Figure 2.1 General scheme for the metagenomic approach. Screening can either be sequence- or function-driven in order to find a clone with a gene encoding the desired enzyme or biomolecule.
Chapter 3: Robust Biocatalysts – Routes to New Diversity
Figure 3.1 Principle of end-to-end gene fusion. Left: Two open reading frames are genetically fused at the DNA level to give a single polypeptide chain with two or multiple functionalities. Protein-encoding genes, respective mRNAs, and parts of a protein are indicated either in gray or in black. Three different methods to generate gene fusion composed of three single genes (shown in light gray, gray, and dark gray) are shown on the right side. Different methods are boxed with dashed lines. Top: Three genes are fused using polymerase chain reaction. Overlapping primer sequences are indicated as dashed, oscillating lines with the same color as hybridizing sequences in gene fragments. Two PCRs are needed to fuse three genes. Flanking sequences containing restriction recognition sites are given as black boxes. Middle: Three amplicons containing flanking recognition sites for appropriate restriction enzymes need to be generated by PCR to be used in a traditional cloning procedure. Genes are ligated step-by-step into a vector using restriction enzymes capable of hydrolyzing the plasmid within its multiple cloning site (MCS). Genes remain interrupted by restriction enzyme recognition sites after ligation. Bottom: Three genes are amplified including overlapping sequences as indicated earlier to be linked via recombination approaches.
Figure 3.2 Substrate channeling by two single enzymes and a bifunctional protein chimera. (a) Enzyme 1 is catalyzing a reaction to produce “Product 1” from “Substrate 1.” The reaction product is used by enzyme 2 as “Substrate 2” and can be converted in a second reaction step to give “Product 2.” (b) A bifunctional fusion enzyme is produced by two enzyme moieties that are coupled by a peptide linker. “Substrate 1” is processed into “Product 2” in a two-step reaction cascade mediated by a single polypeptide chain.
Chapter 4: Application of High-Throughput Screening in Biocatalysis
Scheme 4.1 Screening of alcohol oxidases using horse radish peroxidase/2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (HRP/ABTS).
Figure 4.1 Screening of alcohol dehydrogenases by
in vitro
expression system.
Scheme 4.2 Screening of ketoreductases for the preparation of befloxatone.
Scheme 4.3 Screening of nitrilases using the luminescent substrate
o
-hydroxybenzonitrile.
Figure 4.2 Screening of nitrilases in solid-phase format.
Scheme 4.4 Screening of nitrilases using cobalt ion spectrophotometric method.
Scheme 4.5 Screening of nitrilases using
o
-phthaldialdehyde spectrophotometric method.
Scheme 4.6 Screening of cytochromes P450s using 7-methoxy-4-(aminomethyl)-coumarin as the substrate.
Scheme 4.7 High-throughput screening of cytochromes P450s using 4-nitro-1-phenylethanone as the substrate.
Scheme 4.8 Enlarging the substrate spectrum of galactose oxidases by high-throughput screening using an efficient colorimetric solid-phase assay.
Scheme 4.9 Screening of hydrolases by detecting the generation of NADH.
Scheme 4.10 Screening of hydrolases by cell-surface display and HRP-assisted attachment of fluorescent label technologies.
Scheme 4.11 Substrates with different fluorescent dyes used to improve the enantioselectivity of the EstA.
Scheme 4.12 Enantiomers affecting cell growth differently as the substrate to screen esterases.
Scheme 4.13 Screening of transaminases using copper ion spectrophotometric method.
Scheme 4.14 Threonine aldolases catalyzing the cleavage of threonine to produce glycine and acetaldehyde and its reverse reaction.
Scheme 4.15 2-Deoxy-
d
-ribose 5-phosphate aldolase (DERA) catalyzing the synthesis of (3
R
,5
S
)-6-chloro-2,4,6-trideoxyhexonolactone as a key intermediate for atorvastatin.
Figure 4.3 Screening of hydroxynitrile lyases by a colorimetric assay.
Figure 4.4 Assay workflow in screening of glycoside hydrolases by fluorescence-activated droplet sorter (FADS) system.
Figure 4.5 Screening of glycosyltransferases by fluorescence-activated cell sorting (FACS) system.
Figure 4.6 Screening of glycosyltransferases by phage display technology.
Chapter 5: Supporting Biocatalysis Research with Structural Bioinformatics
Figure 5.1 Schematic depiction of DoGSite's (sub)pocket detection. (a) Grid representation of the protein binding site (light blue). (b) Filtering of the grid using a DoG filter. (c) Merging of favorable grid points to subpocket cores. (d) Dilation to subpockets and merging to one pocket.
Figure 5.2 3D structures of the active site of urokinase-type plasminogen activator (PDB code 1c5q) including three exemplarily calculated descriptors. The protein surface is shown in gray. The co-crystallized ligand is depicted in ball-and-stick mode. (a) Volumes of the three subpockets sketched in orange, yellow, and red. (b) Depth of the pocket, color coding from yellow (solvent exposed) to red (buried). (c) Ellipsoidal shapes calculated for all three subpockets.
Figure 5.3 Thermodynamic cycle of water,
C
p
= heat capacity of water.
Figure 5.4 Saturation factor
F
sat
(
T
) at different temperatures.
Figure 5.5 Schematic depiction of the binding process, modeled in the HYDE scoring function.
Figure 5.6 Exemplified function prediction pipeline via docking of substrates into the active site of a new enzyme. Left: Example substrates from four different enzyme classes. Middle: The binding site of an acetylcholinesterase (1acl, green surface), exemplarily with a co-crystallized ligand (DME) is depicted in light green and the docked ligand (EBW from 1e3q) in gray. Right: Histogram of the achieved docking scores. The red curve shows the scores of the actives (LED substrates) and the gray curve the scores of the inactives (MDR, ThDP, CYP substrates).
Figure 5.7 (a) Subset of clusters resulting from the descriptor-based binding site comparison of 210 glycerol oxidases and five alditol oxidases. The shown subtree includes the five alditol oxidases (green) and the most similar glycerol oxidases (black). (b) Comparison of the active sites of the glycerol-binding oxidase (1d6z) and the alditol oxidase (2vfr). Shown are amino acids that are in a similar position within both binding sites. A salt bridge between Glu702 and Lys709 is depicted as dotted line. Only C
1
to C
3
of xylitol in alditol oxidase (2vfr) are shown.
Figure 5.8 Competitive substrate inhibition by buffering agents. (Top) Reaction catalyzed by phosphoglucose isomerase and (Middle) buffering agents tested for interference with the binding of fructose-6-phosphate. (Bottom) Effect on the activity of phosphoglucose isomerase triggered by different buffering agents.
Figure 5.9 Binding modes of phosphoglucose isomerase substrate (fructose-6-phosphate, co-crystallized) (1hox) and two buffering agents, diglycine and PIPES (docking poses). Images were generated with Chimera [75].
Figure 5.10 Classification of protein-protein contacts using the hydrophobic dehydration energy of the HYDE scoring function. Three different protein-protein contacts were discriminated: permanent and transient complexes, both biologically relevant assemblies, as well as artificial contacts caused due to crystal packing.
