Molecular characterization of a lipase-producing <Emphasis Type="Italic">Bacillus pumilus</Emphasis> strain (NMSN-1d) utilizing colloidal water-dispersible polyurethane

Bacillus pumilus strain NMSN-1d isolated from polyurethane-contaminated water was found to grow in high salt concentration (NaCl 10%, w/v) and degrade Impranil-DLN, water-dispersible polyurethane. The genetic relatedness of the isolate has been established by standard molecular biological techniques and the enzyme(s) involved in polyurethane degradation were also studied. A total of nine bacterial strains were isolated from polyurethane-polluted sites and characterized by conventional, microbiological and biochemical methods. These isolates were subjected to 16S ribosomal RNA gene amplification by PCR using specific primers. The genetic relatedness of the isolates was also ascertained by ribotyping and BLAST analysis of the 16S ribosomal RNA gene sequences. The bacterial isolates were grown in yeast extract-salts minimal broth medium supplemented with water-dispersible polyurethane (Impranil DLN) as a sole source of carbon. The promising isolate utilizing polyurethane and producing lipase was identified as Bacillus pumilus NMSN-1d. The polyurethane degradation has been studied in polyurethane-Rhodamine-B and Luria-Bertani-polyurethane plate assays. The activity of hydrolytic enzymes such as lipase and esterase was confirmed on 2xYT-olive oil and tributyrin-Tween 20 plate assay. The newly isolated Bacillus pumilus appears promising in the management of polyurethane waste and in production of industrially important enzymes.     

Anti-pathogenic Potential of Coral Associated Bacteria Isolated from Gulf of Mannar Against Pseudomonas aeruginosa

Infections of Pseudomonas aeruginosa are of great concern because of its increasing resistance towards conventional antibiotics. Quorum sensing system of P. aeruginosa acts as a global regulator of almost all the virulence factors and majorly its biofilm formation. In the present study, quenching of QS system of P. aeruginosa has been explained with bioactives from bacteria associated with the coral Acropora digitifera. Isolated bioactives inhibited the expression of various virulence traits of P. aeruginosa like biofilm formation, and the production of extracellular enzymes like protease and elastase. This study also emphasises the potential of coral associated bacteria in producing bioactive agents with anti-pathogenic properties.     

Effect of pH and Temperature on Enzyme Activity of Chitosanase Produced Under Solid Stated Fermentation by <Emphasis Type="Italic">Trichoderma</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

Trichoderma strains were extensively studied as biocontrol agents due to their ability of producing hydrolytic enzymes, which are considered key enzymes because they attack the insect exoskeleton allowing the fungi infection. The present work aimed to evaluate the ability of chitosanase production by four Trichoderma strains (T. harzianum, T. koningii, T. viride and T. polysporum) under solid stated fermentation and to evaluate the effect of pH and temperature on enzyme activity. pH strongly affected the enzyme activity from all tested strains. Chitosanase from T. harzianum and T. viride presented optimum activity at pH 5.0 and chitosanase from T. koningii and T. polysporum presented optimum activity at pH 5.5. Temperature in the range of 40–50°C did not affect enzyme activity. T. polysporum was found as the most promising strain to produce chitosanase with maximal enzyme activity of about 1.4 IU/gds, followed by T. viride (~1.2 IU/gds) and T. harzianum (1.06 IU/gds).     

Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake,Iran

Screening of bacteria from different areas of Howz Soltan playa, a hypersaline lake in the central desert zone of Iran, led to the isolation of 231 moderately halophilic bacteria, which were able to grow optimally in media with 5–15% of salt, and 49 extremely halophilic microorganisms that required 20–25% of salt for optimal growth. These isolates produced a great variety of extracellular hydrolytic enzymes. A total of 195, 177, 100, 95, 92, 68, 65, 33, and 28 strains produced lipases, amylases, proteases, inulinases, xylanases, cellulases, pullulanases, DNases, and pectinases, respectively. In comparison with gram-negative bacteria, the gram-positive halophilic rods, showed more hydrolytic activities. Several combined activities were showed by some of these isolates. One strain presented 9 hydrolytic activities, 4 strains presented 8 hydrolytic activities, 10 strains presented 7 hydrolytic activities and 29 strains presented 6 hydrolytic activities. No halophilic isolate without hydrolytic activity has been found in this study. According to their phenotypic characteristics and comparative partial 16S rRNA sequence analysis, the halophilic strains were identified as members of the genera: Salicola, Halovibrio, Halomonas, Oceanobacillus, Thalassobacillus, Halobacillus, Virgibacillus, Gracilibacillus, Salinicoccus, and Piscibacillus. Most lipase and DNase producers were members of the genera Gracilibacillus and Halomonas, respectively, whereas most of the isolates able to produce hydrolytic enzymes such as amylase, protease, cellulose (CMCase) and inulinase, belonged to gram-positive genera, like Gracilibacillus, Thalassobacillus, Virgibacillus, and Halobacillus.     

Clarification of microbial polysaccharides with enzymes secreted fromLysobacter species

Summary Different species of Gram-negativeLysobacter grown in liquid culture secrete a variety of hydrolytic enzymes. The natural enzyme mixtures lyse several Gram-negative polysaccharide-producing bacteria of commercial importance, especiallyXanthomonas campestris. We describe optimal methods for producing and using the lytic enzymes to clarify microbial polysaccharides and show that the proteases are responsible for lysingX. campestris.     

Enumeration of gut associated extracellular enzyme‐producing yeasts in some freshwater fishes

Extracellular enzyme‐producing yeasts might be involved in the supplementation of enzymes within the gastrointestinal tract of fish. The present study was intended to detect yeasts in the intestine of three Indian major carps (Labeo rohita, Catla catla, Cirrhinus mrigala), three exotic carps (Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio), as well as Nile tilapia (Oreochromis niloticus), and to identify the most promising extracellular enzyme‐producing (e.g. amylase, protease, lipase, cellulase, xylanase and phytase) yeast strains by 18S rDNA sequence analysis. Selected for qualitative enzyme assay were 121 yeast strains, from which 28 were further studied for quantitative enzyme assay. The strain CMH6A isolated from C. mrigala exhibited the best extracellular enzyme activities except for amylase and cellulase. The strain ONF19B isolated from O. niloticus was noted as the best extracellular enzyme producer among the strains that produced all of the extracellular enzymes studied. Sequencing of the 18S rDNA fragment followed by nucleotide blast in the National Centre for Biotechnology Information (NCBI) GenBank revealed that strains CMH6A and ONF19B were similar to Pichia kudriavzevii (Accession no. KF479403 ) and Candida rugosa (Accession no. KF479404 ), respectively. The test of antagonism (in vitro) revealed that the isolated yeasts could not affect the growth of the autochthonous gut bacteria. This might indicate likely co‐existence of autochthonous yeasts and bacteria in the fish gut. Further research is necessary to explore the possibilities of utilizing the extracellular enzyme‐producing yeasts detected in the present study for commercial aquaculture.     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an α-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50°C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5–3 M NaCl. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A First Record of Obligate Halophilic Aspergilli from the Dead Sea

The isolation of obligate halophilic aspergilli from the Dead Sea and the range of salt tolerance of halophilic fungi isolated, are reported here for the first time. The mycobiota of the Dead Sea isolated in this study, was dominated by Aspergillus and Penicillium species; Cladosporium were found in lesser numbers. All three genera were obtained from the water sample; however, Aspergillus was the only genus obtained from the sediment. There was significant difference in growth of each isolate at different salt concentrations and intraspecies analysis revealed dissimilarity in response of strains to different salt concentrations in the growth medium The isolates were euryhaline, with halotolerance up to 20–25% solar salt, Aspergillus and Penicillium species showing a higher level of halotolerance, as compared to that of Cladosporium. Halophilic fungi were found in greater numbers in the sediment sample as compared to that in the water sample. Penicillium and Cladosporium species were exclusively facultative halophiles, while some species of Aspergillus were facultative halophiles. All the obligate halophiles isolated, belonged to the genus Aspergillus and were identified as A. penicillioides and A unguis, the latter being a first record of the species from the Dead Sea.     

