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PubMed2001

Industrial biocatalysis.

The number of industrial processes for the synthesis of fine and commodity chemicals, pharmaceutical and agrochemical intermediates and drug substances utilizing biological catalysts continues to grow. The combination of new molecular biology techniques, such as directed evolution and pathway engineering, with new and efficient high-throughput screening methods is poised to bolster this field and further advance the contribution of biocatalysis to the chemical and the pharmaceutical industries.

Catalysis↗
PubMedDate not supplied

Immobilization of lipase on chitin and its use in nonconventional biocatalysis.

Chitin was functionalized with hexamethylenediamine followed by glutaraldehyde activation, and its capacity to bind Candida rugosa lipase was investigated. The loading of 250 units g(-1) support showed to be effective, resulting in a uniform enzyme fixation with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, and thermal stability. For the immobilized lipase, the influence of the reaction temperature and substrate polarity in nonconventional biocatalysis was also analyzed. Production of butyl esters was found to be dependent on the substrate partition coefficient, which accounts the greatest value for the system butanol and butyric acid. The highest enzyme activity was found for the system butanol and caprylic acid at a reaction temperature of 40 degrees C. Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 12 batches, revealing a biocatalyst half-life of 426.7 h.

Butanols↗
PubMed2005

Laminar flow-based electrochemical microreactor for efficient regeneration of nicotinamide cofactors for biocatalysis.

One of the long-standing challenges in biocatalysis is the search for methods to continuously regenerate essential cofactors such as NADH that would enable a wide range of enzymes to be used in the more environmentally friendly synthesis of chiral fine chemicals including pharmaceuticals, cosmetics, and food additives. This communication reports a microreactor-based cofactor regeneration method that exploits the microfluidic phenomenon of laminar flow: a reactant stream and a buffer stream are introduced in a microchannel and continue to flow side by side without turbulent mixing between two electrodes that cover opposing channel walls. Adjustment of the flow rate ratio of the two streams in laminar flow enables focusing of the reactant stream close to the cathode, thereby reversing a normally unfavorable reaction equilibrium essential for cofactor regeneration. The absence of a bulk phase in these microreactors prevents the undesired reverse reaction to take place, which has prevented the use of electrochemical cofactor regeneration in macroscale processes. Here, we demonstrate the regeneration of NADH with conversion efficiencies as high as 31%. We also show the subsequent in situ conversion of an achiral substrate, pyruvate, into a chiral product, l-lactate, within this microreactor.

Bioreactors↗
PubMed2006

Directed assembly of carbon nanotubes at liquid-liquid interfaces: nanoscale conveyors for interfacial biocatalysis.

We report that single-walled carbon nanotubes (SWNTs) can be directed to aqueous-organic interfaces with the aid of surfactants. This phenomenon can also be used to transport enzymes to the interface to effect biphasic biotransformations. Consequently, SWNT-enzyme conjugates enhance the rate of catalysis by up to 3 orders of magnitude relative to the rates obtained with native enzymes in similar biphasic systems. Furthermore, we demonstrate that this concept can be extended to other nanomaterials and other enzymes, thereby providing a general strategy for efficient interfacial biocatalysis. The ability to direct the assembly of nanotubes at the interface also provides an attractive route to organizing these nanomaterials into 2D architectures.

Alcohol Dehydrogenase↗
PubMed2002

Ionic liquid-coated enzyme for biocatalysis in organic solvent.

Ionic liquid-coated enzyme (ILCE) is described as a useful catalyst for biocatalysis in organic solvent. An ionic liquid, [PPMIM]-[PF(6)] (1, [PPMIM] = 1-(3'-phenylpropyl)-3-methylimidazolium), which is solid at room temperature and becomes liquid above 53 degrees C, was synthesized in two steps from N-methylimidazole. The coating of enzyme was done by simply mixing commercially available enzyme with 1 in the liquid phase above 53 degrees C and then allowing the mixture to cool. A representative ILCE, prepared with a lipase from Pseudomonas cepacia, showed markedly enhanced enantioselectivity without losing any significant activity.

Journal Article↗
PubMedDate not supplied

Biocatalysis, biodegradation and bioinformatics.

Biocatalysis, biodegradation and bioinformatics are prominent scientific fields in industrial microbiology and biotechnology. This paper describes developments in these fields with a focus on the role of David T Gibson as a researcher and mentor. He has pioneered studies on the mechanisms by which aerobic microorganisms transform aromatic hydrocarbons. In addition, his research has served as a model for further investigations into bacterial atrazine and dichloromethane catabolism described here. Microbial catabolism research requires information on organic chemistry, microorganisms, metabolic pathways, catabolic genes, and enzymes. These information needs are now being met more comprehensively by development of the University of Minnesota Biocatalysis/Biodegradation Database.

Bacteria↗
PubMed2004

Characteristics of nearly dry enzymes in organic solvents: implications for biocatalysis in the absence of water.