Figure 5.11 DoGSiteScorer web server example for a glycosyltransferase (PDB: 2c9z). (Left) Extraction of result tables of (sub)pocket prediction and descriptor calculation. (Right) Subpockets of the largest pocket of 2c9z are shown in different colored isosurfaces. The colors correspond to the subpocket table on the left side. The protein backbone is shown in blue. The co-crystallized ligands (QUE, UDP) are represented in ball-and-stick mode.
Chapter 6: Engineering Proteases for Industrial Applications
Figure 6.1 Scheme of the active site of a typical subtilisin protease. The catalytic triad is shown in red and the oxyanion hole in yellow. The nonspecific peptide-binding pocket is shown in green, whereas the specificity pocket is in blue. (Branden and Tooze [14]. Reproduced with permission of Taylor and Francis.) The enzyme and the relevant residues are represented in white; the substrate peptide is represented in green.
Figure 6.2 Summary of the engineering campaign of
B. lentus
alkaline protease for increased reversible inhibition by benzylmalonic acid. (a) Overview of a typical directed evolution experiment comprising three steps: (I) gene diversity generation, (II) screening to identify improved mutants out of mutant libraries, and (III) isolating the gene encoding for the improved protein variants. These genes are subsequently used in iterative cycles of diversity generation and screening. (b) Three-dimensional structures of
B. lentus
alkaline protease parent, variant V8 (Ser160Gly, Gln185Arg) and variant V9 (Ala181Asp, Phe183Arg). Models were generated using the homology model routine from YASARA (Yet Another Scientific Artificial Reality Application [21]) based on the coordinates of
B. lentus
alkaline protease (PDB 1ST3) [22]. The active site is represented in red, the oxyanion hole in yellow. The identified key residues are shown in blue and the introduced mutations in variant V8 and V9 are represented in cyan. (c) Comparison of measured IC
50
values for BLAP and its variants in the presence of boric acid (black bars) and BMA (gray bars). The IC
50
value represents the required inhibitor concentration to reduce the initial activity to 50%. The reported values are the average value of three independent measurements (The unpublished data were generated by Dr. Felix Jakob).
Figure 6.3 Overview of the engineering of subtilisin Carlsberg variants toward increased oxidative resistance. (a) The parent variant (Thr58Ala, Leu216Trp, and Met221 as in wild type) was used as starting variant for site saturation mutagenesis (SSM) and site-directed mutagenesis (SDM) studies. Amino acid positions Trp216 and Met221 were identified as key positions for oxidative resistance of subtilisin Carlsberg. Two amino acid positions – Trp216 and Met221 – were saturated and the resultant libraries were screened using the suc-AAPF-pNA assay in MTP format. (b) Three-dimensional structures of subtilisin Carlsberg, parent, and the identified variant M4 (Thr58Ala, Trp216Met, Met221) with the highest oxidative resistance (2.6-fold compared with subtilisin Carlsberg). Models were generated using the homology model routine from YASARA (Yet Another Scientific Artificial Reality Application [21]) based on the coordinates of subtilisin Carlsberg (PDB code 1YU6) crystal structure obtained from RCSB protein data bank. The active site is represented in red, the oxyanion hole in yellow. The identified key residues are shown in blue and the introduced mutations in parent and M4 are represented in cyan.
Figure 6.4 Overview of the mutational campaign of subtilisin E toward increased resistance to GdmCl. (a) After three rounds of directed evolution, variant M4 (Ser62Ile, Ala153Val, Gly166Ser, Ile205Val) was identified. Each of the amino acid substitutions found in M4 were individually saturated, revealing Gly166Met and Ser62Ile as responsible for resistance to GdmCl, which were combined into M2. Subsequently, Ser62 was introduced into M4, resulting in M5. From 10 other identified and saturated positions, Asn218Ser and Thr224Ala were identified and introduced into M4, generating M6. (b) The relative proteolytic activity of wild-type (WT) subtilisin E and the generated variants M4, M5, and M6 over increasing GdmCl concentration showed a substantial improvement, with M6 being the most resistant variant. (c) A structural model based in the crystal structure of subtilisin E (PDB 1SCJ) showing the identified positions 62, 205, 153, 166 (cyan), and 97, 103, 218, and 224 (blue) identified in this work are all close to the active site of subtilisin E (red), the oxyanion hole (yellow) and to the substrate suc-AAPF-
p
NA (green), suggesting that amino acid substitutions responsible for adaptation to high GdmCl or SDS concentrations are related to substrate interaction and reaction mechanism, rather than in the protein surface [10, 40].
Chapter 7: Biocatalytic Synthesis of Natural Products by O-Methyltransferases
Figure 7.1 Examples for the influence of regioselective methylation on the biological activity of natural compounds [1, 2]. The crucial additional methyl group is shown in boldface.
Figure 7.2 OMT-catalyzed transfer reaction of the methyl group of SAM to caffeic acid. The transferred methyl group is shown in boldface.
Figure 7.3 O
-
Methylation steps in the biosynthesis of 3,5-dimethoxytoluene (DMT) catalyzed by orcinol
O-
methyltransferase (OOMT). The crucial methyl groups are highlighted in boldface.
Figure 7.4 Biosynthesis of SAM.
l
-Methionine (Met) and ATP act as substrates for MAT (SAMS) under the driving force of the hydrolysis of the inorganic triphosphate anhydride. SAM, phosphate (Pi), and diphosphate (PPi) result as products.
Figure 7.5 SAM cycle. The reactions of the cycle are connected to the tetrahydrofolate (THF) or the betaine pathway by the methylation of Hcy to Met by methionine synthase (IVa) or betaine-homocysteine MT (IVb) [30]. Met is converted to SAM by MAT (I) [29]. The transmethylation by (O)MTs (II), where SAM acts as cofactor, is illustrated in the dashed line (II); the transferred methyl group is drawn in boldface. Hcy can be salvaged from SAH by hydrolysis by SAH hydrolase (III) [27].
Figure 7.6 Cleavage of SAM under acidic (>pH 1.5) and basic (>pH 6) conditions. Higher stability is achieved in very acidic (<pH 1.5) and nonsolvolytic medium. Basic (nucleophilic) cleavage can also cleave methyl leaving SAM as methanol. Racemization at sulfur (not shown) is more dependent on temperature than pH [33].
Figure 7.7 Phenylpropanoid pathway in plants. The sequence starts with tyrosine (not shown) converted by a tyrosine ammonia lyase (TAL) to
p
-coumaric acid as precursor for flavonoids and stilbenoids [13]. The first step of a
de novo
synthesis that was engineered into microbial systems was the activation by 4-coumarate: CoA ligase (CL) forming the coumaric acid CoA ester. The flavonoid or stilbenoid skeletons, respectively, are synthesized by condensation of three units of malonyl CoA catalyzed by the corresponding type III polyketide synthase, for example, stilbene synthase (STS) in case of resveratrol or chalcone synthase (CHS) in case of naringenin. The latter compound is the central intermediate in the biosynthesis of almost all flavonoids. It is modified by specific flavonoid hydroxylases (FHs) and OMTs and other “decorating” processes to produce a huge variety of flavonoids. Resveratrol as a basic metabolite for stilbenoids can be modified accordingly.
