Evolutionary history and activity towards oligosaccharides and polysaccharides of GH3 glycosidases from an Antarctic marine bacterium.

Glycoside hydrolases (GHs) are pivotal in the hydrolysis of the glycosidic bonds of sugars, which are the main carbon and energy sources. The genome of Marinomonas sp. ef1, an Antarctic bacterium, contains three GHs belonging to family 3. These enzymes have distinct architectures and low sequence identity, suggesting that they originated from separate horizontal gene transfer events. M-GH3_A and M-GH3_B, were found to differ in cold adaptation and substrate specificity. M-GH3_A is a bona fide cold-active enzyme since it retains 20 % activity at 10 °C and exhibits poor long-term thermal stability. On the other hand, M-GH3_B shows mesophilic traits with very low activity at 10 °C (< 5 %) and higher long-term thermal stability. Substrate specificity assays highlight that M-GH3_A is a promiscuous ß-glucosidase mainly active on cellobiose and cellotetraose, whereas M-GH3_B is a ß-xylosidase active on xylan and arabinoxylan. Structural analysis suggests that such functional differences are due to their differently shaped active sites. The active site of M-GH3_A is wider but has a narrower entrance compared to that of M-GH3_B. Genome-based prediction of metabolic pathways suggests that Marinomonas sp. ef1 can use monosaccharides derived from the GH3-catalyzed hydrolysis of oligosaccharides either as a carbon source or for producing osmolytes.

A method for selective and efficient isolation of gray matter astrocytes from the spinal cord of adult mice.

A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.

Glucose Catabolite Repression Participates in the Regulation of Sialidase Biosynthesis by Antarctic Strain Penicillium griseofulvum P29.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

Low temperature acclimation of electroactive microorganisms may be an effective strategy to enhance the toxicity sensing performance of microbial fuel cell sensors.

Microbial fuel cell (MFC) sensing is a promising method for real-time detection of water biotoxicity, however, the low sensing sensitivity limits its application. This study adopted low temperature acclimation as a strategy to enhance the toxicity sensing performance of MFC biosensor. Two types of MFC biosensors were started up at low (10 °C) or warm (25 °C) temperature, denoted as MFC-Ls and MFC-Ws respectively, using Pb2+ as the toxic substance. MFC-Ls exhibited superior sensing sensitivities towards Pb2+ compared with MFC-Ws at both low (10 °C) and warm (25 °C) operation temperatures. For example, the inhibition rate of voltage of MFC-Ls was 22.81 % with 1 mg/L Pb2+ shock at 10 °C, while that of MFC-Ws was only 5.9 %. The morphological observation showed the anode biofilm of MFC-Ls had appropriate amount of extracellular polymer substances, thinner thickness (28.95 µm for MFC-Ls and 41.58 µm for MFC-Ws) and higher proportion of living cells (90.65 % for MFC-Ls and 86.01 % for MFC-Ws) compared to that of MFC-Ws. Microbial analysis indicated the enrichment of psychrophilic electroactive microorganisms and cold-active enzymes as well as their sensitivity to Pb2+ shock was the foundation for the effective operation and good performance of MFC-Ls biosensors. In conclusion, low temperature acclimation of electroactive microorganisms enhanced not only the sensitivity but also the temperature adaptability of MFC biosensors.

Biochemical and in silico structural characterization of a cold-active arginase from the psychrophilic yeast, Glaciozyma antarctica PI12.

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.

Structural determinants of cold activity and glucose tolerance of a family 1 glycoside hydrolase (GH1) from Antarctic Marinomonas sp. ef1.

Cold-active enzymes support life at low temperatures due to their ability to maintain high activity in the cold and can be useful in several biotechnological applications. Although information on the mechanisms of enzyme cold adaptation is still too limited to devise general rules, it appears that very diverse structural and functional changes are exploited in different protein families and within the same family. In this context, we studied the cold adaptation mechanism and the functional properties of a member of the glycoside hydrolase family 1 (GH1) from the Antarctic bacterium Marinomonas sp. ef1. This enzyme exhibits all typical functional hallmarks of cold adaptation, including high catalytic activity at 5 °C, broad substrate specificity, low thermal stability, and higher lability of the active site compared to the overall structure. Analysis of the here-reported crystal structure (1.8 Å resolution) and molecular dynamics simulations suggest that cold activity and thermolability may be due to a flexible region around the active site (residues 298-331), whereas the dynamic behavior of loops flanking the active site (residues 47-61 and 407-413) may favor enzyme-substrate interactions at the optimal temperature of catalysis (Topt) by tethering together protein regions lining the active site. Stapling of the N-terminus onto the surface of the ß-barrel is suggested to partly counterbalance protein flexibility, thus providing a stabilizing effect. The tolerance of the enzyme to glucose and galactose is accounted for by the presence of a "gatekeeping" hydrophobic residue (Leu178), located at the entrance of the active site.

