Improved production of alkaline and solvent-stable proteases from a halotolerant Exiguobacterium isolate through heterologous expression.

Halophiles are excellent sources of detergent proteases that are attributed to stability in alkaline pH, salts, surfactants, and hydrophobic solvents. The lower enzymatic yields and tedious downstream processes necessitate the search for newer halophilic sources. We have previously reported a halotolerant Exiguobacterium sp. TBG-PICH-001, which secretes solvent-tolerant alkaline protease/s. The present study describes the heterologous expression of two protease genes, namely, rsep metalloprotease (WP_195864791, 1.23 Kb) and tpa serine protease (WP_195864453, 0.879 Kb) genes. These were cloned into the pET 22b + plasmid vector and expressed in Escherichia coli BL21(DE3). The recombinant proteases rsep and tpa showed respective yields of 6.3 and 6.7 IU/mg, 11 and 12-fold higher than the crude native protease/s from TBG-PICH-001. These showed soluble expression at 46 and 32 KDa, respectively. These were purified to homogeneity through Ni-NTA-affinity chromatography. The purified proteases were characterized for properties like pH & temperature optima and stability, substrate specificity, kinetic parameters, and detergent attributes. They showed affinity towards various substrates with a respective Km of 392 and 301 µM towards casein. The recombinant proteases exhibited stability in the alkaline pH (7-10), surfactants, metal ions, detergents, and hydrophobic solvents, rendering their suitability as detergent additives.

Exiguobacterium sp. as a bioinoculant for plant-growth promotion and Selenium biofortification strategies in horticultural plants.

Plant growth-promoting rhizobacteria (PGPR) have a positive effect on plant development and being a promising way to enhance crop productivity and as substitution of chemical fertilizers. Selenium (Se) is an important trace element and its intake is usually lower than the daily minimum amount required for humans; hence, there is a demand on the design of Se biofortification strategies. Here, the genetic traits known to be associated with Plant-Growth Promotion (PGP) and Se biotransformation of Exiguobacterium sp. S17 were evaluated through genome analysis. Its growth-promoting capacity was tested through plant-growth promotion assays in laboratory and field conditions, using Brassica juncea (indian mustard), Beta vulgaris (chard), and Lactuca sativa (lettuce). Additionally, the Se biotransformation ability of Exiguobacterium sp. S17 was evaluated and the obtained selenized bacteria were tested in mustard plants. The sequenced bacteria genome revealed the presence of multiple genes involved in important functions regarding soil and plant colonization, PGP and Se biotransformation. Moreover, it was demonstrated that Exiguobacterium sp. S17 enhanced plant growth and could be useful to produce Se accumulation and biofortification in accumulator plants such as mustard. Thereby, Exiguobacterium sp. S17 might be used for developing new, sustainable, and environmentally friendly agro-technological strategies.

Biodegradation of crude oil by immobilized Exiguobacterium sp. AO-11 and shelf life evaluation.

Exiguobacterium sp. AO-11 was immobilized on bio-cord at 109 CFU g-1 carrier for the removal of crude oil from marine environments. To prepare a ready-to-use bioremediation product, the shelf life of the immobilized cells was calculated. Approximately 90% of 0.25% (v/v) crude oil removal was achieved within 9 days when the starved state of immobilized cells was used. The oil removal activity of the immobilized cells was maintained in the presence of oil dispersant (89%) and at pH values of 7-9. Meanwhile, pH, oil concentration and salinity affected the oil removal efficacy. The immobilized cells could be reused for at least 5 cycles. The Arrhenius equation describing the relationship between the rate of reaction and temperature was validated as a useful model of the kinetics of retention of activity by an immobilized biocatalyst. It was estimated that the immobilized cells could be stored in a non-vacuum bag containing phosphate buffer (pH 7.0) at 30 °C for 39 days to retain the cells at 107 CFU g-1 carrier and more than 50% degradation activity. These results indicated the potential of using bio-cord-immobilized crude oil-degrading Exiguobacterium sp. AO-11 as a bioremediation product in a marine environment.

In vitro biosynthesis of Ag, Au and Te-containing nanostructures by Exiguobacterium cell-free extracts.

BACKGROUND: The bacterial genus Exiguobacterium includes several species that inhabit environments with a wide range of temperature, salinity, and pH. This is why the microorganisms from this genus are known generically as polyextremophiles. Several environmental isolates have been explored and characterized for enzyme production as well as for bioremediation purposes. In this line, toxic metal(loid) reduction by these microorganisms represents an approach to decontaminate soluble metal ions via their transformation into less toxic, insoluble derivatives. Microbial-mediated metal(loid) reduction frequently results in the synthesis of nanoscale structures-nanostructures (NS) -. Thus, microorganisms could be used as an ecofriendly way to get NS. RESULTS: We analyzed the tolerance of Exiguobacterium acetylicum MF03, E. aurantiacum MF06, and E. profundum MF08 to Silver (I), gold (III), and tellurium (IV) compounds. Specifically, we explored the ability of cell-free extracts from these bacteria to reduce these toxicants and synthesize NS in vitro, both in the presence or absence of oxygen. All isolates exhibited higher tolerance to these toxicants in anaerobiosis. While in the absence of oxygen they showed high tellurite- and silver-reducing activity at pH 9.0, whereas AuCl4- which was reduced at pH 7.0 in both conditions. Given these results, cell-free extracts were used to synthesize NS containing silver, gold or tellurium, characterizing their size, morphology and chemical composition. Silver and tellurium NS exhibited smaller size under anaerobiosis and their morphology was circular (silver NS), starred (tellurium NS) or amorphous (gold NS). CONCLUSIONS: This nanostructure-synthesizing ability makes these isolates interesting candidates to get NS with biotechnological potential.

Efficient extraction of chitin from shrimp waste by mutagenized strain fermentation using atmospheric and room-temperature plasma.

Chitin extraction from shrimp waste by protease-producing microorganisms was a positive and simple method. To improve the protease activity of microorganism used for the extraction of chitin, atmospheric and room temperature plasma technology was adopted to induce mutations in Exiguobacterium profundum, a protease-producing bacterium, which was isolated from traditional fermented shrimp paste. After several rounds of screening, the mutant numbered 10017 was screened. The hereditary properties of the mutant were found to be stable after a series of passages. This strain was subsequently used in the deproteinization process, which could remove 91.48% ± 2.60% protein, and the chitin recovery was 70.18 ± 2.68%. Fourier transform infrared spectrometry, X-ray diffraction, and scanning electron microscopy was adopted to compare the characteristics of the chitin extracted from mutagenized and wild-type strain fermentation. The crystallinity indices were 80.72% and 82.46%, and the degrees of deacetylation were 15.78% and 27.84%. These results indicated that the deproteinization by mutagenized strain fermentation might be applied to the production of chitin. Thus, the present study provides an appropriate strategy to develop an efficient method to improve protease activity in microbial fermentation.