Figure 7.8 Examples of alkaloids depending on at least one OMT during their biosynthesis. In several cases, the
O
-methyl moiety is transient, that is, it is eventually transformed (e.g., for
C–E
, and partially
B
) or disappears during the biosynthesis (
A
). (
A
) Morphine and (
B
) berberine are natural benzyl isoquinoline alkaloids (BIAs) synthesized from reticuline [64]. (
C
) Safrol, (
D
) piperine, and (
E
) sanguinarine feature a methylenedioxo-bridge formed by oxidation of a 2-methoxyphenol moiety [65].
Figure 7.9 Insertion of a methyl group at position 4 of
l
-dihydroxyphenylalanine (
l
-DOPA) as essential step in the biosynthesis of saframycin [73] (right). The isovanilloid motif is highlighted in red and indispensable for the biological activity of this antitumor compound [74].
Chapter 8: Biocatalytic Phosphorylation of Metabolites
Figure 8.1 Reaction schemes illustrating types of phosphorylation reactions.
Figure 8.2 Comparison of stability of selected phosphorylated metabolites in aqueous solution depending on the pH value. The blue areas are indicating the pH regions the compounds are stable.
Figure 8.3 Selection of emerging phosphorylation reactions.
Figure 8.4 Optimum pH conditions and pyrophosphate concentration levels for acid phosphatase- and alkaline phosphatase-catalyzed phosphorylation and reverse hydrolysis reactions.
Figure 8.5 Schematic diagram of biocatalytic phosphorylation reaction system optimization.
Figure 8.6 The activity of ATP:glycerol phosphotransferase E.C. 2.7.1.30 as a function of Mg
2+
to ATP ratio: the reaction conditions are 50 mM glycerol, 50 mM ATP, and variable 0 to 100 mM Mg
2+
catalyzed by ATP: glycerol phosphotransferase from
Cellulomonas
sp. (0.03 mg ml
−1
) in 100 mM Tris at pH 8.5 and 25 °C.
Figure 8.7 Design of stirred-tank reactor (STR) and the substrate and product concentration profile over time and space.
Figure 8.8 Design of plug-flow reactor (PFR) and the substrate and product concentration profile over time and space.
Figure 8.9 Design of continuous stirred-tank reactor (CSTR) and the substrate and product concentration profile over time and space.
Figure 8.10 Schematic diagram of bioprocess optimization.
Chapter 9: Flavonoid Biotechnology – New Ways to High-Added-Value Compounds
Figure 9.1 Main group of compounds based on the phenylbenzopyran moiety.
Figure 9.2 Flavan core structure and the most significant flavonoid groups.
Figure 9.3 Reference pathway of the flavonoid biosynthesis, with emphasis on the most important intermediates and the roots to the biosynthesis of related compounds (gray). The most interesting enzymes are denoted in black boxes, while the biotransformations that need more than one step (enzyme) are denoted with dashed arrows.
Figure 9.4 The flavonoid catabolic pathway of
Eubacterium ramulus
as certified from [45, 51] for at least naringenin (R
1
: –OH, R
2
: –H), eriodictyol: (R
1
: –OH, R
2
: –OH), and homoeriodictyol (R
1
: –OH, R
2
: –OCH
3
). CHI: chalcone isomerase, ERED: enoate reductase, PHY: phloretin hydrolase. Note that the equilibrium of the CHI reaction is lying toward the formation of the flavonone. Anaerobic conditions are necessary for the activity of ERED.
Figure 9.5 The alternative catabolic pathway of flavonoids in eukaryotes. Rha-Glu is the abbreviation for the disaccharide composed of rhamnose and glucose.
Chapter 10: Transaminases – A Biosynthetic Route for Chiral Amines
Figure 10.1 General structure of chiral amines.
Figure 10.2 Enantioselective asymmetric synthesis of chiral amines by chemical hydrogenation and biocatalytic transamination.
Figure 10.3 Merck and Codexis developed a more efficient process for the production of sitagliptin, using a protein-engineered transaminase ATA117. The wild-type (WT) transaminase ATA117 is inactive on the pro-sitagliptin ketone due to steric constraints in the active site. In contrast, the tailored biocatalyst provided the desired activity and productivity. DMSO – dimethylsulfoxide, PLP – pyridoxal-5′-phosphate, iPr-amine – isopropylamine.
Figure 10.4 Principle of the transamination reaction catalyzed by a transaminase.
Figure 10.5 Reaction scheme of the transamination. Illustrated is the first half reaction of the catalytic cycle. Sequentially, the PMP attacks the amino acceptor and finally the amine product is released and the PLP recycled. The circled “P” denotes the phosphate group.
Figure 10.6 Strategies for protein engineering. (a) Possible strategies to engineer an (
R
)-selective ATA (center). One option is the modification of the amino acids in the carboxyl group–binding pocket of an α-transaminase (left), such as an
l
-branched chain transaminase of the PLP-dependent fold class IV, or by engineering of the binding pockets of an (
S
)-selective ATA (right) from PLP-dependent fold class I. It was assumed that according to the CIP rule, the large substituent (
R
L
) has a higher priority than the small substituent (
R
S
). (b) Flow scheme of the
in silico
approach for the identification of transaminases with inverted enantiopreference, with steps A–E.
Figure 10.7 Production of chiral amines using amine transaminases. In a kinetic resolution (a), the stereoselective ATA converts only one of the amine enantiomers to the corresponding ketone. The remaining enantiomer can be isolated in optical purity at a maximum yield of 50%. In an asymmetric synthesis (b), a pro-stereogenic ketone is enantioselectively aminated, yielding the chiral amine. The most common cosubstrates for ATA are pyruvate/alanine. Since in this case the equilibrium favors ketone formation, high yields in asymmetric synthesis can be achieved only by shifting the equilibrium, for example, by enzymatic removal of the coproduct pyruvate.
Figure 10.8 Dynamic kinetic resolution (DKR) of chiral aldehydes employing transaminases for the production of Niraparib and smoothened receptor inhibitor (SMO) in industrially relevant scale. ATA – amine transaminase and IPA – isopropylamine.
Figure 10.9 Bottleneck analysis of asymmetric reductive amination with ATAs, showing challenges in biocatalytic transamination and solutions.
Figure 10.10 Recent advances in the industrially relevant production of drugs employing ATA-catalyzed asymmetric synthesis of chiral amines.
Figure 10.11 Principle of the enzymatic cascade in the (
R
)-ATA screening kit. During the reaction,
d
-alanine is converted to pyruvate, which is removed from the reaction by a two-enzyme cascade system. Lactate dehydrogenase (LDH) converts pyruvate to lactate. The LDH requires NADH as cofactor, which is recycled by conversion of glucose to gluconic acid by glucose dehydrogenase (GDH).
Chapter 11: Biocatalytic Processes for the Synthesis of Chiral Alcohols
Figure 11.1 Structure of HMG-CoA reductase inhibitors.
Figure 11.2 A two-step, three-enzyme process for the synthesis of hydroxynitrile
3
.
Figure 11.3 Enzymatic reduction of diketo ester by
Lb
ADH.