Microbial metabolomics: essential definitions and the importance of cultivation conditions for utilizing Bacillus species as bionematicides

Root-knot nematodes are destructive phytopathogens that damage agricultural crops globally, and there is growing interest in the use of biocontrol based on rhizobacteria such as Bacillus to combat Meloidogyne species. It is hypothesized that nematicidal activity of Bacillus can be attributed to the production of secondary metabolites and hydrolytic enzymes. Yet, few studies have characterized these metabolites and their identities remain unknown. Others are speculative or fail to elaborate on how secondary metabolites were detected or distinguished from primary metabolites. Metabolites can be classified based on their origin as either intracellular or extracellular and based on their function, as either primary or secondary. Although this classification is in general use, the boundaries are not always well defined. An understanding of the secondary metabolite and hydrolytic enzyme classification of Bacillus species will facilitate investigations aimed at bionematicide development. This review summarizes the significance of Bacillus hydrolytic enzymes and secondary metabolites in bionematicide research and provides an overview of known classifications. The importance of appropriate cultivation conditions for optimum metabolite and enzyme production is also discussed. Finally, the use of metabolomics for the detection and identification of nematicidal compounds is considered.     

Enzyme mechanisms involved in cellulose hydrolysis by the rot fungus Sporotrichum pulverulentum

This article presents a review of the enzyme mechanisms involved in degradation of cellulose by the white-rot fungus Sporotrichum poulverulentum. The hydrolytic enzymes involved include: (1) five endo-1,4-β-glucanases; (2) one exo-1,4-β-glucanase, and (3) one or several 1,4-β-glucosidases. A recently discovered oxidative enzyme of importance in in vitro cellulose degradation seems to be a cellobiose oxidase. An oxidoreductase, cellobiose:quinone oxidoreductase, is of importance both in cellulose and in lignin degradation. Regulatory mechanisms of the extracellular enzyme activities, such as monosugar levels causing catabolite repression of the endoglucanases, have also been investigated. The enzymes used by S. pulverulentum in cellulose hydrolysis are compared to those used by Trichoderma viride. Very similar types of enzymes are used in both cases. However, no oxidative enzyme has so far been found to be involved in extracellular cellulose degradation in the case of T. viride. Recommendations for further research are given.     

Characterization of esterase activity from an Acetomicrobium hydrogeniformans enzyme with high structural stability in extreme conditions

The biotechnological and industrial uses of thermostable and organic solvent-tolerant enzymes are extensive and the investigation of such enzymes from microbiota present in oil reservoirs is a promising approach. Searching sequence databases for esterases from such microbiota, we have identified in silico a potentially secreted esterase from Acetomicrobium hydrogeniformans, named AhEst. The recombinant enzyme was produced in E. coli to be used in biochemical and biophysical characterization studies. AhEst presented hydrolytic activity on short-acyl-chain p-nitrophenyl ester substrates. AhEst activity was high and stable in temperatures up to 75 °C. Interestingly, high salt concentration induced a significant increase of catalytic activity. AhEst still retained ~ 50% of its activity in 30% concentration of several organic solvents. Synchrotron radiation circular dichroism and fluorescence spectroscopies confirmed that AhEst displays high structural stability in extreme conditions of temperature, salinity, and organic solvents. The enzyme is a good emulsifier agent and is able to partially reverse the wettability of an oil-wet carbonate substrate, making it of potential interest for use in enhanced oil recovery. All the traits observed in AhEst make it an interesting candidate for many industrial applications, such as those in which a significant hydrolytic activity at high temperatures is required.

