Biotransformation using plant cultured cells

This review outlines the recent progress during the last 25 years concerning the biotransformation of exogenous substrates by plant cultured cells. The plant cultured cells have abilities of the regio- and stereoselective hydroxylation, oxido-reduction, hydrogenation, glycosylation, and hydrolysis for various organic compounds as well as microorganisms. The reaction types and the stereochemistry of the products involved in the biotransformations are described. The development of techniques using immobilized plant cells are also delineated.     

Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases

Many flavoenzymes catalyze hydroxylation of aromatic compounds especially phenolic compounds have been isolated and characterized. These enzymes can be classified as either single‐component or two‐component flavin‐dependent hydroxylases (monooxygenases). The hydroxylation reactions catalyzed by the enzymes in this group are useful for modifying the biological properties of phenolic compounds. This review aims to provide an in‐depth discussion of the current mechanistic understanding of representative flavin‐dependent monooxygenases including 3‐hydroxy‐benzoate 4‐hydroxylase (PHBH, a single‐component hydroxylase), 3‐hydroxyphenylacetate 4‐hydroxylase (HPAH, a two‐component hydroxylase), and other monooxygenases which catalyze reactions in addition to hydroxylation, including 2‐methyl‐3‐hydroxypyridine‐5‐carboxylate oxygenase (MHPCO, a single‐component enzyme that catalyzes aromatic‐ring cleavage), and HadA monooxygenase (a two‐component enzyme that catalyzes additional group elimination reaction). These enzymes have different unique structural features which dictate their reactivity toward various substrates and influence their ability to stabilize flavin intermediates such as C4a‐hydroperoxyflavin. Understanding the key catalytic residues and the active site environments important for governing enzyme reactivity will undoubtedly facilitate future work in enzyme engineering or enzyme redesign for the development of biocatalytic methods for the synthesis of valuable compounds.     

Catabolism of citronellol and related acyclic terpenoids in pseudomonads

Terpenes are a huge group of natural compounds characterised by their predominantly pleasant smell. They are built up by isoprene units in cyclic or acyclic form and can be functionalised by carbonyl, hydroxyl or carboxyl groups and by presence of additional carbon–carbon double bonds (terpenoids). Currently, much more than 10,000 terpenoid compounds are known, and many thereof are present in different iso- and stereoforms. Terpenoids are secondary metabolites and can have important biological functions in living organisms. In many cases, the biological functions of terpenoids are not known at all. Nevertheless, terpenoids are used in large quantities as perfumes and aroma compounds for food additives. Terpenoids can be also precursors and building blocks for synthesis of complex chiral compounds in chemical and pharmaceutical industry. Unfortunately, only few terpenoids are available in large quantities at reasonable costs. Therefore, characterisation of suited biocatalysts specific for terpenoid compounds and development of biotransformation processes of abundant terpenoids to commercially interesting derivates becomes more and more important. This minireview summarises knowledge on catabolic pathways and biotransformations of acyclic monoterpenes that have received only little attention. Terpenoids with 20 or more carbon atoms are not a subject of this study.     

Characteristics and biotechnology applications of aliphatic amino acid hydroxylases belonging to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily

The asymmetric hydroxylation of inactive carbon atoms is still an important reaction in the industrial synthesis of valuable chiral compounds such as pharmaceuticals and fine chemicals. Applications of monooxygenation enzymes, like cytochrome P450 monooxygenases, flavin-containing monooxygenases, and Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe/αKG-DOs), are strongly desired as hydroxylation biocatalysts because they have great advantages in regio- and stereoselectivity of the reactions. Recently, several novel Fe/αKG-DOs have been found to catalyze the asymmetric hydroxylation of aliphatic amino acids. Depending on their amino acid sequences, these Fe/αKG-DOs catalyze different types of regioselective hydroxylations, or C3–, C4–, and C5-hydroxylation. Additionally, most also have stereoselective sulfoxidation activities. Here, we have reviewed the characterization and process development of this novel functioning group of Fe/αKG-DOs.     

