Production Of Cold-active Enzymes Research Articles

A cold-active esterase enhances mesophilic properties through Mn2+ binding.

A key aspect of adaptation to cold environments is the production of cold-active enzymes by psychrophilic organisms. These enzymes not only have high activity at low temperatures, but also exhibit remarkable structural flexibility and thermolability. In this context, the role of metal ions has been little explored, and the few available studies seem to suggest that metal binding counteracts structural flexibility. This article reports an investigation into the role of the binding of manganese ion (Mn2+ ) in the thermal adaptation of an esterase (M-Est) of the GDSx family, identified in the genome of the Antarctic bacterium Marinomonas sp. ef1. M-Est is specific for esters containing acetate groups and turned out to be a highly thermolabile cold-active enzyme, with a catalysis optimum temperature of 5 °C and a melting temperature of 31.7 °C. A combination of biochemical and computational analyses, including molecular dynamics simulations, revealed that M-Est binds Mn2+ ions via a single binding site located on the surface of the enzyme, close to the active site. Although the interaction between M-Est and Mn2+ induces only local conformational changes involving the active site, quite surprisingly they trigger an improvement in both thermal stability and catalytic efficiency under mild temperature conditions. These results, together with the conservation of the Mn2+ binding site among psychrophilic and psychrotolerant homologues, suggest that Mn2+ binding may be a useful, albeit atypical, strategy to mitigate the detrimental effects of temperature on true cold-active enzymes.

A Comprehensive Investigation of Potential Novel Marine Psychrotolerant Actinomycetes sp. Isolated from the Bay-of-Bengal.

Background: This study was carried out to classify the diversity of the deep marine psychrotolerant actinomycetes sp. nov., in the Bay of Bengal and exploit the production of cold-active industrial and pharmaceutical biomolecules.Objective: 1) Characterization, optimum the growth conditions and classify the diversity of the novel isolated deep marine psychrotolerant actinomycetes sp from the Bay-of-Bengal. 2) Screening for industrially important biocatalysts and determine the antimicrobial activities against the five dreadful pathogens. 3) The differential expression profiling of the candidate genes to regulate the biosynthesis of selected enzymes.Methods: The cold-adapted actinomycetes were isolated from the deep marine water collections at 1200 mts below the surface in Bay-of-Bengal. The phenotypic and genotypic characterizations have been carried out to understand the persistent diversity of this novel marine psychrotolerant actinomycetes species. The production of cold-active enzymes, such as amylase, cellulase, lipase, pectinase, and L-asparaginase, were screened and the expression profiling genes were determined by using qRT PCR. The antibacterial and antifungal activities have also been investigated.Results: A total number of 37 novel actinomycetes were isolated and the phenotypic and genotypic characterizations identified the genus, dominated by Streptomyces (17 distinct sub-groups) as the major group, followed by Micromonospora, Actinopolyspora, Actinosynnema, Streptoverticillium, Saccharopolyspora, Nocardiopsis, and Nocardia. The optimum growth and abundant mycelium formation are observed at 15°C to 20°C and also capability for thriving at 4°C. All the isolates exhibited a significant role in the production of biocatalysts, and the antagonistic activities were also noted against five major selected pathogens.Conclusion: The Streptomyces from the Bay-of-Bengal have high biosynthetic potential and can serve as a good resource for the exploration of bioactive natural products.

Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes

BackgroundHeterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes.ResultsWe tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difficult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion.ConclusionsOur results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

Draft Genome Sequence of Permafrost Bacterium Nesterenkonia sp. Strain PF2B19, Revealing a Cold Adaptation Strategy and Diverse Biotechnological Potential.

ABSTRACTNesterenkonia sp. strain PF2B19, a psychrophilic bacterium, was isolated from 44,800-year-old permafrost. The draft genome sequence of this strain revealed the presence of genes involved in the production of cold active enzymes, carotenoid biosynthesis, fatty acid biosynthesis, and resistance to heavy metals. These results show the immense potential of the strain.