Figure 11.4 Synthesis of
syn
-3,5-dihydroxy ester via KRED-catalyzed reduction of hydroxyketoester.
Figure 11.5 DKRED-catalyzed direduction for the synthesis of
syn
-3,5-dihydroxy ester.
Figure 11.6 Nitrilase-catalyzed desymmetrization for the synthesis of hydroxynitrile
11
.
Figure 11.7 DERA-catalyzed aldol condensation for the synthesis of chiral chlorolactol
14
.
Figure 11.8 Lipase-catalyzed hydrolysis of
rac
-4-chloro-3-hydroxybutanoate ester.
Figure 11.9 Lipase-catalyzed transesterification resolution of racemic alcohol
19
.
Figure 11.10 Lipase-catalyzed enantioselective ring opening of
rac
-4-bromomethyl-β-lactone.
Figure 11.11 Structure of clopidogrel and its key chiral intermediates.
Figure 11.12 Chemoenzymatic synthesis of (
R
)-
o
-chloromandelic acid.
Figure 11.13 Synthesis of (
R
)-
o
-chloromandelic acid from
o
-chloromandelonitrile by DKR.
Figure 11.14 Asymmetric synthesis of (
R
)-CMM from CBFM by reduction using ketoreductase and GDH.
Figure 11.15 Lipase-catalyzed kinetic resolution of racemic HPBE for the synthesis of (
R
)-HPBE.
Figure 11.16 Synthesis of (
R
)- or (
S
)-HPBE by asymmetric reduction of OPBE using the reductase combined with a GDH for the regeneration of cofactor.
Figure 11.17 Structure of
l
-carnitine,
l
-GABOB and their key chiral intermediate (
R
)-CHBE.
Figure 11.18 Asymmetric synthesis of (
R
)-CHBE from COBE by reduction using ketoreductase and GDH.
Figure 11.19 Synthesis of (
R
)-CHBM by kinetic resolution of
rac
-CHBM using halohydrin dehalogenase.
Figure 11.20 Structure of (
R
)- and (
S
)-3-quinuclidinol, talsaclidine, cevimeline, revatropate, 8018, and ADR-932.
Figure 11.21 A practical chemoenzymatic route for the synthesis of (
R
)-3-quinuclidinol.
Figure 11.22 Synthesis of optically pure 3-quinuclidinol
via
asymmetric reduction of 3-quinuclidinone with ketoreductase.
Figure 11.23 Structures of Prozac™ (fluoxetine), Straterra™ (atomoxetine), Ergamisol (levamisole) and nisoxetine.
Figure 11.24 Synthesis of (
R
)- or (
S
)-HPPN by lipase-catalyzed kinetic resolution of racemic HPPN.
Figure 11.25 Synthesis of (
R
)-HPPN by enantioselective hydrolysis of racemic HPPN using nitrilase.
Figure 11.26 Synthesis of (
R
)- or (
S
)-HPPN by asymmetric reduction of benzoylacetonitrile using reductase.
Figure 11.27 α-Ethylation of β-ketonitrile in reduction of benzoylacetonitrile using whole-cell biocatalyst.
Figure 11.28 Synthesis of (−)-menthol via enantioselective hydrolysis of (±)-menthyl benzoate by lipase.
Figure 11.29 Synthesis of (−)-menthol via enantioselective hydrolysis of (±)-menthyl acetate by lipase/esterase.
Figure 11.30 Synthesis of (−)-menthol via enantioselective transesterification by lipase.
Figure 11.31 Synthesis of (
R
)-1-(4′-fluorophenyl)ethanol via asymmetric reduction of 4′-fluoro acetophenone using reductase.
Figure 11.32 LsADH-catalyzed hydrogen-transfer reduction of 2,2,2-trifluoro-1-phenylethanone for the synthesis of (
S
)-2,2,2-trifluoro-1-phenylethanol.
Figure 11.33 Structures of emend (aprepitant), fosaprepitant, vestipitant, and the key chiral intermediate (
R
)-1-[3′,5′-bis(trifluoromethyl)phenyl]ethanol.
Figure 11.34 Synthesis of (
R
)- or (
S
)-1-[3′,5′-bis(trifluoromethyl)phenyl]ethanol via asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone using reductase.
Figure 11.35 Synthesis of (
R
)- or (
S
)-2-chloro-1-phenylethanol
via
asymmetric reduction of α-chloroacetophenone using reductase.
Figure 11.36 Synthesis of (
R
)-1-(4′-chlorophenyl)ethanol via asymmetric reduction of 4-chloroacetophenone using reductase.
Chapter 12: Inorganic Adsorbents in Enzymatic Processes
Figure 12.1 Binding capacities of Puralox® KR 160 (a) and Siral® 40 (b) for α-chymotrypsin and penicillin-G-amidase at pH values from 4 to 11.
Figure 12.2 Cross-sectional view of a cellulose-based depth filter from Sartorius Stedim Biotech.
Figure 12.3 Comparison of loading capacities of granulated and depth filter integrated Puralox® KR 160 and Siral® 40 for α-chymotrypsin.
Figure 12.4 (a) Comparison of purification factors of silicic-acid-based materials for adsorption of various lipases. (b) Comparison of remaining volume activities of various lipases after purification with silicic-acid-based materials.
Figure 12.5 General structure of a phospholipid, where R = hydrogen, choline, ethanolamine, inositol, and so on. The various hydrolyzing sites of the different phospholipases (A1, A2, C, and D) are shown by dashed lines.
Figure 12.6 Phosphorous (P) values before and after degumming of soya oil with free and immobilized PLA1 on two different silicate carriers. The immobilized PLA1 was used for up to five repeated recycling steps. The left row shows the P-value of the crude soya oil. PLA1 (free) shows the value of the PLA1-degummed soya oil with nonimmobilized enzyme at 37 °C, pH 4.5 for 16 h at 400 rpm. First degumming shows the value of the PLA1-degummed soya oil with immobilized enzyme at 37 °C, pH 4.5 for 16 h. This immobilized enzyme was again used in the recycling steps 1–5 at same conditions.
Figure 12.7 Phosphorous (P) values before and after degumming of soya soybean oil with free and immobilized PLA2 on two different silicate carriers. The immobilized PLA2 was used for up to 10 repeated recycling steps. The left row shows the P-value of the crude soya oil. PLA2 (free) shows the value of the PLA2 degummed soya oil with nonimmobilized enzyme at 37 °C, pH 4.5 for 16 h at 400 rpm. First degumming shows the value of the PLA2-degummed soya oil with immobilized enzyme at 37 °C, pH 4.5 for 16 h. This immobilized enzyme was again used in the recycling steps 1–10 at same conditions.
Figure 12.8 Phosphorous (P) values before and after degumming of rapeseed oil with free and immobilized PLA2 on two different silicate carriers. The immobilized PLA2 was used for up to nine repeated recycling steps. The left row shows the P-value of the crude rapeseed oil. PLA2 (free) shows the value of the PLA2-degummed rapeseed oil with nonimmobilized enzyme at 37 °C, pH 4.5 for 16 h at 400 rpm. First degumming shows the value of the PLA2-degummed soybean oil with immobilized enzyme at 37 °C, pH 4.5 for 16 h. This immobilized enzyme was again used in the recycling steps 1–9 at same conditions.
