Screening Of Microorganisms Research Articles

Targeted selection of antagonistic microorganisms for control ofBotrytis cinereaof strawberry in New Zealand

A range of microorganisms, with previously identified antagonistic properties towards a variety of plant pathogens, were screened for their biological control activity against the foliar necrotroph Botrytis cinerea. On detached, senescing strawberry leaves the most successful isolate, Trichoderma atroviride LU132, was able to inhibit B. cinerea lesion development and significantly reduce sporulation compared to the control treatment. T. atroviride LU132 was further evaluated under field conditions and, over a 2-year period, on three different strawberry cultivars, was able to significantly suppress B. cinerea sporulation on leaves and flower parts giving a similar level of disease control as the fungicide fenhexamid. The mechanisms of action of T. atroviride LU132were postulated to be a combination of competition for sugars, production of non-volatile compounds and possible mycoparasitism. The strategic approach described in this paper demonstrates an efficient way of screening microorganisms for biocontrol activity and validates the notion that certain microorganisms can be antagonistic to a variety of plant pathogens.

Purification and Gene Cloning of α-Methylserine Aldolase from Ralstonia sp. Strain AJ110405 and Application of the Enzyme in the Synthesis of α-Methyl- l -Serine

By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.

Enrichment of new alkane oxidizing bacterial strains for human drug metabolite production

Abstract Human metabolites of active pharmaceutical ingredients (APIs) are of high interest for pharmacokinetic and toxicological studies. Moreover, some API metabolites are also under investigation as possible next generation drugs. However, often highly selective chemical reactions, e.g. stereo- and regioselective hydroxylations are required to enable the production of these compounds and biocatalysis offers the only feasible synthetic approach. Screening microorganisms to discover new catalytic activities is usually a time consuming task and the outcome is unpredictable. Aerobic alkane oxidizing bacteria depend on their ability to hydroxylate hydrocarbons to introduce such non-activated carbon sources to their central metabolic pathways. Hence, we expected that alkane metabolizers generally might also enable hydroxylation of other substrates, e.g. APIs. Based on this working hypothesis a collection of alkane metabolizing strains was established, roughly classified by 16S rDNA sequencing and screened for metabolite production of seven different pharmacological compounds. Several active strains were found, metabolizing dextromethorphan, harmine, phenacetin and tolbutamide. Overall, almost 30% of the new isolated strains showed activity with at least one of the 7 substrates.

Novel Dehydrogenase Catalyzes Oxidative Hydrolysis of Carbon-Nitrogen Double Bonds for Hydrazone Degradation

Hydrazines and their derivatives are versatile artificial and natural compounds that are metabolized by elusive biological systems. Here we identified microorganisms that assimilate hydrazones and isolated the yeast, Candida palmioleophila MK883. When cultured with adipic acid bis(ethylidene hydrazide) as the sole source of carbon, C. palmioleophila MK883 degraded hydrazones and accumulated adipic acid dihydrazide. Cytosolic NAD+- or NADP+-dependent hydrazone dehydrogenase (Hdh) activity was detectable under these conditions. The production of Hdh was inducible by adipic acid bis(ethylidene hydrazide) and the hydrazone, varelic acid ethylidene hydrazide, under the control of carbon catabolite repression. Purified Hdh oxidized and hydrated the C=N double bond of acetaldehyde hydrazones by reducing NAD+ or NADP+ to produce relevant hydrazides and acetate, the latter of which the yeast assimilated. The deduced amino acid sequence revealed that Hdh belongs to the aldehyde dehydrogenase (Aldh) superfamily. Kinetic and mutagenesis studies showed that Hdh formed a ternary complex with the substrates and that conserved Cys is essential for the activity. The mechanism of Hdh is similar to that of Aldh, except that it catalyzed oxidative hydrolysis of hydrazones that requires adding a water molecule to the reaction catalyzed by conventional Aldh. Surprisingly, both Hdh and Aldh from baker's yeast (Ald4p) catalyzed the Hdh reaction as well as aldehyde oxidation. Our findings are unique in that we discovered a biological mechanism for hydrazone utilization and a novel function of proteins in the Aldh family that act on C=N compounds.

