Ha Ju Park

Isolation and characterization of humic substances-degrading bacteria from the subarctic Alaska grasslands

Humic substances (HS), an important fraction of soil organic carbon, are distributed widely throughout cold environments. A total of cold‐adapted 122 bacterial strains were isolated from 66 Alaska grassland soil samples based on their ability to grow on humic acids (HA), a main fraction of HS, as a carbon and energy source. These isolates were identified based on 16S rRNA gene sequencing, with class Bacilli (79.5%) and γ‐Proteobacteria (17.1%) comprising the largest groups. Among them, 45 strains, mainly Paenibacillus (27 strains) and Pseudomonas (15 strains), were selected for further screening. Two strains (Pseudomonas sp. PAMC 26793 and Paenibacillus sp. PAMC 26794) most efficiently degraded HA, but showed significant differences in their ability to grow on various monocyclic aromatics, which are putative degradative metabolites of HS. Fourier transform infrared spectra also showed substantial but different changes in HA chemical structure after incubation with each strain. Gel permeation chromatography demonstrated that depolymerization and polymerization of HA occurred during HS degradation by these newly isolated microbes.

Cryoprotective properties and preliminary characterization of exopolysaccharide (P-Arcpo 15) produced by the Arctic bacterium Pseudoalteromonas elyakovii Arcpo 15

ABSTRACT Twenty-two bacterial strains that secrete exopolysaccharides (EPS) were isolated from marine samples obtained from the Chukchi Sea in the Arctic Ocean; of these, seven strains were found to be capable of producing cryoprotective EPS. The ArcPo 15 strain was isolated based on its ability to secrete large amounts of EPS, and was identified as Pseudoalteromonas elyakovii based on 16S rDNA analysis. The EPS, P-ArcPo 15, was purified by protease treatment and gel filtration chromatography. The purified EPS (P-ArcPo 15) had a molecular mass of 1.7 × 107 Da, and its infrared spectrum showed absorption bands of hydroxyl and carboxyl groups. The principal sugar components of P-ArcPo 15 were determined to be mannose and galacturonic acid, in the ratio of 3.3:1.0. The cryoprotective properties of P-ArcPo 15 were characterized by an Escherichia coli viability test. In the presence of 0.5% (w/v) EPS, the survival percentage of E. coli cells was as high as 94.19 ± 7.81% over five repeated freeze–thaw cycles. These biochemical characteristics suggest that the EPS P-ArcPo 15 may be useful in the development of cryoprotectants for biotechnological purposes, and we therefore assessed the utility of this novel cryoprotective EPS.

Draft Genome Sequence of Arctic Marine Bacterium Pseudoalteromonas issachenkonii PAMC 22718

The psychrotolerant Pseudoalteromonas issachenkonii PAMC 22718 was isolated for its higher chitinase and protease activities from cold seawater in the Kara Sea, Arctic. Here, we present the draft genome sequence of PAMC 22718 to provide further information for the ecological function of the genus Pseudoalteromonas in a cold marine environment.

Enhanced production of protease by Pseudoalteromonas arctica PAMC 21717 via statistical optimization of mineral components and fed-batch fermentation

ABSTRACT The objective of this study was to statistically optimize the mineral components of the nutritional medium required for enhancing the production of a cold-active extracellular serine-type protease, W-Pro21717, by the Antarctic bacterium Pseudoalteromonas arctica PAMC 21717. Skim milk was identified as the major efficient inducer. Among the 12 components included in the unoptimized medium, skim milk, NaCl, Na2SO4, Fe(C6H5O7) (ferric citrate), and KCl were determined, by the Plackett–Burman and Box–Behnken design, to have a major effect on W-Pro21717 production. Fed-batch fermentation (5 L scale) using the mineral-optimized medium supplemented with concentrated skim milk (critical medium component) resulted in a W-Pro21717 activity of 53.4 U/L, a 15-fold increment in production over that obtained using unoptimized flask culture conditions. These findings could be applied to scale up the production of cold-active protease.