Complete genome sequence of "Bacillaceae sp. strain IKA-2": a cold-active, amylase-producing bacterium from ikaite columns in SW Greenland.

Bacillaceae sp. strain IKA-2 is a bacterium isolated from the permanently cold and alkaline ikaite columns in the Ikka Fjord in SW Greenland (61°12'05″N; 48°00'50″W). The bacterium grows well at 10°C in a substrate buffered to pH 10. It has a genome size of 4,424,890 bp and a guanine-cytosine (GC) content of 36.2%. The genome harbors genes involved in hydrolysis of long carbohydrates and in protection against cold shock.

Structural and functional characterization of cold-active sialidase isolated from Antarctic fungus Penicillium griseofulvum P29.

The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 â†’ 3) sialyl linkages than those containing α(2 â†’ 6) linkages. Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date.

Genomic Analysis of Two Cold-Active Pseudoalteromonas Phages Isolated from the Continental Shelf in the Arctic Ocean.

Cold-active bacteriophages are bacterial viruses that infect and replicate at low temperatures (≤4 °C). Understanding remains limited of how cold-active phage-host systems sustain high viral abundance despite the persistently low temperatures in pelagic sediments in polar seas. In this study, two Pseudoalteromonas phages, ACA1 and ACA2, were isolated from sediment core samples of the continental shelf in the western Arctic Ocean. These phages exhibited successful propagation at a low temperature of 1 °C and displayed typical myovirus morphology with isometric icosahedral heads and contractile tails. The complete genome sequences of phages ACA1 and ACA2 were 36,825 bp and 36,826 bp in size, respectively, sharing almost the same gene content. These are temperate phages encoding lysogeny-related proteins such as anti-repressor, immunity repressor and integrase. The absence of cross-infection between the host strains, which were genomically distinct Pseudoalteromonas species, can likely be attributed to heavy divergence in the anti-receptor apparently mediated by an associated diversity-generating retroelement. HHpred searching identified genes for all of the structural components of a P2-like phage (family Peduoviridae), although the whole of the Peduoviridae family appeared to be divided between two anciently diverged tail modules. In contrast, Blast matching and whole genome tree analysis are dominated by a nonstructural gene module sharing high similarity with Pseudoalteromonas phage C5a (founder of genus Catalunyavirus). This study expands the knowledge of diversity of P2-like phages known to inhabit Peudoalteromonas and demonstrates their presence in the Arctic niche.

Heterologous protein production using Psychrobacter sp. PAMC 21119 analyzed with a green fluorescent protein-based reporter system.

Insolubility and low expression are typical bottlenecks in the production of proteins for studying their function and structure using X-ray crystallography or nuclear magnetic resonance spectroscopy. Cold-active enzymes from polar microorganisms have unique structural features that render them unstable and thermolabile, and are responsible for decreased protein yield in heterologous expression systems. To address these challenges, we developed a heterologous protein expression system using a psychrophilic organism, Psychrobacter sp. PAMC 21119, as a protein expression host with its own promoter. We screened 11 promoters and evaluated their strength using quantitative real-time polymerase chain reaction and a reporter system harboring the SfGFP gene. The highest expression was achieved using promoters RH96_RS13655 (P21119_20930) and RH96_RS15090 (P21119_23410), regardless of the temperature used. The p20930 strain exhibited a maximum expression level 19.6-fold higher than that of its control at 20 °C and produced approximately 0.5 mg of protein per gram of dry cell weight. To our knowledge, this is the first report of a low-temperature recombinant protein expression system developed using Psychrobacter sp. that can be used to express various difficult-to-express and cold-active proteins.

Structure-Guided Engineering of a Protease to Improve Its Activity under Cold Conditions.

Bacillus proteases commonly exhibit remarkably reduced activity under cold conditions. Herein, we employed a tailored combination of a loop engineering strategy and iterative saturation mutagenesis method to engineer two loops for substrate binding at the entrance of the substrate tunnel of a protease (bcPRO) from Bacillus clausii to improve its activity under cold conditions. The variant MT6 (G95P/A96D/S99W/S101T/P127S/S126T) exhibited an 18.3-fold greater catalytic efficiency than the wild-type (WT) variant at 10 °C. Molecular dynamics simulations and dynamic tunnel analysis indicated that the introduced mutations extended the substrate-binding pocket volume and facilitated extra interactions with the substrate, promoting catalysis through binding in a more favorable conformation. This study provides insights and strategies relevant to improving the activities of proteases and supplies a novel protease with enhanced activity under cold conditions for the food industry to maintain the initial flavor and color of food and reduce energy consumption.