Development of an efficient method for screening microorganisms by using symbiotic association between Nasutitermes takasagoensis and intestinal microorganisms

Screening method of microorganisms that utilized the symbiotic association between insect (Nasutitermes takasagoensis: Nt) and intestinal microorganisms was developed. The existence of desired microorganisms that grew by degrading difficult-to-degrade materials in the gut was detected using survivability of Nt as an indicator. The desired microorganisms were isolated from the survived Nt. It was thought that guts of Nt behave as continuous culture systems whereby microorganisms that cannot degrade diet components are washed out, whereas those that can degrade it are retained and concentrated in the gut. About 60% of Nt fed with phenol artificial diet (PAD) died within 7 days, while 4% of termites survived for 9 days. The structure of intestinal microorganisms of the survived Nt fed with PAD differed from the bacterial communities obtained from enrichment culture (which contained phenol) of wood-feeding Nt. Relatively high colonies (650-times) were detected in the gut of Nt fed on phenol artificial diet compared with those obtained when Nt was fed on wood. Seven denaturing gradient gel electrophoresis (DGGE) bands were detected from gut of wood-feeding Nt, whereas 11 DGGE-bands were detected from that of phenol-feeding Nt. Out of 11 DGGE-bands, 5 of them were sequenced, and bacterial species including phenol-degrading bacteria were identified.

Development of novel method for screening microorganisms using symbiotic association between insect (Coptotermes formosanus Shiraki) and intestinal microorganisms

It is becoming increasingly difficult to isolate novel useful microorganisms from the natural environment using conventional screening methods based on pure culture techniques. A novel method for screening microorganisms in symbiotic association with insects was developed. This method involves the following two steps. In the first step, the existence of desired microorganisms that grow well by degrading difficult-to-degrade materials in the gut of insects is detected using the survivability of insects as an indicator. In the second step, the desired microorganisms are selected from the surviving insects. The second step is based on an idea that the guts of insects act as continuous-culture systems whereby microorganisms that cannot degrade diet components are washed out whereas those that can degrade diet components are retained and made to multiply in the gut. Coptotermes formosanus Shiraki was fed with an artificial diet containing phenol as a model of lignin-derived and difficult-to-degrade compound. Each C. formosanus feeding on an artificial diet containing 100 mg/l phenol had different levels of adaptation to the toxicity of phenol. About 20% of C. formosanus fed with an artificial diet containing 100 mg/l phenol died within a few days whereas others survived for more than 10 d. The structure of the intestinal microorganisms of the surviving C. formosanus fed with the 100 mg/l phenol artificial diet gradually changed and was very different from that of the bacterial communities obtained from the enrichment culture of wood-feeding C. formosanus using an artificial medium containing phenol as a sole carbon source. Furthermore, Only three species (as DGGE band) were detected from the gut of wood-feeding C. formosanus, whereas 200 times more phenol-degrading microorganisms were detected in the gut of C. formosanus feeding on a phenol artificial diet. Out of these nine species (as DGGE band) of phenol-degrading microorganisms were isolated. The screening method developed in this study can also be applied to various insects, leading to the isolation of various microorganisms that can degrade difficult-to-degrade compounds.

