Academic literature on the topic 'Biocatalysis – Industrial applications'

Biocatalysis has undergone a remarkable transition in the last two decades, from being considered a niche technology to playing a much more relevant role in organic synthesis today. Advances in molecular biology and bioinformatics, and the decreasing costs for gene synthesis and sequencing contribute to the growing success of engineered biocatalysts in industrial applications. However, the incorporation of biocatalytic process steps in new or established manufacturing routes is not always straightforward. To realize the full synthetic potential of biocatalysis for the sustainable manufacture of chemical building blocks, it is therefore important to regularly analyze the success factors and existing hurdles for the implementation of enzymes in large scale small molecule synthesis. Building on our previous analysis of biocatalysis in the Swiss manufacturing environment, we present a follow-up study on how the industrial biocatalysis situation in Switzerland has evolved in the last four years. Considering the current industrial landscape, we record recent advances in biocatalysis in Switzerland as well as give suggestions where enzymatic transformations may be valuably employed to address some of the societal challenges we face today, particularly in the context of the current Coronavirus disease 2019 (COVID-19) pandemic.

Biocatalysis refers to the use of microorganisms and enzymes in chemical reactions, has become increasingly popular and is frequently used in industrial applications due to the high efficiency and selectivity of biocatalysts [...]

Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in “extreme” conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

The class EC 5.xx, a group of enzymes that interconvert optical, geometric, or positional isomers are interesting biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. This class, named “isomerases,” can transform cheap biomolecules into expensive isomers with suitable stereochemistry useful in synthetic medicinal chemistry, and interesting cases of production of l-ribose, d-psicose, lactulose, and d-phenylalanine are known. However, in two published reports about potential biocatalysts of marine origin, isomerases are hardly mentioned. Therefore, it is of interest to deepen the knowledge of these biocatalysts from the marine environment with this specialized in-depth analysis conducted using a literature search without time limit constraints. In this review, the focus is dedicated mainly to example applications in biocatalysis that are not numerous confirming the general view previously reported. However, from this overall literature analysis, curiosity-driven scientific interest for marine isomerases seems to have been long-standing. However, the major fields in which application examples are framed are placed at the cutting edge of current biotechnological development. Since these enzymes can offer properties of industrial interest, this will act as a promoter for future studies of marine-originating isomerases in applied biocatalysis.

AbstractEnzymes, the driving biocatalysts in living organisms, are typically not suited for large-scale industrial use. In the last decade, enzyme engineering has evolved into the key technology to design tailor-made enzymes for chemical and pharmaceutical applications. We highlight current trends in enzyme engineering and biocatalysis based on outstanding examples from the pharmaceutical industry.

Biocatalysts represent an efficient, highly selective and greener alternative to metal catalysts in both industry and academia. In the last two decades, the interest in biocatalytic transformations has increased due to an urgent need for more sustainable industrial processes that comply with the principles of green chemistry. Thanks to the recent advances in biotechnologies, protein engineering and the Nobel prize awarded concept of direct enzymatic evolution, the synthetic enzymatic toolbox has expanded significantly. In particular, the implementation of biocatalysts in continuous flow systems has attracted much attention, especially from industry. The advantages of flow chemistry enable biosynthesis to overcome well-known limitations of “classic” enzymatic catalysis, such as time-consuming work-ups and enzyme inhibition, as well as difficult scale-up and process intensifications. Moreover, continuous flow biocatalysis provides access to practical, economical and more sustainable synthetic pathways, an important aspect for the future of pharmaceutical companies if they want to compete in the market while complying with European Medicines Agency (EMA), Food and Drug Administration (FDA) and green chemistry requirements. This review focuses on the most recent advances in the use of flow biocatalysis for the synthesis of active pharmaceutical ingredients (APIs), pharmaceuticals and natural products, and the advantages and limitations are discussed.

ABSTRACTThe application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase forin situproduct removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especiallyPseudomonasstrains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified inPseudomonastaiwanensisVLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion ofttgV, coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants ofP. taiwanensisVLB120 and VLB120ΔC. Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of theP. taiwanensisVLB120ΔCparent strain, the maximum specific epoxidation activity ofP. taiwanensisVLB120ΔCΔttgVdoubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