Identification of proteolytic bacteria from the Arctic Chukchi Sea expedition cruise and characterization of cold-active proteases

Following collection of seawater samples during an Arctic Chukchi Sea expedition cruise of the Korean icebreaker Araon in 2012, a total of 15,696 bacteria were randomly isolated from Marine Broth 2216 agar plates. Of these, 2,526 (16%) showed proteolytic activity and were identified as mainly Alteromonas (31%), Staphylococcus (27%), and Pseudoalteromonas (14%). Among the proteolytic strains, seven were selected based on their significant ability to grow and produce a halo on skim milk plates at low temperatures (<5°C) owing to cold-active proteases. These strains were affiliated with the genus Pseudoalteromonas and were divided into three groups based on phylogenetic analysis of the 16S rRNA genes. Profiling cell membrane fatty acids confirmed the 16S rRNA-based differentiation and revealed the accordance between the two analyses. Seven genes for serine protease precursors were amplified from the corresponding strains, and based on sequence similarities, these genes were divided into three groups that were identical to those identified by the 16S rRNA phylogenetic analysis. Three protease genes from the representative strains of each group were composed of 2,127–2,130 bp, encoding 708–709 amino acids, and these genes yielded products with calculated molecular weights of approximately 72.3–72.8 kDa. Amino acid sequence analysis suggested that the precursors are members of the subtilase serine endo- and exo-peptidase clan and contain four domains (signal peptide, N-terminal prosequence, catalytic domain, and two pre-peptidase C-terminal domains). Upon expression in E. coli, each recombinant protease exhibited proteolytic activity on zymogram gels.

Enhancing Extracellular Lipolytic Enzyme Production In An Arctic Bacterium, Psychrobacter sp. ArcL13, By Using Statistical Optimization And Fed-Batch Fermentation

A strain isolated from seawater samples in the Chuckchi Sea and exhibiting extracellular lipolytic activity was identified using 16S rRNA gene sequence analysis as Psychrobacter sp. ArcL13. The lipolytic enzyme exhibited cold-active properties and high hydrolytic activity toward p-nitrophenyl caprylate (C8), p-nitrophenyl decanoate (C10), and sunflower oil. Statistical optimization of the medium components was performed to enhance the production of cold-active extracellular lipolytic activity. Glucose, yeast extract (YE), and NaCl were selected as the main efficient nutrient sources. Fed-batch fermentation using optimized medium with concentrated YE as the main feeding material showed a maximum lipolytic activity of 10.7 U/mL, which was a 21-fold increase in production over unoptimized flask culture conditions. The information obtained in the present study could prove applicable to the production of cold-active lipase on a large scale.

Characterization of β-N-acetylglucosaminidase from a marine Pseudoalteromonas sp. for application in N-acetyl-glucosamine production

ABSTRACT The psychrotolerant Pseudoalteromonas issachenkonii PAMC 22718 was isolated for its high exo-acting chitinase activity in the Kara Sea, Arctic. An exo-acting chitinase (W-Chi22718) was homogeneously purified from the culture supernatant of PAMC 22718, the molecular weight of which was estimated to be approximately 112 kDa. Due to its β-N-acetylglucosaminidase activity, W-Chi22718 was able to produce N-acetyl-D-glucosamine (GlcNAc) monomers from chitin oligosaccharide substrates. W-Chi22718 displayed chitinase activity from 0 to 37°C (optimal temperature of 30°C) and maintained activity from pH 6.0 to 9.0 (optimal pH of 7.6). W-Chi22718 exhibited a relative activity of 13 and 35% of maximal activity at 0 and 10°C, respectively, which is comparable to the activities of previously characterized, cold-adapted bacterial chitinases. W-Chi22718 activity was enhanced by K+, Ca2+, and Fe2+, but completely inhibited by Cu2+ and SDS. We found that W-Chi22718 can produce much more (GlcNAcs) from colloidal chitin, working together with previously characterized cold-active endochitinase W-Chi21702. Genome sequencing revealed that the corresponding gene (chi22718_IV) was 2,856 bp encoding a 951 amino acid protein with a calculated molecular weight of approximately 102 kDa.