Food-processing enzymes from recombinant microorganisms—a review

Enzymes are commonly used in food processing and in the production of food ingredients. Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian tissues are often not well-adapted to the conditions used in modern food production methods. The use of recombinant DNA technology has made it possible to manufacture novel enzymes suitable for specific food-processing conditions. Such enzymes may be discovered by screening microorganisms sampled from diverse environments or developed by modification of known enzymes using modern methods of protein engineering or molecular evolution. As a result, several important food-processing enzymes such as amylases and lipases with properties tailored to particular food applications have become available. Another important achievement is improvement of microbial production strains. For example, several microbial strains recently developed for enzyme production have been engineered to increase enzyme yield by deleting native genes encoding extracellular proteases. Moreover, certain fungal production strains have been modified to reduce or eliminate their potential for production of toxic secondary metabolites. In this article, we discuss the safety of microorganisms used as hosts for enzyme-encoding genes, the construction of recombinant production strains, and methods of improving enzyme properties. We also briefly describe the manufacture and safety assessment of enzyme preparations and summarize options for submitting information on enzyme preparations to the US Food and Drug Administration.

Utilization of n-alkanes by a newly isolated strain of Acinetobacter venetianus: the role of two AlkB-type alkane hydroxylases

A bacterial strain capable of utilizing n-alkanes with chain lengths ranging from decane (C10H22) to tetracontane (C40H82) as a sole carbon source was isolated using a system for screening microorganisms able to grow on paraffin (mixed long-chain n-alkanes). The isolate, identified according to its 16S rRNA sequence as Acinetobacter venetianus, was designated A. venetianus 6A2. Two DNA fragments encoding parts of AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, were polymerase chain reaction-amplified from the genome of A. venetianus 6A2. To study the roles of these two alkM paralogues in n-alkane utilization in A. venetianus 6A2, we constructed alkMa, alkMb, and alkMa/alkMb disruption mutants. Studies on the growth patterns of the disruption mutants using n-alkanes with different chain lengths as sole carbon source demonstrated central roles for the alkMa and alkMb genes in utilization of C10 to C18 n-alkanes. Comparative analysis of these patterns also suggested different substrate preferences for AlkMa and AlkMb in n-alkane utilization. Because both single and double mutants were able to grow on n-alkanes with chain lengths of C20 and longer, we concluded that yet another enzyme(s) for the utilization of these n-alkanes must exist in A. venetianus 6A2.

Enzymes de l’extrême et alimentation: Application des propriétés enzymatiques spécifiques aux procédés alimentaires

Les activités catalytiques des enzymes sont largement utilisées dans les procédés industriels de l’industrie agroalimentaire. Les applications de ces substances naturelles dans l’alimentation sont très diversifiées puisqu’elles peuvent être utilisées lors du procédé de fabrication des aliments pour, par exemple, améliorer leur digestibilité, mais aussi pour accélérer les transformations physicochimiques leur conférant des propriétés organoleptiques particulières. De nombreux compléments alimentaires « naturels » sont aujourd’hui fabriqués à l’aide d’enzymes, de même des protéines sont hydrolysées de façon enzymatique pour augmenter un pouvoir émulsifiant, pour développer les arômes ou pour donner une saveur particulière à un aliment. Ces procédés industriels ont été longtemps développés en fonction des enzymes disponibles. Aujourd’hui, les technologies les plus récentes de microbiologie et de biologie moléculaire permettent de mettre au point des enzymes adaptées à l’application visée. Les enzymes impliquées doivent pouvoir catalyser les réactions à des pH et des températures particuliers, résister aux conditions de formulation et présenter une très grande spécificité. La découverte et l’exploitation des microorganismes extrêmophiles a donné accès à une large diversité d’enzymes aux propriétés nouvelles. Protéus possède une collection unique de microorganismes et dispose du savoir-faire et des technologies propriétaires nécessaires pour les cribler et identifier puis isoler les enzymes d’intérêt. Par ailleurs, les technologies d’évolution dirigée de protéines permettent aujourd’hui d’affiner les propriétés des enzymes pour qu’elles soient parfaitement adaptées aux applications visées. Pour déplacer un optimum de pH ou de température ou pour augmenter une spécificité, la technologie de L-Shuffling™ brevetée par Protéus permet de mettre au point très rapidement de nombreuses nouvelles enzymes aux caractéristiques améliorées et parfaitement adaptées au process visé. La technologie de L-Shuffling™ permet d’ajuster aussi bien les caractéristiques physicochimiques de enzymes (telles que par exemple leur pH optimum, leur température optimale, ou leur thermostabilité), que leurs caractéristiques catalytiques (activité spécifique, spécificité de substrat, sensibilité aux inhibiteurs). The catalytic activities of the enzymes are widely used in industrial processes of feed and food industry. The applications of these natural substances in this industry are very diversified since they can be used during manufacturing process of food, for example to improve their digestibility, and also to accelerate the physicochemical transformations giving particular organoleptic properties. Many natural food complements are already manufactured using enzymes and proteins are enzymatically hydrolyzed in order to increase their emulsifying property, develop flavors or give a particular savor to food. Until recently, industrial processes developed were limited to those possible using the enzymes available. Today, state-of-the-art technologies in microbiology and in molecular biology enable the development of enzymes perfectly suited to the targeted application. Selected enzymes must be able to catalyze the reactions under specific pH and temperature conditions. They should resist to the conditions of formulation and have a very high specificity. The use of extremophilic micro-organisms has given access to a broad diversity of enzymes with new properties. Protéus has a unique collection of micro-organisms, and has developed a unique know-how and proprietary technologies to use these micro-organisms for screening and selecting the enzymes of interest. Additionally, technologies of directed evolution have made possible to refine the properties of the enzymes so that they perfectly fit with the targeted applications. The L-Shuffling™ technology, developed and patented by Protéus, is a dramatically efficient tool for adjusting both the physico-chemical characteristics of the enzymes (e.g. optimal pH or temperature, thermostability, etc.) and their catalytic properties (e.g. specific activities, substrate specificity, sensitivity to inhibitors, etc.). L-Shuffling™ therefore allows to quickly design new enzymes with improved characteristics and performances in the targeted process.