We have examined enzymes in nearly anhydrous organic solvents spanning a wide range of dielectric constants using a combination of electron paramagnetic resonance (EPR) spectroscopy, molecular dynamics simulations, high-pressure kinetic studies and the electrostatic model of Kirkwood. This approach enabled us to investigate the relationship between catalytic activity, protein flexibility and solvent polarity for an enzymatic reaction proceeding through a highly polar transition state in the near absence of water. Further insights into water-protein interactions and the involvement of water in enzyme structure and function have been obtained by EPR and multinuclear nuclear magnetic resonance studies of enzymes suspended and immobilized in organic solvents with and without added water. In these systems, correlations were observed between the water content and enzyme activity, flexibility, and active-site polarity, although the structural properties of suspended and immobilized enzymes differed markedly. These results have helped to elucidate the role of water in molecular events at the enzymic active site leading to improved biocatalysis in low-water environments.

Catalysis↗
PubMed1998

Directed evolution of new enzymes and pathways for environmental biocatalysis.

Biocatalysis is important in both natural and engineered environments. The major global reactions in the biospheric cycling of carbon, nitrogen, and other elements are catalyzed by microorganisms. The global carbon cycle includes millions of organic compounds that are made by plants, microorganisms, and organic chemists. Most of those compounds are transformed by microbial enzymes. Degradative metabolism is known as catabolism and yields principally carbon dioxide, methane, or biomass. Microbial catabolic enzymes are a great resource for biotechnology. They are the building blocks for engineering novel metabolic pathways and evolving improved enzymes in the laboratory. Two multicomponent bacterial oxygeneases, cytochrome P450cam and toluene dioxygenase, catalyze the dechlorination of polyhalogenated C2 compounds. Seven genes encoding those functional enzyme complexes were coexpressed in a Pseudomonas and shown to metabolize pentachloreothane to nonhalogenated organic acids that were metabolized further to carbon dioxide. In another example, the enzyme catalyzing the dechlorination of the herbicide atrazine was subjected to iterative DNA shuffling to produce mutations. By using a plate screening assay, mutated atrazine chlorohydrolase that catalyzed a more rapid dechlorination of atrazine was obtained. The mutant genes were sequences and found to encode up to 11 amino acid changes. Atrazine chlorohydrolase is currently being used in a model municipal water treatment system to test the feasibility of using enzymes for atrazine decontamination. These data suggest that the natural diversity of bacterial catabolic enzymes provides the starting point for improved biocatalytic systems that meet the needs of commercial applications.

Biodegradation, Environmental↗
PubMed2026

Genomic prospecting and biochemical characterization of a novel thermostable 3-quinuclidinone reductase from hot spring metagenomes for efficient biocatalysis.

This study presents the discovery and characterization of a novel thermophilic 3-quinuclidinone reductase (ScQR) identified through metagenomic mining of hot spring environments. ScQR, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, was heterologously expressed in Escherichia coli, and its catalytic properties were systematically characterized. The enzyme demonstrates exceptional thermal stability, retaining 86% of its activity after 48 hours at 70°C. Furthermore, K+ and Mg²+ ions significantly enhanced ScQR's activity at specific concentrations. Structural analysis revealed that ScQR adopts a typical SDR fold with a conserved catalytic triad (S141-Y155-K159), and it is NAD(H) dependent. Enzyme assays indicated that ScQR is highly stereoselective for (R)-3-quinuclidinol, with no activity against its enantiomer, (S)-3-quinuclidinol. The enzyme exhibits optimal activity at pH 9 and 85°C, making it a promising candidate for industrial applications requiring high thermal stability. Molecular dynamics simulations further revealed that ScQR preserves global structural integrity up to 360 K, whereas higher temperatures induce destabilization, predominantly in the C-terminal region and residues 95-100. In addition, structure-guided computational design enabled by LigandMPNN and UniKP yielded three ScQR variants with improved substrate affinity and catalytic efficiency while maintaining the overall fold and function. This work underscores the power of metagenomics with structure-driven protein design in discovering novel enzymes with unique catalytic properties from extreme environments and establishes ScQR as a promising biocatalyst for biotechnological and pharmaceutical applications.IMPORTANCEThis study reports the discovery of ScQR, a novel thermophilic 3-quinuclidinone reductase identified via metagenomic mining. ScQR represents one of the most heat-resistant members of the SDR superfamily discovered to date, maintaining 86% activity after 48 hours at 70°C. These findings establish ScQR as a robust biocatalyst for high-temperature pharmaceutical applications and demonstrate a scalable workflow for optimizing enzymes from extreme environments, offering significant value to the fields of biocatalysis and protein engineering.

computational design↗
PubMed2004

Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis.

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50 degrees C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.

Base Sequence↗
PubMed1998

Tour de paclitaxel: biocatalysis for semisynthesis.