EXTRACELLULAR COLD ACTIVE ENDOGLUCANASE AND PIGMENT PRODUCING PSYCHROTOLERANT PENICILLIUM PINOPHILUM

<p><strong>Objective: </strong>The objective of the present study was on <em>Penicillium pinophilum </em>strain F2 from soil samples of Jammu city having the potentiality to produce alkaline cold active endoglucanase and pigment.</p><p><strong>Methods: </strong><em>Penicillium pinophilum </em>strain F2,<em> </em>a<em> </em>psychrotolerant micro-fungus was isolated from soil of Jammu city, India by taking Czapek’s Dox agar incubated at 15 °C. The strain was screened for production of cold active enzymes by taking various substrates at 15 °C. Final production was done for cold active endoglucanase by using sugarcane bagasse and ground nut shell as substrates. Besides, the strain was also able to produce red color pigment at a low temperature which was further studied to optimize its production by changing pH and growth medium. The produced pigment was used for dyeing of wool and silk, and absorption percentages were also calculated.</p><p><strong>Results: </strong>Screening for the production of cold active enzymes revealed it as a good producer of cellulose followed by lipase and amylase. Endoglucanase production revealed the total enzyme titer (total enzyme activity) was found to be 5.032 folds higher in sugarcane bagasse (38.91 units) than groundnut shell (7.732 units). Endoglucanase activity was maximum 9.82±0.33 units/ml and 2.29±0.31 units/ml after 120 h of incubation at 15 °C by sugarcane bagasse and groundnut shells, respectively. Red color pigment production was maxima at pH 5 in Czapek’s Dox broth. Maximum absorption percentage was seen by the treatment soaked with mordant, i.e. 5% CuSO<sub>4 </sub>(51.52%) and without a mordant, it showed about 45.54%.</p><p><strong>Conclusion: </strong>Due to the above unique features and capability to produce cold active endoglucanase and pigment by strain F2, can be used significantly in various industries.</p>

Psychrophilic and psychrotrophic fungi: a comprehensive review

This article reviews the comparative diversity of psychrophilic and psychrotrophic fungi, their adaptability mechanisms for survival and potential applications in biotechnology and pharmaceuticals. Fungi are able to grow and survive at low temperature and exist widely in polar and non-polar habitats. These cold regions are known for very low temperature, high ultra violet-B radiation, frequent freeze and thaw cycles and low water and nutrient availability. Most of the fungi adapt to such harsh conditions by evolving various strategies in their metabolism and physiology. Psychrophilic and psychrotrophic fungi are of importance in biotechnological and pharmaceutical fields due to their diverse characteristics developed or evolved due to their adaptation and survival in extreme environments, like; production of cold-active enzymes, pharmaceutical or bioactive metabolites and exo-polysaccharides, have potential for bioremediation and can also be used as biofertilizer.

Biodiversity and physiological characteristics of Antarctic and Arctic lichens-associated bacteria

The diversity and physiological characteristics of culturable bacteria associated with lichens from different habitats of the Arctic and Antarctica were investigated. The 68 retrieved isolates could be grouped on the basis of their 16S rRNA gene sequences into 26 phylotypes affiliated with the phyla Actinobacteria, Bacteroidetes, Deinococcus-Thermus, and Firmicutes and with the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Isolates belonging to the Alphaproteobacteria were the most abundant, followed by those belonging to Actinobacteria, Betaproteobacteria, Gammaproteobacteria, Bacteroidetes, Firmicutes, and Deinococcus-Thermus. Phylogenetic analysis showed that approximately 21 % of the total isolates represented a potentially novel species or genus (≤97 % sequence similarity). Strains belonging to the genera Sphingomonas, Frondihabitans, Hymenobacter, and Burkholderia were recovered from lichen samples from both geographic locations, implying common and important bacterial functions within lichens. Extracellular protease activities were detected in six isolates, affiliated with Burkholderia, Frondihabitans, Hymenobacter, Pseudomonas, and Rhodanobacter. Extracellular lipase activities were detected in 37 isolates of the genera Burkholderia, Deinococcus, Frondihabitans, Pseudomonas, Rhodanobacter, Sphingomonas, and Subtercola. This is the first report on the culturable bacterial diversity present within lichens from Arctic and Antarctica and the isolates described herein are valuable resources to decode the functional and ecological roles of bacteria within lichens. In addition, the low similarity (≤97 %) of the recovered isolates to known species and their production of cold-active enzymes together suggest that lichens are noteworthy sources of novel bacterial strains for use in biotechnological applications.