Plate assay for endo-β- N-acetylglucosaminidase activity using a chromogenic substrate synthesized by transglycosylation with Arthrobacter Endo-β- N-acetylglucosaminidase

The transglycosylation activity of Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A) was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-5-bromo-4-chloro-3-indolyl-beta-glucoside (Man6GlcNAc-Glc-beta-X). Various endo-beta-N-acetylglucosaminidases hydrolyzed this oligosaccharide, producing Man6GlcNAc and Glc-beta-X. The E. coli strains coexpressing Endo-A and beta-glucosidase formed blue colonies in the presence of Man6GlcNAc-Glc-beta-X. Therefore, endo-beta-N-acetylglucosaminidase activity could be directly detected by the plate assay. This simple plate assay is useful for screening microorganisms for endo-beta-N-acetylglucosaminidase activity.

Plate Assay for Endo-.BETA.-N-Acetylglucosaminidase Activity Using a Chromogenic Substrate Synthesized by Transglycosylation with Arthrobacter Endo-.BETA.-N-Acetylglucosaminidase.

The transglycosylation activity of Arthrobacter endo-β-N-acetylglucosaminidase (Endo-A) was used for the enzymatic synthesis of a novel oligosaccharide, Man6GlcNAc-5-bromo-4-chloro-3-indolyl-β-glucoside (Man6GlcNAc-Glc-β-X). Various endo-β-N-acetylglucosaminidases hydrolyzed this oligosaccharide, producing Man6GlcNAc and Glc-β-X. The E. coli strains coexpressing Endo-A and β-glucosidase formed blue colonies in the presence of Man6GlcNAc-Glc-β-X. Therefore, endo-β-N-acetylglucosaminidase activity could be directly detected by the plate assay. This simple plate assay is useful for screening microorganisms for endo-β-N-acetylglucosaminidase activity.