Nitrile biocatalysts are of use in the chemical and pharmaceutical industries for the synthesis of carboxyamides and carboxylic acids. In particular, the application of biocatalysts in the synthesis of single enantiomer compounds is of increasing interest, but requires novel substrate specific highly stereoselective biocatalysts. Addition to the limited toolbox of known nitrile biocatalysts requires definitive characterisation of the biocatalysts through accurate determination of the substrate profiles and quantification of activity. The accurate quantification of stereoisomers chiral mixtures to determine biocatalyst stereoselectivity remains a significant challenge due to the difficulty in separating stereoisomers by physical methods. The known nitrile metabolising organism, Rhodococcus rhodochrous ATCC BAA-870, was grown in a defined medium and harvested, providing whole cell biocatalyst. Additional biomass was disrupted to provide a cell free enzyme extract, which was put through an enzyme purification protocol to provide a solution with specific activity of 351 U.mg⁻¹. A portion of the enzyme was self immobilised using the SphereZyme™ technique. The nitrile hydratase SphereZymes™ (1.2 U.mg⁻¹ initial activity) that were prepared had pH and temperature optima of 6 and 30°C respectively, and could be recovered by repeated washing. The particles retained activity in the presence of the organic solvents isooctane and n-hexadecane saturated with 50 mM phosphate buffer (pH 7.5). An initial analytical system was devised for quantification of the nitrile hydratase activity using the non-chiral substrate benzonitrile. An improved reversed phase high performance liquid chromatography method was developed to separate and quantify benzamide, benzoic acid and benzonitrile. The mobile phase consisting of 0.1% trifluoroacetic acid in H₂O and acetonitrile (70:30, %v/v), at a flow rate of 0.5 ml.ml⁻¹, 25°C, resolved all three analytes in 3.5 minutes on a Waters X-Terra MS C18 3.5μm column. UV detection was carried out at 210 nm. Analytical methods to determine activity and enantioselectivity of the whole cell biocatalyst were subsequently developed for both β-amino nitriles and β-hydroxy nitrile substrates and hydrolysis products.

Enzymes are proteins necessary to maintain essential biochemical processes taking place in all living organisms. Many of them have found applications in industrial sectors such as biotechnology, production of pharmaceuticals and other fine chemicals. The search for new and efficient biocatalysts requires specialised methodology designed for biochemical characterisation and selection of those with potential applications.

Thesis submitted in partial fulfilment of the requirements for the degree Doctor of Technology: Biomedical Technology In the Faculty of Health and Wellness Sciences At the Cape Peninsula University of Technology 2014
Peroxidases are ubiquitous catalysts that oxidise a wide variety of organic and inorganic compounds employing peroxide as the electron acceptor. They are an important class of oxidative enzymes which are found in nature, where they perform diverse physiological functions. Apart from the white rot fungi, actinomycetes are the only other known source of extracellular peroxidases. In this study, the production of extracellular peroxidase in wild type actinomycete strains was investigated, for the purpose of large-scale production and finding suitable applications. The adjustment of environmental parameters (medium components, pH, temperature and inducers) to optimise extracellular peroxidase production in five different strains was carried out. Five Streptomyces strains isolated from various natural habitats were initially selected for optimisation of their peroxidase production. Streptomyces sp. strain BSII#1 and Streptomyces sp. strain GSIII#1 exhibited the highest peroxidase activities (1.30±0.04 U ml-1 and 0.757±0.01 U ml-1, respectively) in a complex production medium at 37°C and pH 8.0 in both cases. Maximum enzyme production for Streptomyces strain BSII#1 was obtained in the presence of 0.1 mM veratryl alcohol or pyrogallol, while 0.1 mM guaiacol induced the highest peroxidase production in Streptomyces sp. strain GSIII#1. As the highest peroxidase producer, Streptomyces sp. strain BSII#1 was selected for further studies. The strain was first characterised by a polyphasic approach, and was shown to belong to the genus Streptomyces using various chemotaxonomic, genotypic and phenotypic tests. Production of peroxidase was scaled up to larger volumes in different bioreactor formats. The airlift configuration was optimal for peroxidase production, with Streptomyces sp. strain BSII#1 achieving maximum production (4.76±0.46 U ml-1) in the 3 l culture volume within 60 hrs of incubation. A protocol for the purification of the peroxidase was developed, which involved sequential steps of acid and acetone precipitation, as well as ultrafiltration. A purification factor of at least 46-fold was achieved using this method and the protein was further analysed by LC-MS. The protein was shown to be a 46 kDa protein, and further biochemical characterisation showed that the peroxidase had a narrower spectrum of substrates as compared to reports on other peroxidases derived from actinomycetes. With 2,4-dichlorophenol as the substrate, the Km and Vmax for this enzyme were 0.893 mM and 1.081 μmol min-1, respectively. The purified peroxidase was also capable of catalysing coupling reactions between several phenolic monomer pairs. Overall, the peroxidase from Streptomyces sp. strain BSII#1 could feasibly be produced in larger scales and there remains further room to investigate other potential applications for this enzyme.