     

Ecophysiological Variabilities in Ectohydrolytic Enzyme Activities of Some <Emphasis Type="Italic">Pseudoalteromonas</Emphasis> Species, <Emphasis Type="Italic">P. citrea,P. issachenkonii</Emphasis>, and <Emphasis Type="Italic">P. nigrifaciens</Emphasis>

The ecophysiological variabilities in the ectohydrolytic enzyme profiles of the three species of Pseudoalteromonas, P. citrea, P. issachenkonii, and P. nigrifaciens, have been investigated. Forty-one bacteria isolated from several invertebrates, macroalgae, sea grass, and the surrounding water exhibited different patterns of hydrolytic enzyme activities measured as the hydrolysis of either native biopolymers or fluorogenic substrates. The activities of the following enzymes were assayed: proteinase, tyrosinase, lipase, amylase, chitinase, agarase, fucoidan hydrolase, laminaranase, alginase, pustulanase, cellulase, β-glucosidase, α- and β-galactosidases, β-N-acetylglucosaminidase, β-glucosaminidase, β-xylosidase, and α-mannosidase. The occurrence and cell-specific activities of all enzymes varied over a broad range (from 0 to 44 μmol EU per hour) and depended not only on taxonomic affiliation of the strain, but also on the source/place of its isolation. This suggests ‘specialization’ of different species for different types of polymeric substrates as, for example, all strains of P. citrea and P. issachenkonii hydrolyzed alginate and laminaran, while strains of P. nigrifaciens were lacking the ability to hydrolyze most of the algal polysaccharides. The incidence of certain enzymes such as fucoidan hydrolases, alginate lyases, agarases, and α-galactosidases might be strain specific and reflect its particular ecological habitat. Received: 15 February 2002 / Accepted: 27 March 2002     

Identification and characterization of H10 enzymes isolated from <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">cereus</Emphasis> H10 with keratinolytic and proteolytic activities

Two types of extracellular proteases with molecular mass of 50.0 and 44.8 kDa were found in H10 enzymes partially purified from Bacillus cereus H10. Further identification using liquid chromatography-tandem mass spectrometry, the enzyme with 50.0 kDa was identified as being similar to leucine dehydrogenase; while the enzyme with 44.8 kDa might be a novel keratinolytic enzyme with little similarity to other proteins. To maximize the keratinolytic and proteolytic abilities in the H10 enzymes, a combination of response surface methodology and sequential quadratic programming technique was used to study the hydrolytic pH and temperature. Results showed that the H10 enzymes could produce optimal proteolytic and keratinolytic activities at a hydrolysis temperature of 59°C at pH 7.57. Testing the protease activity on various protein substrates and temperatures indicated that the H10 enzymes showed high thermal stability and were very effective in porcine hair.     

伊犁河流域春季可培养细菌多样性及其胞外酶检测

利用可培养法对新疆伊犁河流域水体和沉积物中细菌多样性进行分析，以期初步阐明流域河流可培养细菌群落结构。采用5种琼脂培养基分离纯化可培养细菌，依据其16S rRNA基因序列进行系统发育分析，并运用平板法对纯化菌株的胞外酶产生情况进行检测。序列分析结果表明，225株细菌分别属于变形菌门γ亚群(Gamma-pseudomonadota, 56.44%)、放线菌门(Actinomycetota, 18.22%)、厚壁菌门(Firmicutes, 14.22%)、变形菌门α亚群(Alpha-pseudomonadota, 4.89%)、变形菌门β亚群(Beta-pseudomonadota, 4%)、拟杆菌门(Bacteroidota, 0.44%)和异常球菌-栖热菌门(Deinococcota, 0.44%)等7个大的系统发育类群，41个属84个种。其中假单胞菌属(Pseudomonas,42.22%)、不动杆菌属(Acinetobacter,9.33%)和芽胞杆菌属(Bacillus,9.33%)为优势菌属。菌种分布结果显示，伊犁河流域主要支流和干流中可培养细菌地域分布性强。分离菌株产胞外酶...     

Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases

Organic co-solvents can expand the use of enzymes in lignocellulose deconstruction through making substrates more soluble and thus more accessible. In choosing the most adequate co-solvent for feruloyl esterases, hydrolysis of methyl p-hydroxycinnamates by three pure enzymes (and a multi-enzyme preparation) was evaluated. Low concentrations of dimethylsulfoxide (DMSO) enhanced hydrolysis by two of the enzymes while at levels >20%, activity was reduced. DMSO also enhanced acetyl esterase-type activity of the enzymes. The co-solvent effect was different for each enzyme-substrate couple, indicating that other factors are also involved. Kinetic studies with a Talaromyces stipitatus feruloyl esterase showed low concentrations of dimethylsulfoxide enhanced the hydrolytic rate while K_m also increased. Moreover, long-term incubation (96 h) of an Aspergillus niger feruloyl esterase in dimethylsulfoxide:water provided to the enzyme the ability to hydrolyze methyl p-coumarate, suggesting an active-site re-arrangement. Dimethylsulfoxide (10-30%) is proposed as an adequate co-solvent for feruloyl esterase treatment of water-insoluble substrates.     

Is there competition among ciliates and nematodes?

1. Biotic interaction between the ciliate Cyclidium glaucoma and the nematode Caenorhabditis elegans was investigated by manipulating the densities of the organisms in microcosms with and without sediment. 2. After 11 days the abundance of ciliates, nematodes and bacteria as well as extracellular enzyme activity were determined. Ciliates had a negative effect on nematode abundance in microcosms without sediment and in microcosms with sandy sediment, whereas in muddy sediment the effect was less distinctive. An effect of nematodes on ciliates was not observed. 3. The common resource bacteria were not affected negatively by the activity of the grazers. Overall grazer biomass increased with the addition of sediment to the microcosms, suggesting a rise of the carrying capacity in the experimental system. Especially in muddy sediment the abundance of bacteria and extracellular enzyme activity was higher compared to the microcosms without sediment. 4. The results of the experiment suggest a strong interspecific competition between nematodes and ciliates, where nematodes are, at least temporary, strongly affected.     

Interactions between Pseudomonas putida UW4 and Gigaspora rosea BEG9 and their consequences for the growth of cucumber under salt-stress conditions

Aims: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1‐aminocyclopropane‐1‐carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. Methods and Results: In the first experiment, cucumber seedlings were exposed to 0·1, 50, 100 or 200 mmol l^?1 NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l^?1 NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l^?1 salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase‐producing strain Pseudomonas putida UW4 (AcdS⁺), its mutant unable to produce the enzyme (AcdS^?), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l^?1 salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro‐organism on the photosynthetic efficiency were evaluated. The AcdS⁺ strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. Conclusions: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase‐producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. Significance and Impact of the Study: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS⁺ increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.     

Extracellular hydrolytic enzyme screening of culturable heterotrophic bacteria from deep-sea sediments of the Southern Okinawa Trough

The Southern Okinawa Trough is an area of focused sedimentation due to particulate matter export from the shelf of the East China Sea and the island of Taiwan. In order to understand the geomicrobiological characteristics of this unique sedimentary environment, bacterial cultivations were carried out for an 8.61 m CASQ core sediment sample. A total of 98 heterotrophic bacterial isolates were characterized based on 16S rRNA gene phylogenetic analysis. These isolates can be grouped into four bacterial divisions, including 13 genera and more than 20 species. Bacteria of the γ-Proteobacteria lineage, especially those from the Halomonas (27 isolates) and Psychrobacter (20 isolates) groups, dominate in the culturable bacteria assemblage. They also have the broadest distribution along the depth of the sediment. More than 72.4% of the isolates showed extracellular hydrolytic enzyme activities, such as amylases, proteases, lipases and Dnases, and nearly 59.2% were cold-adapted exoenzyme-producers. Several Halomonas strains show almost all the tested hydrolases activities. The wide distribution of exoenzyme activities in the isolates may indicate their important ecological role of element biogeochemical cycling in the studied deep-sea sedimentary environment.