Microbial models of drug metabolism: microbial transformations of trimegestone® (RU27987), a 3-Keto-Δ4,9(10)-19-norsteroid drug

Screening microorganisms for the biotransformation of the 3-keto-delta(4,9(10))-19-norsteroid RU27987 (Trimegestone) resulted in the isolation of nine identified metabolites, some of them being selectively produced by different strains. Eight metabolites were found to be hydroxylated on various positions of the rings, and one was additionally epoxidized. These microbial metabolites could be used as chromatographic standards and two of them were found identical to the unknown major human metabolites. Moreover, most microbial metabolites were produced in sufficient amounts to be tested for their biological activities. All these features demonstrate the usefulness and versatility of microbial biotransformation systems as a tool for early identification and convenient production of potentially active mammalian and non-mammalian metabolites.

A second polycaprolactone depolymerase from Fusarium , a lipase distinct from cutinase

Polycaprolactone (PCL), a synthetic polyester with applications in biodegradable plastics, is degraded by a variety of microorganisms, including fungal phytopathogens. These pathogens secrete cutinase, which hydrolyzes cutin, the polyester structural component of plant cuticle, releasing ω-hydroxy fatty acids that induce cutinase synthesis. Our laboratory previously reported that growth of Fusarium solani on PCL requires cutinase, which is active as a PCL depolymerase and induced by the products of its action on PCL. A mutant strain of F. solani in which the cutinase gene is deleted was unable to grow on PCL and did not secrete PCL depolymerase activity in the media tested. It is now shown that this mutant produces a PCL depolymerase in media containing lipase inducers. Wild-type strains also produce this second PCL depolymerase, which is induced by Tween 80 and tributyrin, but not by PCL or cutin. The second depolymerase shows interfacial activation, indicating that it is a lipase. PCL may thus be a substrate but not an inducer of depolymerases that degrade it, and screening microorganisms on medium with PCL as the sole source of carbon and energy may fail to reveal strains with active PCL depolymerases, because of the absence of an inducer. Surprisingly, Tween 80 induces both cutinase and lipase activities in wild-type F. solani.

A Rapid Microplate Callus Bioassay for Assessment of Rhizobacteria for Biocontrol of Leafy Spurge ( Euphorbia esula L.)

Screening large collections of microorganisms for potential biological control activity on economically important weeds is often diY cult. Seeds required as indicator species are often not available in large supply and are highly variable in viability and germination, resulting in uneven seedling development. A bioassay system for rapidly assessing the phytotoxic eVects of rhizobacteria was developed based on leafy spurge ( Euphorbia esula L. ) callus tissue culture in multiple-well plates. Callus pieces (0.5 g) were placed in 24-well plates containing Gamborg's B5 medium, inoculated with rhizobacterial suspension and incubated at 27oC for 48 h. By rating inoculated callus for cellular damage, about 30% of rhizobacteria isolated from weedy Euphorbia spp. collected in Europe and North America were identified as being highly phytotoxic. Symptoms of phytotoxicity included growth reduction, discoloration and extensive cellular leakage. A high proportion of isolates found to be phytotoxic in tissue culture bioassays were similarly eVective in standard leafy spurge seedling bioassays. The method is rapid, host specific and more uniform compared with assays using seedlings, and should be adaptable to other weed species for screening microorganisms for potential biocontrol activity.

Rules for bioengineered microorganisms issued

The Environmental Protection Agency has proposed a long-awaited rule that would, for the first time, define how it will regulate genetically modified organisms under the Toxic Substances Control Act (TSCA). The agency also has issued a final rule governing field testing of microbial pesticides developed by genetic modification. In a rare moment of unanimity, environmental and industry groups applauded both actions.The proposed rule formalizes EPA's current system for screening microorganisms. It defines as new chemical substances, subject to TSCA notification requirements, microorganisms formed by combining genetic materials from organisms in different genera. The rule requires persons intending to manufacture, import, or process intergeneric organisms for commercial purposes to file a Microbial Commercial Activity Notice (MCAN) with EPA at least 90 days before undertaking such activity. Introduction of such organisms into the environment for commercial R&D purposes requires 60 days' advance notific...