Figure 12.9 Principle of layer-by-layer technology.
Figure 12.10 The properties of the immobilized enzymes are given in Table 12.4. Relative activity of CALB (blue) and ADH-‘A’ (red) immobilized by LbL technology on precipitated silica.
Figure 12.11 The three different stages of protonation of the polyethylenimine.
Figure 12.12 Simulation of
ADH
-‘
A
’ positioned 12 nm away from the polyelectrolyte surface. (ADH (infini)).
Figure 12.13 Starting structure of the simulation ADH-ym (orientation of ADH with the negative
y
-principal axis of inertia to the polyethylenimine surface).
Figure 12.14 Stabilization energies of the various
ADH
-‘
A
’ orientations to the Polyethylenimine surface.
Figure 12.15 The ADH-‘A’ in
y
p-orientation adsorbed at the Polyethylenimine surface.
Figure 12.16 Zn
2+
ions and active sites (yellow) of the adsorbed ADH-‘A’ in the energetically most stable
y
p-orientation to the Polyethylenimine surface.
Figure 12.17 Lysine molecules in ADH-‘A’.
Figure 12.18 Isotherms for the adsorption of acetophenone (▪) and phenylethanol (□) on natural kerolite clay in water [64].
Figure 12.19 Molecular structure of the studied substrate–product pairs: acetophenone–phenylethanol (a,b) and 2,5-hexanedione–2,5-hexanediol (c,d).
Figure 12.20 Asymmetric synthesis of (
S
,
S
)-2,5-hexandiol with ADH-‘A’ in homogenous solution (Δ) and in the presence of 50 g L
−1
β-zeolite () [62].
Figure 12.21 Screening experiments of different adsorbents on the basis of alumina oxide and alumina silicate. Investigation of adsorption of 2,5-hexanedione/2,5-hexanediol from the solvent ethyl acetate at ambient temperature after 30 min treatment in a lab shaker, 0.5 g adsorbent in 15 mL of solution (concentrations of the solutes in the supernatant were determined by gas chromatography).
Figure 12.22 Adsorption isotherm of 2.5-hexanedione, 2,5-hexanediol from the organic solvent MTBE, adsorbent: granulated adsorber: ExM 2014 (alumina oxide) 200 mg, room temperature,
t
= 60 min.
Figure 12.23 Breakthrough curve of 2.5-hexanedione/2,5-hexanediol (
V
max
= 60 mM) from organic solvent MTBE, granulated adsorbent: Puralox KR-160, 6.51 g (γ-alumina oxide).
Figure 12.24 Scheme of enzymatic reduction of prochiral ketones with enzyme ADH-‘A’ and substrate-coupled cofactor regeneration of the cofactor NADH.
Figure 12.25 Scheme of presaturation of the enzyme carrier with cofactor-buffer solution. The water is needed to sustain the enzyme activity (Figure not at scale; for simplicity the porosity of the carrier was omitted).
Figure 12.26 Scheme of the production setup for continuous diol-production with discontinuous adsorption of the product alcohol.
Figure 12.27 Experiment for continuous production of 2
S
,5
S
-hexanediol in a fixed-bed reactor (bed height 12.5 cm, immobilisate 100 µm Sipernat® with polyethylene imine 25 000 layer, enzyme ADH-‘A’), substrate 60 mM 2,5-hexanedione, 5% V V
−1
isopropanol in
t
BME, flow rate 0.1 mL min
−1
, 30 °C,
τ
= 37.5 min, STY (space time yield) 36.6 g L
−1
day
−1
.
Chapter 13: Industrial Application of Membrane Chromatography for the Purification of Enzymes
Figure 13.1 Comparison of double-porous structure versus grafted hydrogel structure.
Figure 13.2 Flow pattern in Sartobind void volume optimized capsules: flow of the fluid is from the outside flow channel through the membrane layers to the inside flow channel in a radial manner.
Figure 13.3 Void volume optimized capsules with 3 ml, 150 ml, 1.2 l, and 5 l bed volume [20].
Figure 13.4 Scalability of void volume optimized capsules with 3 ml, 150 ml, 1.2 l, and 5 l bed volume. Comparison of breakthrough curves and elution peaks [20].
Figure 13.5 Sartobind® 96-well plates.
Figure 13.6 (a) FPLC run of the one-step purification process of PGA from real cell lysate from
E. coli
5KpHM12 culture by milliliter-scale CEX membrane adsorbers (collected fractions are indicated by numbered rectangles). (b) Corresponding SDS-PAGE of the purification of PGA from cell lysate, S-membrane, loaded at pH 6 and eluted at pH 6 with 50 mM NaCl. Lane M: molecular weight marker, lane 1–3: flow-through fractions, lane 4: wash fraction, lane 5–9: elution at pH 6 with 50 mM NaCl (12% acrylamide gel).
Figure 13.7 Adsorption isotherms for PGA binding to hydrogel-engrafted and double-porous S-membranes.
Figure 13.8 HCP depletion at various pH values and conductivities using Sartobind® STIC PA (a) and Sartobind® Q (b).
Figure 13.9 SDS-PAGE gels after isolation of cellulase Cel5A with IDA membranes under variation of the coordinating cation. Binding was performed directly from cell lysate at pH 6.0, for Co
2+
and Zn
2+
in the presence of 20 mM imidazole. Elution was carried out at pH 8 with an imidazole gradient. FT = flow through, WF = wash fraction, E1–E4 for Co
2+
and Zn
2+
: 50, 75, 100, and 200 mM imidazole, E1–E5 for Ni
2+
and Cu
2+
: 20, 50, 75, 100, and 200 mM cellulose (12% acrylamide gel silver stained).
Figure 13.10 (a) Dynamic binding capacity
Q
max
of cellulase Cel5A to IDA membranes decorated with various transition metal ions. (b) Residual cellulase Cel5A activity after purification over IDA membranes decorated with various transition metal ions.
Figure 13.11 (a) FPLC run of the one-step purification process of cellulase Cel5A from cell lysate from
E. coli
M15Cel5A culture at milliliter-scale Zn
2+
-functionalized IDA-membrane adsorbers (collected fractions are indicated by numbered rectangles), flow rate: 1 ml min
−1
, sample 10 ml cell lysate, pH 6 + 20 mM imidazole. (b) Corresponding SDS-PAGE of the purification of cellulase Cel5A from cell lysate, Zn
2+
-functionalized IDA-membrane, lane 1–3 flow-through fractions, lane 4,5 wash fractions, lane 6–8 elution fractions (12% acrylamide gel with silver staining).
Figure 13.12 Adsorption isotherms for Cel5A purification from cell lysate with hydrogel-engrafted and double-porous IDA membrane.
Figure 13.13 (a) Chromatogram of lipase aGTL purification with mixed-mode membrane. Flow rate: 1 ml min
−1
, sample: 12 ml cell lysate pH 5. Washing was performed at same pH with 8 ml binding buffer (25 mM NaAc, pH 5; flow rate: 2 ml min
−1
). Subsequent elution with a three-step gradient and 4 ml elution volume per stage (first step: 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6, second step: 25 mM MES, 50 mM NaCl, pH 6, third step: 25 mM Tris, pH 8). (b) SDS-PAGE gel of fractions as indicated in Figure 13.8a. Lane 1: flow-through fraction, lane 2: wash fraction, lane 3: first elution fraction at pH 6, and lane 4 + 5: third elution at pH 8 (12% gel).