In collaboration with the National Cancer Institute, Bristol-Myers Squibb has developed paclitaxel for treatment of various cancers; it has been approved by the Food and Drug Administration for the treatment of ovarian and metastatic breast cancer. Originally paclitaxel was isolated and purified from the bark of Pacific yew trees. This source of paclitaxel was considered to be economically and ecologically unsuitable as it required the destruction of the yew trees. This review article describes alternate methods for the production of paclitaxel, specifically, a semisynthetic approach and the application of biocatalysis in enabling the semisynthesis of paclitaxel. Three novel enzymes were discovered in our laboratory that converted the variety of taxanes to a single molecule, namely 10-deacetylbaccatin III (paclitaxel without C-13 side chain and C-10 acetate), a precursor for paclitaxel semisynthesis. These enzymes are C-13 taxolase (catalyzes the cleavage of C-13 side chain of various taxanes), C-10 deacetylase (catalyzes the cleavage of C-10 acetate of various taxanes), and C-7 xylosidase (catalyzes the cleavage of C-7 xylose from various xylosyltaxanes). Using a biocatalytic approach, paclitaxel and a variety of taxane in extracts of a variety of Taxus cultivars were converted to a 10-deacetylbaccatin III. The concentration of 10-deacetylbaccatin III was increased by 5.5- to 24-fold in the extracts treated with the enzymes, depending upon the type of Taxus cultivars used. Biocatalytic processes have also been described for the preparation of C-13 paclitaxel side chain synthons. The chemical coupling of 10-deacetylbaccatin III or baccatin III to C-13 paclitaxel side chain has been summarized to prepare paclitaxel by semisynthesis.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis.

BACKGROUND: Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. RESULTS: To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA) from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP) anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC). The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37 degrees C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. CONCLUSION: In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

Amino Acid Motifs↗
PubMed2003

Enhancing marine natural product structural diversity and bioactivity through semisynthesis and biocatalysis.

In the last several decades the plants, animals and microbes from the marine environment have revealed a portion of what is clearly a tremendous resource for structurally diverse and bioactive secondary metabolites. Many of these extraordinarily sophisticated and bioactive natural products can be isolated in significant quantities without great difficulty. As a result these readily available bioactive natural products provide valuable starting materials for the rational generation of libraries of compounds prepared through semisynthesis and biocatalysis. A review of our work using marine natural products to generate rationally designed compound libraries and their biological activity against infectious diseases, cancer and neurological targets is presented. The marine natural products utilized to date as starting materials consist of compounds from a variety of structural classes and include: aureol, puupehenone, sarcophine, palinurin, and the manzamine alkaloids. The possibility to generate diverse bioactive products beginning with a marine natural product scaffold is a direct result of improvements made in the technologies to harvest samples from the ocean, purify and characterize complex natural products quickly and complete chemical reactions and biotransformations in parallel. As a result the vast resources of the ocean can now be utilized routinely to design and produce countless products to be evaluated as part of drug discovery and development programs.

Animals↗
PubMedDate not supplied

[Biocatalysis using immobilized cells or enzymes as a method of water and air purification in habitable enclosed environment].

This paper shows that the method of water and air purification using immobilized cells and enzymes can be applied in regenerative life support systems in a habitable enclosed environment. This method is based on selective and adaptive functions of enzymic systems of microorganisms to assimilate organic components of the medium to be eliminated. Advantages of biocatalysis are low energy requirements and mild temperatures of purification leading to practically complete elimination of the substrate. Due to immobilization, cells and enzymes constitute an independent component which can be added to any continuously operating system of purification without generating a specific interface which is crucially important in microgravity. This allows the process of purification to be controllable. The resulting water and air meet biological requirements because they are formed under the influence of living organisms as in natural biogeocenoses. The production of ecologically pure water and air is highly important for long-duration space missions, especially for flights to Mars.

Air Conditioning↗
PubMed2026

Generative artificial intelligence for enzyme design and biocatalysis.

Sparked by innovations in generative artificial intelligence (AI), the field of protein design has undergone a paradigm shift with an explosion of new models for optimizing existing enzymes or creating them from scratch. After more than one decade of low success rates for computationally designed enzymes, generative AI models are now frequently used for designing proficient enzymes. Here, we provide a comprehensive overview and classification of generative AI models for enzyme design, highlighting models with experimental validation relevant to real-world settings and outlining their respective limitations. We argue that generative AI models now have the maturity to create and optimize enzymes for industrial applications. Wider adoption of generative AI models with experimental feedback loops can speed up the development of biocatalysts and serve as a community assessment to inform the next generation of models.

Biocatalysis↗
PubMed1999

Alkaline biocatalysis for the direct synthesis of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc).

Integration between the alkaline epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-Acetyl-D-mannosamine (ManNAc) and the N-acetyl-D-neuraminic acid (Neu5Ac) aldolase-catalyzed biotransformation has been assessed experimentally. GlcNAc epimerization took place above pH 9.0, and the initial rate of ManNAc formation increased exponentially to 10.37 mmol/L per hour at pH 12. However, above this pH, severe degradation of pyruvate occurred. A value of 31.3% molar conversion on Pyr was achieved in an integrated biotransformation. The "pseudo"-steady state at the end of the reaction was comparable to the equilibrium achieved with a combination of an epimerase and aldolase enzymes. The integrated reaction proved feasible, but at the expense of pyruvate and Neu5Ac aldolase degradation.

Acetylglucosamine↗