La lactosa és un dissacàrid de la llet produït per gaire bé tots els mamífers. Per a la seva digestió cal que sigui hidrolitzada per la lactasa (EC. 3.3.1.23), que és produïda per les cèl·lules epitelials de l’intestí prim, donant lloc a galactosa i glucosa. La seva deficiència o baixa quantitat (hipolactàsia) pot produir diversos símptomes incloent inflor, dolor abdominal, flatulències i diarrea. L’avaluació de la deficiència de la lactasa és principalment important en pediatria i en gastroenterologia a causa de l’elevada incidència de l’alteració genètica de la lactasa en aquests grups poblacionals (65%). La majoria dels mètodes de diagnòstic són invasius i dràstics i no són adequats per a ser aplicats a infants. Davant d’aquesta situació, un nou mètode no invasiu va ser desenvolupat. Aquest es basa en l’administració de 4-O-β-D-galactopiranosil-D-xilose (gaxilosa), un anàleg estructural de la lactosa. Aquest compost també es substrat per la lactasa i és hidrolitzat donant lloc a galactosa i xilosa. Aquest segon és absorbit passivament per l’intestí prim i és eliminat per orina on pot ser fàcilment detectat per un mètode colorimètric senzill. La síntesi química de la gaxilosa necessita de l’ús de grups protectors i de llargs i complexes etapes sintètiques per arribar a rendiments de producció baixos (9%). La ruta biosintètica utilitzant galactosiltransferases implica diverses dificultats tècniques i costos elevats. Les glicosidases tenen la capacitat de formar enllaços o-glicosídics per transglicosidació amb costos baixos. Els desavantatges d’aquesta metodologia són els baixos rendiments limitats per la competència de l’activitat hidrolítica i els problemes de purificació per la formació de regioisòmers o altres productes. La β–galactosidasa d’Escherichia coli va ser seleccioionada entre altres enzims per la producció de gaxilosa a nivell industrial. Després de diversos passos de purificació el rendiment obtingut és del 20-23%. Aquest treball pretén augmentar el rendiment en la producció de gaxilosa. Primer, les condicions de reacció es van modificar per tal d’augmentar l’activitat enzimàtica de transglicosidació. D’altra banda, es va buscar un altre enzim per ser utilitzat en la producció de gaxilosa. Estudis bibliogràfics i experimentals van permetre la selecció d’un nou enzim capaç de sintetitzar més gaxilosa, arribant a un rendiment final del 35% utiiltzant 3,3 cops menys enzim (amb la corresponent disminució dels costos de producció). Tot i que el nou enzim presentava una activitat de transglicosidació superior a l’enzim d’E. coli, la seva activitat hidrolítica romanent no permetia augmentar-ne més el rendiment. Per modificar l’activitat enzimàtica i augmentar la síntesi de gaxilosa es va optar per utilitzar modificar l’enzim amb enginyeria de proteïnes (racional i aleatòria).
La lactosa es un disacárido de la leche producido por casi todos los mamíferos. Para su digestión debe ser hidrolizado per la enzima lactasa (EC. 3.3.1.23), que es producida por las células epiteliales del intestino delgado, dando lugar a glucosa i galactosa. Su deficiencia o baja concentración (hipolactasia) puede producir diversos síntomas como son hinchazón, dolor abdominal, flatulencias y diarrea. La evaluación de la deficiencia de la lactasa es importante en pediatría y gastroenterología por la elevada frecuencia de esta alteración genética (65%). La mayoría de los métodos de diagnóstico son invasivos y drásticos y no son adecuados para su uso en población infantil. Frente a esta situación, se desarolló un nuevo método no invasivo. Éste se basa en la administración de 4-O-β-D-galactopiranosil-D-xilose (gaxilosa), un análogo estructural de la lactosa. Este compuesto también es sustrato de la lactasa y es hidrolizado dando lugar a galactosa y xilosa. El segundo se absorbe pasivamente a través del intetsino delgado y se elimina a través de la orina donde puede ser detectado por un método colorimétrico senzillo. La síntesi química de la gaxilosa requiere del uso de grupos protectores y de largos y complejos pasos de síntesis para llegar a rendimientos de producción bajos (9%). La ruta biosintética utilizando galactosyltransferasas comporta dificultades técnicas y costes elevados. Por otro lado, las glicosidasas tienen la capacidad de síntesis de glicósidos por transglicosidación con costes bajos. La principal desventaja de esta metodologia son los bajos rendimientos por la actividad hidrolítica de la enzima y los problemas de purificación por la formación de regioisómeros y/u otros productos. La β-galactosidasa de Escherichia coli fue seleccionada, entre otras enzimas, para la producción de gaxilosa a nivel industrial. Después de varios pasos de