Stepwise conversion of flavonoids by engineered dioxygenases and dehydrogenase: Characterization of novel biotransformation products

Flavonoids are a large group of plant secondary metabolites that exert various biological and pharmacological effects. In this context, the generation of derivatives is of considerable interest. The introduction of hydroxy groups is of particular relevance, as they are known to be involved in many of the biological interactions and furthermore enable additional modifications, such as glycosylations. Bacterial aryl-hydroxylating dioxygenases (ARHDOs) have proven to be very useful for the conversion of aromatic structures into versatile building blocks for different kinds of derivatizations. Such enzymes have been used with varying success for the oxidation of flavonoids. In order to find better ARHDOs for the hydroxylation of such substrates, we carried out biotransformation trials with a collection of hybrid ARHDOs of different origin, using resting cells of recombinant strains. This identified enzymes able to transform all of the flavonoids examined, typically in yields above 50%. It also showed that moderately reactive substituents of flavonoids, such as hydroxy or amino groups, can lead to spontaneous follow-up reactions with the dienediol structures generated by dioxygenation. A report of flavanone epoxidation, a reaction never before observed to be catalyzed by an ARHDO, is challenged by our results. All ARHDOs examined converted this substrate into a dehydrogenase-transformable dihydrodiol. All dihydrodiols obtained by dioxygenation of the examined flavonoids were successfully re-aromatized into catechols by a bacterial dehydrogenase. These metabolites were usually stable. However, the catechols formed from flavanone and 2’-hydroxy-chalcone, respectively, were interconvertible under mild conditions. Altogether, we isolated and characterized 13 compounds that have not previously been described. The biotransformations reported here give access to novel flavonoid derivatives that may be applied for biological screens as well as for further modification, such as glycodiversification.     

Microbial Baeyer–Villiger oxidation of 4,4-disubstituted cyclohexan- and cyclohexenones by recombinant whole-cells expressing monooxygenases of bacterial origin

Screening of 4,4-disubstituted and 3,4,5-polysubstituted cyclohexan- and cyclohexenones with eight different overexpression systems of microbial monooxygenases in recombinant Escherichia coli provided valuable information about substrate acceptance and enantioselectivity of this enzyme family, which are responsible for the stereoselective Baeyer–Villiger biooxidation of ketones. For this purpose whole-cell mediated biotransformations were realized to overcome some limitations in the application of cofactor dependent biocatalysts. The different behavior of various enzymes reflects a recent hypothesis about two distinct clusters of biooxidation catalysts. In contrast to isolated enzyme biooxidations, recombinant cells did not yield unsaturated lactone products derived from cycloalkenones. They rather displayed reductase activity to reduce such precursors to saturated ketones, which were subsequently oxidized to the corresponding Baeyer–Villiger products in a sequential two-step biotransformation.     

Biotechnological production of flavours and fragrances

The biotechnological generation of natural aroma compounds is rapidly expanding. Aroma chemicals, such as vanillin, benzaldehyde (bitter almond, cherry) and 4-(R)-decanolide (fruity–fatty) are marketed on a scale of several thousand tons per year. Their possible production by single-step biotransformations, bioconversions and de novo synthesis using microorganisms, plant cells or isolated enzymes is shown. The perspectives of bioprocesses for the oxifunctionalisation of lower terpenes by genetically modified organisms and economic aspects are discussed. Received: 27 May 1997 / Received revision: 25 September 1997 / Accepted: 28 September 1997     

Development and evaluation of a new device to effectively detach micro-organisms from food samples

