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Screening and isolation of halophilic bacteria producing industrially important enzymes.

ABSTRACT: Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

SUBMITTER: Kumar S

PROVIDER: S-EPMC3769037 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Screening and isolation of halophilic bacteria producing industrially important enzymes.

Kumar Sumit S Karan Ram R Kapoor Sanjay S S P Singh S S K Khare K

Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] 20120601 4

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potentia ...[more]

PMID: 24031991

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Project description:This study catalogs production of industrially important enzymes and changes in transcript expression caused by 2-deoxy D-glucose (2-DG) treatment in Arthrinium malaysianum cultures. Carbon Catabolite Repression (CCR) induced by 2-DG in this species is cAMP independent unlike many other organisms. Higher levels of secreted endoglucanase (EG), β-glucosidase (BGL), β-xylosidase (BXL), and filter paper activity assay (FPase) enzymes under 2-DG treatment can be exploited for commercial purposes. An integrated RNA sequencing and quantitative proteomic analysis was performed to investigate the cellular response to 2-DG in A. malaysianum. Analysis of RNASeq data under 2-DG treated and control condition reveals that 56% of the unigenes do not have any known similarity to proteins in non-redundant database. Gene Ontology IDs were assigned to 36% of the transcripts (13260) and about 5207 (14%) were mapped to Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). About 1711 genes encoding 2691 transcripts were differentially expressed in treated vs. control samples. Out of the 2691 differentially expressed transcripts, only 582 have any known function. The most up regulated genes belonged to Pentose Phosphate Pathways and carbohydrate degradation class as expected. In addition, genes involved in protein folding, binding, catalytic activity, DNA repair, and secondary metabolites were up-regulated under 2-DG treatment. Whereas genes encoding glycosylation pathways, growth, nutrient reservoir activity was repressed. Gene ontology analysis of the differentially expressed genes indicates metabolic process (35%) is the pre-dominant class followed by carbohydrate degradation (11%), protein folding, and trafficking (6.2%) and transport (5.3%) classes. Unlike other organisms, conventional unfolded protein response (UPR) was not activated in either control or treated conditions. Major enzymes secreted by A. malaysianum are those degrading plant polysaccharides, the most dominant ones being β-glucosidase, as demonstrated by the 2D gel analysis. A set of 7 differentially expressed mRNAs were validated by qPCR. Transmission electron microscopy analyses demonstrated that the 2-DG treated cell walls of hyphae showed significant differences in the cell-wall thickness. Overall 2-DG treatment in A. malaysianum induced secretion of large amount of commercially viable enzymes compared to other known species.

Project description:BackgroundFluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied.ResultsFlow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability.ConclusionsThis study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.

Dataset Information

Screening and isolation of halophilic bacteria producing industrially important enzymes.

Publications

Screening and isolation of halophilic bacteria producing industrially important enzymes.

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