Chapter 14: Fermentation of Lactic Acid Bacteria: State of the Art and New Perspectives
Figure 14.1 SEM of different lactic acid bacteria (bar 2 µm) (a)
Lactococcus lactis
ssp
. lactis
, (b)
Lactobacillus delbrueckii
ssp.
bulgaricus
, and (c)
Lactobacillus reuteri
.
Figure 14.2 Batch cultivation of
Lactobacillus delbrueckii
ssp.
bulgaricus.
(Data from M. Aasif (TU Hamburg-Harburg), not published.) Concentration of cells, glucose, and lactate as well as pH and volumetric lactic acid productivity versus time. Medium: MRS, 18.53 g l
−1
glucose, initial pH 6.08, temperature: 40 °C. Without pH control: shake flask, volume 100 ml. With pH control: volume 600 mL, stirred tank, magnetic bar 180 rpm, pH controlled at 6.0 by 1 M NaOH.
Figure 14.3 Overview on cultivation systems suggested for cultivation of lactic acid bacteria.
Figure 14.4 Continuous chemostat cultivation of
Lactobacillus delbrueckii
ssp.
bulgaricus.
(Data from M. Aasif (TU Hamburg-Harburg), not published.) (a) Steady-state values for cell concentration and volumetric lactate productivity versus dilution rate D. (b) Cell-specific lactate production rate versus cell-specific growth rate μ. Medium: MRS, 18.53 g l
−1
glucose, initial pH6.08. Temperature: 40 °C. Stirred tank, volume 600 ml, magnetic bar 180 rpm, pH controlled at 6.0 by 1 M NaOH.
Figure 14.5 Scheme of axial and radial-flow fixed bed.
Figure 14.6 Macroporous carrier used for the immobilization of lactic acid bacteria. (a) CERAMTEC EO/90 (Ceramtec), (b) Sponceram (Zellwerk), and (c) Siran (QVF). Top: native carrier. Below: REM of carrier with immobilized cells after fixed-bed cultivation.
Figure 14.7 Fixed-bed bioreactor with (a) axial-flow bed (100 ml, medorex) and (b) radial-flow bed (1 l, medorex) connected to 100 l tank for fresh medium and harvest. (c) Cultivation of
L. lactis
ssp.
lactis
on Ceramtec carrier.
Figure 14.8 Cultivation of
Lactococcus lactis
ssp
. lactis
and
Lactobacillus delbrueckii
ssp.
bulgaricus
in an axial-flow fixed bed (100 ml, medorex) and a radial-flow fixed bed (1 l, medorex) filled with macroporous carrier CERAMTEC EO/90 at different perfusion rates and in different media. Cultivation time 50–100 h per dilution rate.
Lactococcus lactis
ssp
. lactis
– media: CM, M17, MRS, temperature: 37 °C.
Lactobacillus delbrueckii
ssp.
bulgaricus
– Medium: MRS, temperature: 40 °C.
Figure 14.9 Long-term cultivation of
Lactococcus lactis
ssp.
lactis
in an axial-flow fixed bed (100 ml, medorex) filled with macroporous carrier CERAMTEC EO/90 at different perfusion rates and in MRS medium. Medium: MRS + 20% K
2
HPO
4
, 20 g l
−1
glucose, initial pH 5.7, Temperature: 37 °C.
Chapter 15: The Bubble Column Reactor: A Novel Reactor Type for Cosmetic Esters
Figure 15.1 Schematic illustration of a bubble column reactor (BCR) (a) and examples of reaction systems carried out within the bubble column reactor (b) polyglycerol-3/lauric acid (I); myristic alcohol/myristic acid (II); and α-methylglycosid/lauric acid (III) [6].
Figure 15.2 Schematic illustration of a fixed bed reactor (a) and a strirred tank reactor (STR) (b) with water removal by evaporation. Enzymatic reaction takes place in the fixed bed (a) or direct in the stirred tank (b).
Figure 15.3 Viscosities of the reactant polyglycerol-3, the substrate mixture of polyglycerol-3 and lauric acid at mass ratio of 50 (w/w), and the product polyglycerol-3 laurate as a function of temperature.
Figure 15.4 Comparison of the conversion of polyglycerol-3 and lauric acid while water is removed by vacuum (STR) and aeration (BCR) at 95 °C (a). Repetitive batch reaction of polyglycerol-3 and lauric acid catalyzed by Novozym 435 at 75 °C; equimass amounts of reactants; 5% (w/w) Novozym 435; BC; 2.2 l min
−1
compressed air (b).
Figure 15.5 Repetitive batch reaction of myristyl alcohol and myristic acid catalyzed by Novozym 435 at 75 °C; equimolar amounts of reactants; 5% (w/w) Novozym 435; BCR;
D
= 2.1 cm; 2.2 l min
−1
compressed air.
Figure 15.6 Analysis of the enzyme content for the myristyl myristate production in lab scale; equimolar substrate content, 80 g total mass, 75 °C, BCR;
D
= 2.1 cm; 2.2 l min
−1
compressed air.
Figure 15.7 Analysis of the aeration rate for the myristyl myristate production in lab scale; equimolar substrate content, 80 g total mass, 0.5 % (w/w) Novozym 435; 75 °C, BCR;
D
= 2.1 cm; compressed air.
Figure 15.8 Bubble column reactor on pilot scale with a diameter of 30 cm.
Figure 15.9 Analysis of the enzyme content (a) and aeration rate (b) for the myristyl myristate production on pilot scale; equimolar substrate content, 21 kg total mass, 60 °C, BCR;
D
= 30 cm; 5 m
3
·h
−1
nitrogen (a); 0.4% Novozym 435 (b).
Figure 15.10 Comparison of different reactor setups for the synthesis of myristyl myristate with the theoretically calculated reaction time. All reactions with 0.4% (w/w) Novozym 435, 60 °C and equimolar substrate content;
STR
: 400 rpm, 10 mbar vacuum, and 1015 g total mass;
fixed bed reactor
: 60 ml min
−1
flow rate, 10 mbar vacuum and 1015 g total mass; BCR: 13 m
3
h
−1
compressed air, 22 kg total mass; pilot scale with
D
= 30 cm.
Figure 15.11 Schematic illustration of the reaction system consisting three phases, whereby solid α-methyl glycoside is dispersed in the continuous unpolar lauric acid phase.
Figure 15.12 Enzymatic esterification of α-methyl glycoside (MG) and lauric acid catalyzed by Novozym 435 at 70 °C; 80 g lauric acid; 77.5 g MG total mass; 20% (w/w) MG as well as 10 g α-methyl glycoside laurate at the beginning and stepwise addition of 20% (w/w) MG (); 5% (w/w) Novozym 435; BCR;
D
= 2.1 cm; 0.45 l min
−1
compressed air.