Aims: To develop a new instrument of great versatility for recovering micro‐organisms from all types of food samples and to compare the effects with existing sample preparation methods. Methods and Results: To detach micro‐organisms from large‐size unbroken food samples such as apples, carrots, potatoes and tomatoes without preprocessing, the Spindle apparatus was newly developed. The Spindle was used to effectively detach micro‐organisms from large‐size samples. In a comparative study involving 51 food samples, treatment with the Spindle and Stomacher showed that recovery of total aerobic micro‐organisms (naturally occurring mesophilic microflora) and foodborne pathogens (from samples inoculated with Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes) for both methods was highly correlated (R² = 0·98). Furthermore, diluents treated by the Spindle contained much less food debris than those treated by stomaching. Conclusions: These results indicate that Spindle is a novel, effective alternative method for detaching micro‐organisms from food samples including four kinds of large‐size samples without the need for preprocessing. Significance and Impact of Study: The Spindle might be used to widely detaching micro‐organisms from all types of food samples for microbiological assay.     

Aspects for the future of biotransformations

Biotransformations have gained extensive importance in practical use as a support for chemical synthesis or in the conversion of natural products. Biotransformations may present an enlargement, a sequential degradation or a specific modification of synthetic or natural compounds. The tools for biotransformations are principally mammalian, plant or microbial cells and their cell-free enzymes. In technical practice the biocatalysts are so far limited to the use of microorganisms and some cell-free enzymes of low cost. Although numerous microbial or enzymatical reactions were already developed for industrial processes, the capacities of biotransformations offer a broad field of inexhaustible possibilities for the future.

     

Production of fine chemicals using biocatalysis

Presently, a large number of biotransformations are carried out on an industrial scale and are discussed in a fast increasing number of reviews. Besides this, a significant number of biotransformations have been investigated over the past year, from degrading to transforming and synthetic reactions. The development of more specific and stable biocatalysts, either isolated enzymes or whole cells, generated by the new methods of genetic engineering and improved by reaction engineering have led to new industrial biotransformations.     

Biotransformation of terpenes

The main application of terpenes as fragrances and flavors depends on the absolute configuration of the compounds because enantiomers present different organoleptic properties. Biotransformations allow the production of regio- and stereoselective compounds under mild conditions. These products may be labeled as "natural". Commercially useful chemical building-blocks and pharmaceutical stereo isomers can also be produced by bioconversion of terpenes. Enzymes and extracts from bacteria, cyanobacteria, yeasts, microalgae, fungi, plants, and animal cells have been used for the production and/or bioconversion of terpenes. In addition, whole cell catalysis has also been used. A variety of media and reactors have been assessed for these biotransformations and have produced encouraging results, as discussed in this review.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Status and future of disease protection and grape berry quality alteration by micro‐organisms in viticulture

Grapevine is one of the most widely grown fruit crops in the world. At present, however, there is much concern regarding chemical pollution in viticulture due to the application of chemical fungicides and fertilizers. One viticultural practice to resolve this issue is the application of micro‐organisms to grapevine as a substitute for chemicals. Some micro‐organisms act as an enhancer of grape berry quality as well as a suppresser of disease in grapevine through their antagonistic ability and/or systemic resistance inducing ability. Herein, we review current and prospective applications of micro‐organisms in viticulture.

Significance and Impact of the Study

In this review, we evaluate the applicability of micro‐organisms in viticulture. Micro‐organisms can improve grape berry quality through grapevine disease protection and grape berry quality alteration. Because the use of micro‐organisms to protect grapevine from plant diseases is safer than the use of chemical fungicides, the use of biofungicides in viticulture is expected to be enhanced by the increasing consumer concern towards chemical fungicides. Micro‐organisms also modify plant secondary metabolites for use as flavours, pharmaceuticals and food additives. Studies of micro‐organisms that promote polyphenol, anthocyanin and aroma compound biosynthesis are in progress with an eye to improving grape berry quality.     