purificación el rendimiento es del 20-23%. Este trabajo pretende aumentar el rendimiento de la producción de gaxilosa. Primero se modificaron las condiciones de reacción para aumentar la activitat enzimática de transglicosidación. Por otro lado, se buscó otra enzima para la producción industrial. Estudios bibliográficos y experimentales permitieron la selección de una nueva enzima capaz de sintetitzar más gaxilosa, llegando a un rendimiento del 35% utilizando 3,3 veces menos enzima (con la consecuente disminución de los costes de producción). Aunque la nueva enzima presentaba una actividad de transglicosidación mayor, la actividad hidrolítica remanente no permite aumentar el rendimento. Para modificar la actividad enzimática y aumentar la síntesis de gaxilosa se decidió modificar la enzima mediante ingeniería de proteínas (racional y aleatoria).
Lactose is a milk disaccharide produced by nearly all mammalian species. For its digestion, it must be hydrolysed to galactose and glucose by lactase (EC 3.2.1.23), which is normally produced by the cells that line the small intestine. Deficiency or low levels of lactase (hypolactase) can cause common symptoms including bloating, abdominal pain or cramps, flatulence and diarrhea. Evaluation of enzyme deficiency is important in pediatrics and gastroenterology due to the high frequency of genetic predisposition (65%). Many of the standard diagnostic procedures are invasive, quite drastic and not applicable to infants and young childen. A non-invasive method was developed based on the use of 4-O-β-D-galactopyranosyl-D-xylose (gaxilose), a structural analogue of lactose (it only lacks the hydroxymethyl group at position 5). This compound is substrate of the lactase enzyme in vivo, yielding D-galactose and D-xylose. The latter is passively absorbed from the small intestine and is eliminated in the urine where it can be quantified by a colorimetric procedure. Chemical synthesis of gaxilose requires the addition of protective groups and suffers from long, tedious reaction sequences with low overall productivity (9%). The biosynthetic route involving galactosyltransferase enzymes implies technical difficulties (unstable and expensive enzymes) and high costs. Glycosidases have been shown to catalyse the formation of glycosides by transglycosylation at low cost. Disadvantatges of this approach include yield limitations due to competing hydrolysis reactions, and purification problems because of the formation of other regioisomers and by-products. Escherichia coli β-galactosidase was selected to produce gaxilose at industrial level. After an enzymatic reaction and several purification steps, a yield of 20-23% is reached. The present work aims to increase gaxilose production yield by different strategies. First, enzymatic conditions were modified to increase enzymatic gaxilose production. On ther other hand, other enzymes were searched to be used as gaxilose biocatalysts. Bibliographic and experimental studies allowed to find one enzyme able to increase gaxilose yield up to 35% using 3.3-fold less enzyme, and thus diminishing the production costs). Despite the higher transglycosidase activity of the new enzyme, its hydrolase activity did not allow the increase of gaxilose prodution. Protein engineering (random and rational approaches) was used to modify the enzyme activity and increase gaxilose synthesis.

Lipases are a multipurpose enzyme that holds a significant position in industrial applications due to its ability to catalyse a large number of reactions such as hydrolysis, esterification, interesterification, transesterification which makes it a potential candidate. It is also used for the separation of chiral drugs from the racemic mixture and this property of lipase is considered very important in pharmaceutical industries for the synthesis of enantiopure bioactive molecules. Assuming the tremendous importance of lipases, as stereoselective biocatalysts, in pharmaceuticals and various other commercial applications, industrial enzymologists have been forced to search for those microorganisms which are able to produce novel biocatalysts at reasonably high yield. In the present study microbial lipase was isolated from the water sample of pond at Katra, Jammu and Kashmir (India). This enzyme has shown wide specificity and higher enantioselectivity, which make it pharmaceutical important enzyme. To make it economical for industrial application, it was produced on cheap nutrient media using Response Surface Methodology and got maximum production. It was used for resolution of chiral drugs and the significant results obtained during the course of work shall have potential towards pharmaceutical industries.