Figure 15.13 Schematic illustration of a bubble column reactor equipped with an external attenuated total reflection (ATR) probe and a FT-IR device. The box displays the tip of the ATR FT-IR probe and a four-phase system [32], whereby the ATR technique allows the measurement in the continuous phase.
Figure 15.14 Fatty acid conversion for the synthesis of myristyl myristate measured with acid value titration (a). Corresponding time-resolved FT-IR spectra measured with a Mettler Toledo ReactIR 45 m equipped with an AgX FibreConduit diamond ATR probe (b); Reaction condition: 100 g total amount; equimolar substrate content; 60 °C; 1% (w/w) Novozym 435; BCR; 1.5 l min
−1
.
Figure 15.15 Prediction of the reaction course of myristyl myristate by chemometric modeling (detailed information see [27]); Reaction condition: 100 g total amount; equimolar substrate content; 60 °C; 1% (w/w) Novozym 435; BCR; 1.5 l min
−1
; Offline analysis: acid value titration; Online analysis: Mettler Toledo ReactIR 45 m equipped with an AgX FibreConduit diamond ATR probe; Chemometric: iCQuant software for chemometric modeling; Data preprocessing: baseline correction, cutting at 2800 to 1900 and 900 to 650 cm
−1
and centered. Prediction of error <3% fatty acid conversion.
Figure 15.16 Qualitative online monitoring of the water concentration in the liquid phase parallel to the reaction course of the synthesis of myristal myrsitate; Reaction condition: 120 g total amount; equimolar substrate content; 60 °C; 1% (w/w) Novozym 435; BCR; 1.5 l min
−1
; Offline analysis: acid value titration; Online analysis: Mettler Toledo ReactIR 45 m equipped with an AgX FibreConduit diamond ATR probe; Chemometric: MCR-algorithm.
Figure 15.17 Comparison of the accuracy of the acid value determination in a high-viscous multiphase system by titration (a) and the prediction by a chemometric model (detailed information see [27]) based on FT-IR spectra (b) in a bubble column reactor at 75 °C (added amounts of lauric acid, polyglycerol-3, and polyglycerol-3-laurate that correspond to a specific conversion).
Figure 15.18 Prediction of the reaction course of polyglycerol-3-laurate by chemometric modeling (detailed information see [27]); Reaction condition: 100 g total amount; equimolar substrate content; 75 °C; 4% (w/w) Novozym 435; BCR; 1.7 l min
−1
; Offline analysis: acid value titration; Online analysis: Bruker Vertex 70 Spektrometer; Data preprocessing: baseline correction, cutting below 800 and above 1950 cm
−1
and centered.
Figure 15.19 Esterification reaction sequence of glycerol and lauric acid, yielding trilaurin [33]. All possible intermediate glycerides are shown [40].
Figure 15.20 Molar fraction of 1-monolaurin compared to the total monolaurin content (1- and 2-monolaurin and 1,3-dilaurin content compared to the total dilaurin content (1,2- and 1,3-dilaurin) as a function of reaction time (a) and fatty acid conversion (b); measured by GC; 75 g total amount of substrates; 3.1 M ratio of lauric acid and glycerol; 2% (w/w) Novozym 435; 60 °C; BCR; 0.75 l min
−1
.
Figure 15.21 Prediction of the reaction course of trilaurin synthesis by chemometric modeling (detailed information see [22, 33]); 75 g total amount of substrates; 3.1 M ratio of lauric acid and glycerol; 2% (w/w) Novozym 435; 60 °C; BCR; 0.75 l min
−1
; Offline analysis: gas chromatography (GC); Online analysis: Bruker Vertex 70 Spektrometer; Data preprocessing: baseline correction, first derivative, cutting from 1760 to 1600 and 1475 to 890 cm
−1
and centered.
Chapter 16: Pharmaceutical Intermediates by Biocatalysis: From Fundamental Science to Industrial Applications
Figure 16.1 Boceprevir: oxidation of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane by monoamine oxidase.
Figure 16.2 Pregabalin: enzymatic preparation of (
S
)-3-cyano-5-methylhexanoic acid ethyl ester.
Figure 16.3 Glucagon-like peptide-1 (GLP-1): enzymatic synthesis of (
S
)-amino-3-[3-{6-(2-methylphenyl)} pyridyl]-propionic acid.
Figure 16.4 Rhinovirus protease inhibitor: enzymatic preparation of (
R
)-3-(4-fluorophenyl)-2-hydroxy propionic acid.
Figure 16.5 Saxagliptin: enzymatic synthesis of (
S
)-
N
-boc-3-hydroxyadamantylglycine.
Figure 16.6 Sitagliptin: enzymatic synthesis of chiral amine.
Figure 16.7 Montelukast: enzymatic reduction for synthesis of LTD 4 antagonists.
Figure 16.8 Clopidogrel: enzymatic preparation of (
S
)-2-chloromandelic acid esters.
Figure 16.9 Calcitonin gene-related peptide receptors antagonist: enzymatic preparation of (
R
)-2-amino-3-(7-methyl-1H-indazol-5-yl)propanoic acid.
Figure 16.10 Chemokine receptor modulators: enzymatic desymmetrization of dimethyl ester.
Figure 16.11 Regioselective enzymatic acylation of ribavirin.
Figure 16.12 Atorvastatin: enzymatic preparation of (
R
)-4-cyano-3-hydroxybutyrate.
Figure 16.13 Atazanavir, telaprevir, boceprevir: enzymatic synthesis of (
S
)-tertiary-leucine.
Figure 16.14 Relenza (zanamivir): enzymatic synthesis of
N
-acetylneuraminic acid.
Figure 16.15 Atorvastatin, rosuvastatin: aldolase-catalyzed synthesis of chiral lactol intermediates.
Figure 16.16 Anticancer drugs: epothilone B and microbial hydroxylation of epothiolone B.
Figure 16.17 Corticotropin-releasing factor-1 (CRF-1) receptor antagonist: enzymatic synthesis of (
S
)-1-cyclopropyl-2-methoxyethanamine.
Chapter 17: Biocatalysis toward New Biobased Building Blocks for Polymeric Materials
Figure 17.1 Comparison of the innovation potential with fossil building blocks (left curve) and with biobased building blocks (dotted line = prediction). Figure partly taken from [10]. Abbreviations: UF = urea formaldehyde, PF = phenol formaldehyde, PUR = polyurethane, PIB = polyisobutylene, PET = polyethylene terephthalate, PA = polyamide, LDPE = low-density polyethylene, PMMA = polymethyl methacrylate, BR = butadiene rubber, PS = polystyrene, PVC = polyvinylchloride, POM = polyoxymethylene, PTFE = polytetrafluoroethylene, EPM = ethylene propylene rubber, EPDM = ethylene propylene diene monomer rubber, iso. PP = isotactic polypropylene, ABS = acrylonitrile butadiene styrene, PAN = polyacrylonitrile, epoxy, PBT = polybutylene terephtalate, silicone, LLDPE = linear low-density polyethylene, PEEK = polyetheretherketone, PES = polyethersulfone, PI = polyimide, PEI = polyethylene imine, LCP = liquid crystalline polymer, PLA = polylactic acid, PEF = polyethylene furanoate, PDO = 1,3-propanediol, and SA = succinic acid.