Homogeneous biocatalysis in organic solvents and water-organic mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Relevance of microbial coculture fermentations in biotechnology

The purpose of this article is to review coculture fermentations in industrial biotechnology. Examples for the advantageous utilization of cocultures instead of single cultivations include the production of bulk chemicals, enzymes, food additives, antimicrobial substances and microbial fuel cells. Coculture fermentations may result in increased yield, improved control of product qualities and the possibility of utilizing cheaper substrates. Cocultivation of different micro‐organisms may also help to identify and develop new biotechnological substances. The relevance of coculture fermentations and the potential of improving existing processes as well as the production of new chemical compounds in industrial biotechnology are pointed out here by means of more than 35 examples.     

A class of plant glycosyltransferases involved in cellular homeostasis

Many small lipophilic compounds in living cells can be modified by glycosylation. These processes can regulate the bioactivity of the compounds, their intracellular location and their metabolism. The glycosyltransferases involved in biotransformations of small molecules have been grouped into Family 1 of the 69 families that are classified on the basis of substrate recognition and sequence relatedness. In plants, these transfer reactions generally use UDP-glucose with acceptors that include hormones such as auxins and cytokinins, secondary metabolites such as flavonoids, and foreign compounds including herbicides and pesticides. In mammalian organisms, UDP-glucuronic acid is typically used in the transfer reactions to endogenous acceptors, such as steroid and thyroid hormones, bile acids and retinoids, and to xenobiotics, including nonsteroidal anti-inflammatory drugs and dietary metabolites. There is widespread interest in this class of enzyme since they are known to function both in the regulation of cellular homeostasis and in detoxification pathways. This review outlines current knowledge of these glycosyltransferases drawing on information gained from studies of plant and mammalian enzymes.     

Biotransformation of constituents of essential oils by germinating wheat seed

Wheat seeds, when exposed to essential oils, are able to metabolise certain monoterpenes. The actual amounts of the compounds and their derivatives in the endosperm and embryo of wheat seeds, after exposure to the monoterpenes were determined. Neral and geranial, which are the constituents of citral, are reduced and oxidised to the corresponding alcohols and acids. Similarly citronellal, pulegone and carvacrol are converted partly to the corresponding reduction and oxidation products. The aromatic compound vanillin is partly reduced to vanillyl alcohol or oxidised to vanillic acid. In all cases it seems that part of the compounds applied are degraded, as indicated by the inability to account for all the compounds, which were supplied to the germinated seeds. In most cases the derivatives of the essential oil applied were less toxic than the parent compound. The possible role of non-specific enzymes by which the compounds are oxidised or reduced is discussed.     

Norovirus‐binding proteins recovered from activated sludge micro‐organisms with an affinity to a noroviral capsid peptide

Aims: Transmission routes of noroviruses, leading aetiological agents of acute gastroenteritis, are rarely verified when outbreaks occur. Because the destination of norovirus particles being firmly captured by micro‐organisms could be totally different from that of those particles moving freely, micro‐organisms with natural affinity ligands such as virus‐binding proteins would affect the fate of viruses in environment, if such microbial affinity ligands exist. The aim of this study is to identify norovirus‐binding proteins (NoVBPs) that are presumably working as natural ligands for norovirus particles in water environments. Methods and Results: NoVBPs were recovered from activated sludge micro‐organisms by an affinity chromatography technique in which a capsid peptide of norovirus genogroup II (GII) was immobilized. The recovered NoVBPs bind to norovirus‐like particles (NoVLPs) of norovirus GII, and this adsorption was stronger than that to NoVLPs of norovirus genogroup I. The profile of two‐dimensional electrophoresis of NoVBPs showed that the recovered NoVBPs included at least seven spots of protein. The determination of N‐terminal amino acid sequences of these NoVBPs revealed that hydrophobic interactions could contribute to the adsorption between NoVBPs and norovirus particles. Conclusions: NoVBPs conferring a high affinity to norovirus GII were successfully isolated from activated sludge micro‐organisms. Significance and Impact of the Study: NoVBPs could be natural viral ligands and play an important role in the NoV transmission.