Isolation and characterization of a cold-active, detergent-stable protease from Serratia sp. TGS1.

Psychrophiles are cold-adapted microorganisms living in cold regions and are known to generate cold-active enzymes such as proteases, lipases, and peptidases. These types of enzymes are a major part of the market of the food and textile sector. This study aimed to isolate and characterize the cold-active and detergent-stable, extracellular protease from psychotrophic bacteria Serratia sp. TGS1 (OQ654005). Protease was purified by gel permeation chromatography using Sephadex G-75. The specific activity of the purified protease was 250 U/mg at 15°C, with a purification fold of 5.68 and a percentage yield of 60%. The cold active protease was stable within a temperature range of 5-30°C and a pH range of 6-10. Ca+2 and Mg+2 enhanced its activity while chelators like ethylenediaminetetraacetic acid inhibited cold active protease, showing it as metalloprotease in nature. The enzyme was sensitive to Cu+2 , Zn+2 , and Hg+2 , and the proteolytic activity decreased upon treatment with heavy metals. The molecular weight of the protease was estimated to be 47 kDa using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins within a specific range of molecular weight possess desirable properties for industrial enzyme use. By working on a specific range, the researchers intended to examine an enzyme to examine its specific characteristics. The purified protease showed high stability to detergents like SDS, Tween 20, Tween 60, and Triton X. The maximum velocity Vmax and Km values were 59.90 mg/min/mL and 1.53 mg/mL, respectively. The obtained protease exhibited an interesting activity at a broad range of pH (6-10) and stability at low temperatures (5-30°C) and detergents. Such enzymatic features of versatile and potent cold-active enzymes enhance their industrial applications to meet food, dairy, and laundry requirements.

Formulation of synthetic bacteria consortia for enzymatic biodegradation of polyaromatic hydrocarbons contaminated soil: soil column study.

As an efficient method to remove contaminants from highly polluted sites, enzyme biodegradation addresses unresolved issues such as bioremediation inefficiency. In this study, the key enzymes involved in PAH degradation were brought together from different arctic strains for the biodegradation of highly contaminated soil. These enzymes were produced via a multi-culture of psychrophilic Pseudomonas and Rhodococcus strains. As a result of biosurfactant production, the removal of pyrene was sufficiently prompted by Alcanivorax borkumensis. The key enzymes (e.g., naphthalene dioxygenase, pyrene dioxygenase, catechol-2,3 dioxygenase, 1-hydroxy-2-naphthoate hydroxylase, protocatechuic acid 3,4-dioxygenase) obtained via multi-culture were characterized by tandem LC-MS/MS and kinetic studies. To simulate in situ application of produced enzyme solutions, pyrene- and dilbit-contaminated soil was bioremediated in soil columns and flask tests by injecting enzyme cocktails from the most promising consortia. The enzyme cocktail contained about 35.2 U/mg protein pyrene dioxygenase, 61.4 U/mg protein naphthalene dioxygenase, 56.5 U/mg protein catechol-2,3-dioxygenase, 6.1 U/mg protein 1-hydroxy-2-naphthoate hydroxylase, and 33.5 U/mg protein protocatechuic acid (P3,4D) 3,4-dioxygenase enzymes. It was found that after 6 weeks, the average pyrene removal values showed that the enzyme solution could be effective in the soil column system (80-85% degradation of pyrene).

Highly efficient low-temperature biodegradation of polyethylene microplastics by using cold-active laccase cell-surface display system.

To eliminate efficiency restriction of polyethylene microplastics low-temperature biodegradation, a novel InaKN-mediated Escherichia coli surface display platform for cold-active degrading laccase PsLAC production was developed. Display efficiency of 88.0% for engineering bacteria BL21/pET-InaKN-PsLAC was verified via subcellular extraction and protease accessibility, exhibiting an activity load of 29.6 U/mg. Cell growth and membrane integrity revealed BL21/pET-InaKN-PsLAC maintained stable growth and intact membrane structure during the display process. The favorable applicability was confirmed, with 50.0% activity remaining in 4 days at 15 °C, and 39.0% activity recovery retention after 15 batches of activity substrate oxidation reactions. Moreover, BL21/pET-InaKN-PsLAC possessed high polyethylene low-temperature depolymerizing capacity. Bioremediation experiments proved that the degradation rate was 48.0% within 48 h at 15 °C, and reached 66.0% after 144 h. Collectively, cold-active PsLAC functional surface display technology and its significant contributions to polyethylene microplastics low-temperature degradation constitute an effective improvement strategy for biomanufacturing and microplastics cold remediation.

Improving cold-adaptability of mesophilic cellulase complex with a novel mushroom cellobiohydrolase for efficient low-temperature ensiling.

