Psychrophilic microorganisms are cold-adapted with distinct properties from other thermal classes thriving in cold conditions in large areas of the earth's cold environment. Maintenance of functional membranes, evolving cold-adapted enzymes and synthesizing a range of structural features are basic adaptive strategies of psychrophiles. Among the cold-evolved enzymes are the cold-active lipases, a group of microbial lipases with inherent stability-activity-flexibility property that have engaged the interest of researchers over the years. Current knowledge regarding these cold-evolved enzymes in psychrophilic bacteria proves a display of high catalytic efficiency with low thermal stability, which is a differentiating feature with that of their mesophilic and thermophilic counterparts. Improvement strategies of their adaptive structural features have significantly benefited the enzyme industry. Based on their homogeneity and purity, molecular characterizations of these enzymes have been successful and their properties make them unique biocatalysts for various industrial and biotechnological applications. Although, strong association of lipopolysaccharides from Antarctic microorganisms with lipid hydrolases pose a challenge in their purification, heterologous expression of the cold-adapted lipases with affinity tags simplifies purification with higher yield. The review discusses these cold-evolved lipases from bacteria and their peculiar properties, in addition to their potential biotechnological and industrial applications.
A range of small- to moderate-scale studies of patterns in bacterial biodiversity have been conducted in Antarctica over the last two decades, most suggesting strong correlations between the described bacterial communities and elements of local environmental heterogeneity. However, very few of these studies have advanced interpretations in terms of spatially associated patterns, despite increasing evidence of patterns in bacterial biogeography globally. This is likely to be a consequence of restricted sampling coverage, with most studies to date focusing only on a few localities within a specific Antarctic region. Clearly, there is now a need for synthesis over a much larger spatial to consolidate the available data. In this study, we collated Antarctic bacterial culture identities based on the 16S rRNA gene information available in the literature and the GenBank database (n > 2,000 sequences). In contrast to some recent evidence for a distinct Antarctic microbiome, our phylogenetic comparisons show that a majority (~75 %) of Antarctic bacterial isolates were highly similar (≥99 % sequence similarity) to those retrieved from tropical and temperate regions, suggesting widespread distribution of eurythermal mesophiles in Antarctic environments. However, across different Antarctic regions, the dominant bacterial genera exhibit some spatially distinct diversity patterns analogous to those recently proposed for Antarctic terrestrial macroorganisms. Taken together, our results highlight the threat of cross-regional homogenisation in Antarctic biodiversity, and the imperative to include microbiota within the framework of biosecurity measures for Antarctica.
The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.
A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70 degrees C and pH 9, respectively. It was stable up to 65 degrees C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na(+), Ca(2+), Mn(2+), K(+) and Mg(2+ ), but inhibited by Cu(2+), Fe(3+) and Zn(2+). Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10-C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T(m) for T1 lipase was around 72.2 degrees C, as revealed by denatured protein analysis of CD spectra.
The gene encoding for a novel cold-adapted enzyme from family II of bacterial classification (GDSL family) was cloned from the genomic DNA of Photobacterium sp. strain J15 in an Escherichia coli system, yielding a recombinant 36 kDa J15 GDSL esterase which was purified in two steps with a final yield and purification of 38.6 and 15.3 respectively. Characterization of the biochemical properties showed the J15 GDSL esterase had maximum activity at 20 °C and pH 8.0, was stable at 10 °C for 3 h and retained 50 % of its activity after a 6 h incubation at 10 °C. The enzyme was activated by Tween-20, -60 and Triton-X100 and inhibited by 1 mM Sodium dodecyl sulphate (SDS), while β-mercaptoethanol and Dithiothreitol (DTT) enhanced activity by 4.3 and 5.4 fold respectively. These results showed the J15 GDSL esterase was a novel cold-adapted enzyme from family II of lipolytic enzymes. A structural model constructed using autotransporter EstA from Pseudomonas aeruginosa as a template revealed the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine which was verified with site directed mutagenesis on active serine.
The data reported in this paper are among the first relating to the microbiology of hyper-arid, very high altitude deserts and they provide base line information on the structure of actinobacterial communities. The high mountain Cerro Chajnantor landscape of the Central Andes in northern Chile is exposed to the world's most intense levels of solar radiation and its impoverished soils are severely desiccated. The purpose of this research was to define the actinobacterial community structures in soils at altitudes ranging from 3000 to 5000 m above sea level. Pyrosequencing surveys have revealed an extraordinary degree of microbial dark matter at these elevations that includes novel candidate actinobacterial classes, orders and families. Ultraviolet-B irradiance and a range of edaphic factors were found to be highly significant in determining community compositions at family and genus levels of diversity.
Petroleum pollution is a major concern in Antarctica due to the persistent nature of its hydrocarbon components coupled with the region's extreme environmental conditions, which means that bioremediation approaches are largely inapplicable at present. The current study assessed the ability of the psychrotolerant phenol-degrader, Rhodococcus sp. strain AQ5-07, to assimilate diesel fuel as the sole carbon source. Factors expected to influence the efficiency of diesel degradation, including the initial hydrocarbon concentration, nitrogen source concentration and type, temperature, pH and salinity were studied. Strain AQ5-07 displayed optimal cell growth and biodegradation activity at 1% v/v initial diesel concentration, 1 g/L NH4Cl concentration, pH 7 and 1% NaCl during one-factor-at-a-time (OFAT) analyses. Strain AQ5-07 was psychrotolerant based on its optimum growth temperature being near 20 °C. In conventionally optimised media, strain AQ5-07 showed total petroleum hydrocarbons (TPH) mineralisation of 75.83%. However, the optimised condition for TPH mineralisation predicted through statistical response surface methodology (RSM) enhanced the reduction to 90.39% within a 2 days incubation. Our preliminary data support strain AQ5-07 being a potential candidate for real-field soil bioremediation by specifically adopting sludge-phase bioreactor system in chronically cold environments such as Antarctica. The study also confirmed the utility of RSM in medium optimisation.
Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys-His-Asp and a G-x-C-x-G-G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni-NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5-9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s-1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.
Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against L-arginine as its substrate in a reaction at 20 °C, pH 9. At 10-35 °C and pH 7-9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.