Figure 17.2 Fundamental differences in business dynamics for drop-in versus new biobased building blocks.
Figure 17.3 Polymer pyramid grouped according to polymer state and performance. Abbreviations: PS = polystyrene, SAN = styrene acrylonitrile copolymer, PVC = polyvinylchloride, PP = polypropylene, HDPE = high-density polyethylene, LDPE = low-density polyethylene, PC = polycarbonate, PPO = poly(
p
-phenylene oxide), ABS = acrylonitrile butadiene styrene, PMMA = polymethyl methacrylate, PBT = polybutylene terephthalate, PET = polyethylene terephthalate, POM = polyoxymethylene, PA = polyamide, modified PP = modified polypropylene, PE-UHMW = ultra high-molecular-weight polyethylene, PPSU = polyphenylsulfone, PEI = polyethylene imine, PSS = polystyrene sulfonic acid, PES = polyethersulfone, PSU = polysulfone, COC = cyclic olefin copolymer, PPC = polyphthalate carbonate copolymer of polycarbonate, PFA = perfluoroalkoxy, MFA = copolymer of tetrafluoroethylene and perfluoromethyl vinyl ether, ECTFE = ethylene chloro trifluoroethylene, PVDF = polyvinylidene difluoride, PTFE = polytetrafluoroethylene, LCP = liquid crystalline polymer, PPS = poly(
p
-phenylene sulfide), PPA = polyphthalamide, PAMXD6 = polyamide MXD6, PBI = polybenzimidazole, PI = polyimide polymer, SRP = self-reinforcing polyphenylene, TPI = thermoplastic polyimide, HTS = high-temperature sulfone polymer, PAI = polyamide-imide, PEEK = polyether ether ketone, and FDCA = furane dicarboxylic acid.
Figure 17.4 Reaction scheme for long-chain fatty acid ω-oxidation.
Figure 17.5 Reaction scheme for the conversion of HMF to FDCA.
Figure 17.6 Reaction scheme for the conversion of
d
-glucose to gluconic acid with glucose dehydrogenase.
Figure 17.7 Reaction scheme for the regioselective desaturation of saturated fatty acid to polyunsaturated fatty acid.
Figure 17.8 Reaction scheme for the regioselective reduction of dinitrile to amino acid with tandem nitrilase/(putative) nitrile reductase.
Figure 17.9 Reaction scheme for the regioselective dehydrogenation of unactivated double bonds with dihydroxylases.
Figure 17.10 Reaction scheme for the regioselective oxidation of a polyol.
Figure 17.11 Reaction scheme for multiketones undergoing controlled transaminase reactions.
Figure 17.12 Reaction scheme for the biocatalytic resolution of a chiral amide with an amidase.
Figure 17.13 Reaction scheme for the α-amination of nonaromatic α-amino acids with ammonia lyases.
Figure 17.14 Reaction scheme for the α-amination of nonaromatic α-amino acids with ammonia lyases.
Figure 17.15 Reaction scheme illustrating the role of aminomutases.
Figure 17.16 Reaction scheme for the hydratation of double bonds by (regio- and stereoselective) hydratases.
Figure 17.17 Reaction scheme for the cascade reaction of
l
-phenylalanine to 4-(2-carboxyethyl)benzoic acid via cinnamic acid.
Figure 17.18 Reaction scheme for the conversion of isosorbide to isoidide.
Figure 17.19 Reaction scheme for the regioselective conversion of limonene to perillic acid, followed by hydrogenation of the double bond.
List of Tables
Chapter 2: Screening of Enzymes: Novel Screening Technologies to Exploit Noncultivated Microbes for Biotechnology
Table 2.1 Comparison between different culture-independent approaches to assess the diversity of biocatalysts from metagenomes
Chapter 3: Robust Biocatalysts – Routes to New Diversity
Table 3.1 Metagenomic strategies applied for the identification of thermoactive glycoside hydrolases
Table 3.2 Expression systems for selected industrial enzymes, such as hydrolases applied in the starch industry and commercial oxidative enzymes
Chapter 5: Supporting Biocatalysis Research with Structural Bioinformatics
Table 5.1 Summary of computational methods and tools for protein engineering
Chapter 6: Engineering Proteases for Industrial Applications
Table 6.1 Calculated PS
50
values (using single exponential fitting) and kinetic characterization (
K
M
and
k
cat
values) for perhydrolytic (methylbutyrate) and proteolytic activities (suc-AAPF-
p
NA) for subtilisin Carlsberg, parent, and variants (E1, E4, E5, and E6)
a
Table 6.2 Activity and stability of purified subtilisin E variants in the absence and presence of chaotropic compounds GdmCl or SDS
Chapter 7: Biocatalytic Synthesis of Natural Products by O-Methyltransferases
Table 7.1 Industrially relevant natural compounds, synthesized
in planta
involving crucial O-methylation steps
Table 7.2 Biocatalytic synthesis of methylated natural products
Chapter 8: Biocatalytic Phosphorylation of Metabolites
Table 8.1 Standard free energy of some important phosphorylating agents upon hydrolysis [101]
Chapter 11: Biocatalytic Processes for the Synthesis of Chiral Alcohols
Table 11.1 Comparison of
Ar
QR with other reductase in the asymmetric reduction of 3-quinuclidinone to (
R
)-3-quinuclidinol
Chapter 12: Inorganic Adsorbents in Enzymatic Processes
Table 12.1 Survey of the adsorbents employed
Table 12.2 Overview of the investigated materials
Table 12.3 Physical characteristics of used silicate carriers
Table 12.4 Propertied of the immobilized CALB and ADH used for the activity measurements shown in Figure 12.10
Table 12.5 Adsorption coefficients and selectivity of the studied substrate–product pairs
Table 12.6 Characteristics for adsorbent materials under investigation
Table 12.7 Results of thin-layer chromatography investigation of retention factors (
R
f
). Separation efficiency on alumina oxide thin-layer chromatography plates with different organic solvents
Chapter 13: Industrial Application of Membrane Chromatography for the Purification of Enzymes
Table 13.1 Different membrane adsorber variants tested for enzyme purification
Chapter 14: Fermentation of Lactic Acid Bacteria: State of the Art and New Perspectives
Table 14.1 Advantages of LAB in industrial fermentations [8]
Table 14.2 Batch cultivation of
L
.
lactis
without pH control in MRS (glucose), M17 (lactose), and CM medium (sucrose, [44])
Table 14.3 Characteristics of fixed-bed reactors
Table 14.4 Characteristics of carriers to be suiTable for cell immobilization [110, 112]
Table 14.5 Comparison of the kinetic parameters of different long-term continuous cultivations of LAB.
Chapter 15: The Bubble Column Reactor: A Novel Reactor Type for Cosmetic Esters
Table 15.1 Commercially obtainable cosmetic esters produced by Evonik Industries AG
Table 15.2 Limiting factors for the esterification in a bubble column reactor [11]
Chapter 17: Biocatalysis toward New Biobased Building Blocks for Polymeric Materials
Table 17.1 Important molecular parameters in organic monomers and their resulting properties in materials