Low ambient temperature poses a challenge for rice straw-silage processing in cold climate regions, as cold limits enzyme and microbial activity in silages. Here, a novel cold-active cellobiohydrolase (VvCBHI-I) was isolated from Volvariella volvacea, which exhibited outstanding cellobiohydrolase activity at 10-30 °C. The crude cellulase complex in the VvCBHI-I-expressing transformant T1 retained 50% relative activity at 10 °C, while the wildtype Trichoderma reesei showed <5% of the activity. VvCBHI-I greatly improved the saccharification efficiency of the cellulase complex with pretreated rice straw as substrate at 10 °C. In rice straw silage, pH (<4.5) and lactic acid content (>4.6%) remained stable after 15-day ensiling with the cellulase complex from T1 and Lactobacillus plantarum. Moreover, the proportions of cellulose and hemicellulose decreased to 29.84% ± 0.15% and 21.25% ± 0.26% of the dried material. This demonstrates the crucial potential of mushroom-derived cold-active cellobiohydrolases in successful ensiling in cold regions.

A bacterial cold-active dye-decolorizing peroxidase from an Antarctic Pseudomonas strain.

DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: • An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. • The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. • rDyP-AU10 showed high activity at low temperatures. • rDyP-AU10 is potentially useful for biotechnological applications.

Characterization of cold-active trehalose synthase from Pseudarthrobacter sp. for trehalose bioproduction.

Trehalose is a functional sugar that has numerous applications in food, cosmetic, and pharmaceutical products. Production of trehalose from maltose via a single-step enzymatic catalysis using trehalose synthase (TreS) is a promising method compared with the conventional two-step process due to its simplicity with lower formation of byproducts. In this study, a cold-active trehalose synthase (PaTreS) from Pseudarthrobacter sp. TBRC 2005 was heterologously expressed and characterized. PaTreS showed the maximum activity at 20 °C and maintained 87% and 59% of its activity at 10 °C and 4 °C, respectively. The enzyme had remarkable stability over a board pH range of 7.0-9.0 with the highest activity at pH 7.0. The activity was enhanced by divalent metal ions (Mg2+, Mn2+ and Ca2+). Conversion of high-concentration maltose syrup (100-300 g/L) using PaTreS yielded 71.7-225.5 g/L trehalose, with 4.5-16.4 g/L glucose as a byproduct within 16 h. The work demonstrated the potential of PaTreS as a promising biocatalyst for the development of low-temperature trehalose production, with the advantages of reduced risk of microbial contamination with low generation of byproduct.

Cold-Active Enzymes and Their Potential Industrial Applications-A Review.

More than 70% of our planet is covered by extremely cold environments, nourishing a broad diversity of microbial life. Temperature is the most significant parameter that plays a key role in the distribution of microorganisms on our planet. Psychrophilic microorganisms are the most prominent inhabitants of the cold ecosystems, and they possess potential cold-active enzymes with diverse uses in the research and commercial sectors. Psychrophiles are modified to nurture, replicate, and retain their active metabolic activities in low temperatures. Their enzymes possess characteristics of maximal activity at low to adequate temperatures; this feature makes them more appealing and attractive in biotechnology. The high enzymatic activity of psychrozymes at low temperatures implies an important feature for energy saving. These enzymes have proven more advantageous than their mesophilic and thermophilic counterparts. Therefore, it is very important to explore the efficiency and utility of different psychrozymes in food processing, pharmaceuticals, brewing, bioremediation, and molecular biology. In this review, we focused on the properties of cold-active enzymes and their diverse uses in different industries and research areas. This review will provide insight into the areas and characteristics to be improved in cold-active enzymes so that potential and desired enzymes can be made available for commercial purposes.

Structural interpretations of a flexible cold-active AMS8 lipase by combining small-angle X-ray scattering and molecular dynamics simulation (SAXS-MD).

Determining structure of highly flexible protein with multiple conformations can be challenging. This paper aims to combine molecular dynamics (MD) and small angle X-ray diffraction (SAX) techniques as a solution to overcome issues related to protein conformation in hardly crystallized protein. Based on prior studies, a cold-active lipase AMS8 was simulated in solvents showing stability in its N-terminal and high flexibility in its C-terminal. However, MD in its own algorithm could not explain the basis of macromolecule conformational transitions or changes related to protein through folding. Hence, by combining SAXS with MD, it is possible to understand the structure of flexible AMS8 lipase in natural space. Based on the findings, SAXS ab-initio model of AMS8 lipase was identified as a monomeric protein in which the optimized model of cold-active lipase AMS8 derived from SAXS data was found to be aligned with AMS8 homology model under series of MD timeframe.