Screening microorganisms for utilization of furfural and possible intermediates in its degradative pathway

A variety of microorganisms were screened for their ability to utilize furfural and possible intermediates in its degradative pathway. Compounds tested included furfural, furfuryl alcohol, 2-furoic acid, tetrahydrofurfuryl alcohol, di- and tetra-hydrofurans, 1,5-pentanediol and 1,4-butanediol. The objective of the research was to determine whether microbial strains might be found that degraded furfuryl via diols. If so, then it might be possible to block the metabolic pathway at the appropriate point allowing accumulation of diol(s). A promising bacterial strain was identified that could utilize furfural, some of its derivatives and diols, which is a candidate for further studies.

Fatty acid production characteristics of fungi with particular emphasis on gamma linolenic acid production

The fatty acid production characteristics of fungi are described. These characteristics are the relationship between the oil content of the cell and the fatty acid content of the oil. For example, for polyunsaturated fatty acid (PUFA) production by Mucor hiemalis IPD 51, the oil content of the cell and the GLA content of the oil are coupled. For fungal production of some PUFA, synthesized after the rate-limiting step in the fatty acid anabolic chain, a maximum fatty acid production model was developed to link the fatty acid content of the oil and the oil content of the cell. Maximum volumetric productivity of gamma linolenic acid (GLA) by molds was found to occur at a specific GLA content of the oil. For example, for M. hiemalis IPD 51, a maximum volumetric of 4.7 mg GLA/L . h was produced at a GLA content of the oil of 8% to 10%. Similarly for Mucor circinelloides v. Tieghem IPD 155 a maximum volumetric productivity of 4.8 mg GLA/L . h was produced at a GLA content of the oil of 14% to 16%. These results imply that, when screening microorganisms for GLA or other fatty acid production, a number of medium compositions need to be evaluated to determine the tradeoff between oil content of the cell and fatty acid content of the oil.

Evolution of beta-lactamase inhibitors.

The production of beta-lactamase is the most important mechanism of bacterial resistance to beta-lactam antibiotics. Attempts to find an inhibitor of beta-lactamase were made as early as the 1940s and 1950s but without success. In the early 1950s, it was found that certain semisynthetic penicillins could function as beta-lactamase inhibitors, but none found a clinical place in this capacity. A program of screening microorganisms for the production of naturally occurring inhibitors was begun in 1967. This process led to the discovery of the olivanic acids and clavulanic acid. Clavulanic acid, formulated with amoxicillin and later with ticarcillin, became available for clinical use in 1981. Since the introduction of clavulanic acid, other beta-lactamase inhibitors have been developed, including sulbactam and tazobactam. It remains to be seen whether these will have any advantage over clavulanate for clinical use.

Isolation and some cultural conditions of Streptomyces species which produce enzymes lysing Aspergillus niger cell wall

Abstract As the result of screening microorganisms having lytic activity towards Aspergillus niger cell wall, two Streptomyces strains were isolated. One, termed J-28, was identified as Streptomyces cinereoruber and the other, termed J-13-3, was an unknown species belonging to Streptomyces . These strains inducibly produced chitinase and α-glucanase in a medium containing A. niger cell wall; specifically, strain J-28 chiefly produced chitinase and strain J-13-3 mainly α-glucanase. The best medium for chitinase production by the former was 0.5% A. niger cell wall, 0.5% yeast extract, 0.2% K 2 HPO 4 , 0.1% MgSO 4 ·7H 2 O and 0.01% FeSO 4 ·7H 2 O, while for α-glucanase production by the latter it was the same medium except for 1% instead of 0.5% A. niger cell wall, both in shaking cultures. Reaction products in chitin hydrolysis with chitinase were N , N ′-diacetyl chitobiose and chitin oligomers and those of nigeran with α-glucanase were maltose and nigeran oligomers.

4882273 Method of screening microorganisms for the production of extracellular enzymes: Robert O Horwath assigned to Nabisco Brands Inc

4882273 Method of screening microorganisms for the production of extracellular enzymes: Robert O Horwath assigned to Nabisco Brands Inc