Crystal structure of a cold-active protease (Pro21717) from the psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, at 1.4 Å resolution: Structural adaptations to cold and functional analysis of a laundry detergent enzyme

Enzymes isolated from organisms found in cold habitats generally exhibit higher catalytic activity at low temperatures than their mesophilic homologs and are therefore known as cold-active enzymes. Cold-active proteases are very useful in a variety of biotechnological applications, particularly as active ingredients in laundry and dishwashing detergents, where they provide strong protein-degrading activity in cold water. We identified a cold-active protease (Pro21717) from a psychrophilic bacterium, Pseudoalteromonas arctica PAMC 21717, and determined the crystal structure of its catalytic domain (CD) at a resolution of 1.4 Å. The Pro21717-CD structure shows a conserved subtilisin-like fold with a typical catalytic triad (Asp185, His244, and Ser425) and contains four calcium ions and three disulfide bonds. Interestingly, we observed an unexpected electron density at the substrate-binding site from a co-purified peptide. Although the sequence of this peptide is unknown, analysis of the peptide-complexed structure nonetheless provides some indication of the substrate recognition and binding mode of Pro21717. Moreover, various parameters, including a wide substrate pocket size, an abundant active-site loop content, and a flexible structure provide potential explanations for the cold-adapted properties of Pro21717. In conclusion, this is first structural characterization of a cold-adapted subtilisin-like protease, and these findings provide a structural and functional basis for industrial applications of Pro21717 as a cold-active laundry or dishwashing detergent enzyme.

Passive warming effect on soil microbial community and humic substance degradation in maritime Antarctic region

Although the maritime Antarctic has undergone rapid warming, the effects on indigenous soil‐inhabiting microorganisms are not well known. Passive warming experiments using open‐top chamber (OTC) have been performed on the Fildes Peninsula in the maritime Antarctic since 2008. When the soil temperature was measured at a depth of 2–5 cm during the 2013–2015 summer seasons, the mean temperature inside OTC (OTC‐In) increased by approximately 0.8 °C compared with outside OTC (OTC‐Out), while soil chemical and physical characteristics did not change. Soils (2015 summer) from OTC‐In and OTC‐Out were subjected to analysis for change in microbial community and degradation rate of humic substances (HS, the largest pool of recalcitrant organic carbon in soil). Archaeal and bacterial communities in OTC‐In were minimally affected by warming compared with those in OTC‐Out, with archaeal methanogenic Thermoplasmata slightly increased in abundance. The abundance of heterotrophic fungi Ascomycota was significantly altered in OTC‐In. Total bacterial and fungal biomass in OTC‐In increased by 20% compared to OTC‐Out, indicating that this may be due to increased microbial degradation activity for soil organic matter (SOM) including HS, which would result in the release of more low‐molecular‐weight growth substrates from SOM. Despite the effects of warming on the microbial community over the 8‐years‐experiments warming did not induce any detectable change in content or structure of polymeric HS. These results suggest that increased temperature may have significant and direct effects on soil microbial communities inhabiting maritime Antarctic and that soil microbes would subsequently provide more available carbon sources for other indigenous microbes.

Transcriptome analysis of Pseudomonas sp. from subarctic tundra soil: pathway description and gene discovery for humic acids degradation

Although humic acids (HA) are involved in many biological processes in soils and thus their ecological importance has received much attention, the degradative pathways and corresponding catalytic genes underlying the HA degradation by bacteria remain unclear. To unveil those uncertainties, we analyzed transcriptomes extracted from Pseudomonas sp. PAMC 26793 cells time-dependently induced in the presence of HA in a lab flask. Out of 6288 genes, 299 (microarray) and 585 (RNA-seq) were up-regulated by > 2.0-fold in HA-induced cells, compared with controls. A significant portion (9.7% in microarray and 24.1% in RNA-seq) of these genes are predicted to function in the transport and metabolism of small molecule compounds, which could result from microbial HA degradation. To further identify lignin (a surrogate for HA)-degradative genes, 6288 protein sequences were analyzed against carbohydrate-active enzyme database and a self-curated list of putative lignin degradative genes. Out of 19 genes predicted to function in lignin degradation, several genes encoding laccase, dye-decolorizing peroxidase, vanillate O-demethylase oxygenase and reductase, and biphenyl 2,3-dioxygenase were up-regulated > 2.0-fold in RNA-seq. This induction was further confirmed by qRT-PCR, validating the likely involvement of these genes in the degradation of HA.