Cold-active Enzymes Research Articles

Exploring The Protease Diversity of Psychrophilic Yeast, Glaciozyma antarctica Through Genome Mining Analysis

Proteases are one of the most significant classes of enzymes, holding immense physiological relevance and extensive industrial applications. The genome of Glaciozyma antarctica was fully sequenced, showing 7,857 open reading frames that offer an intriguing opportunity to investigate its proteolytic repertoire. This study aims to unveil the protease landscape of G. antarctica, a psychrophilic yeast that produces cold-active enzymes that offer remarkable benefits, particularly in the food and pharmaceutical industries. In this work, we performed a comprehensive analysis to identify the diverse families of proteases encoded within the G. antarctica genome and compare them with proteases from other mesophilic and thermophilic fungi in the MEROPS database. The sequence similarity searches resulted in the identification of 195 open reading frames predicted to encode for proteases in G. antarctica with a high number of intracellular proteases. These findings suggest an evolved system for protein quality control and turnover, essential for cell viability and adaptation to environmental stressors. The MEROPS classification analysis showed an abundance of metalloproteases, constituting 38% of the total protease genes, a proportion surpassing that found in other yeast and fungal genomes studied. This reflects the vital role of metalloproteases in the cold adaptation of microbes in the Antarctic region. This unique profile not only sheds light on the adaptive mechanisms of psychrophilic organisms but also presents a rich reservoir of potential cold-active proteases for various applications. The findings of this study provide a foundation for targeted enzyme discovery and engineering, unlocking new frontiers in industrial biotechnology and extremophile biology.

Biochemical and Structural Characterization of a Novel Psychrophilic Laccase (Multicopper Oxidase) Discovered from Oenococcus oeni 229 (ENOLAB 4002).

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K4[Fe(CN6)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments.

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 β-glucosidase mainly active on cellobiose and cellotetraose, whereas M-GH3_B is a β-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.

Metagenomic exploration of cold-active enzymes for detergent applications: Characterization of a novel, cold-active and alkali-stable GH8 endoglucanase from ikaite columns in SW Greenland.

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture-independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate-modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis. Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli, emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase-family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl2, making it a promising candidate for detergent and washing industry applications.

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.

Cold-active microbial enzymes and their biotechnological applications.

Microorganisms known as psychrophiles/psychrotrophs, which survive in cold climates, constitute majority of the biosphere on Earth. Their capability to produce cold-active enzymes along with other distinguishing characteristics allows them to survive in the cold environments. Due to the relative ease of large-scale production compared to enzymes from plants and animals, commercial uses of microbial enzyme are alluring. The ocean depths, polar, and alpine regions, which make up over 85% of the planet, are inhabited to cold ecosystems. Microbes living in these regions are important for their metabolic contribution to the ecosphere as well as for their enzymes, which may have potential industrial applications. Cold-adapted microorganisms are a possible source of cold-active enzymes that have high catalytic efficacy at low and moderate temperatures at which homologous mesophilic enzymes are not active. Cold-active enzymes can be used in a variety of biotechnological processes, including food processing, additives in the detergent and food industries, textile industry, waste-water treatment, biopulping, environmental bioremediation in cold climates, biotransformation, and molecular biology applications with great potential for energy savings. Genetically manipulated strains that are suitable for producing a particular cold-active enzyme would be crucial in a variety of industrial and biotechnological applications. The potential advantage of cold-adapted enzymes will probably lead to a greater annual market than for thermo-stable enzymes in the near future. This review includes latest updates on various microbial source of cold-active enzymes and their biotechnological applications.

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 β-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.

Immobilized cold-active enzymes onto magnetic chitosan microparticles as a highly stable and reusable carrier for p-xylene biodegradation

Stability and reusability properties are the two most important factors that determine an enzyme’s application in industry. To this end, cold-active crude enzymes from a psychrophile (xylene monooxygenase (XMO) and catechol 1,2-dioxygenase (C1,2D) were immobilized on magnetic chitosan microparticles for the first-time using glutaraldehyde as a linker. The potential application of enzyme-loaded magnetic particles to remove and detoxify dissolved p-xylene from water confirmed the synergistic mechanism of degradation for in-situ bioremediation in soil and water. Immobilization was optimized based on four variables, such as magnetic particle (MPs), chitosan, glutaraldehyde, and enzyme concentrations. The immobilized enzymes were characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). The immobilized enzymes showed improved pH tolerance ranging from 4.0 to 9.0, better temperature stability ranging from 5 to 50, higher storage stability (∼70% activity after 30 days of storage), and more importantly, reusability (∼40% activity after 10 repetitive cycles of usage) compared to their free form. Also, the immobilization of enzymes increased the effectiveness of the enzymatic treatment of p-xylene in soil (10,000 mg/kg) and water (200 mg/L) samples. As a result of the superior catalytic properties of immobilized XMO and C1,2D, they offer great potential for in situ or ex-situ bioremediation of pollutants in soil or water.

Optimization of cold active amylase production by mesophilic Bacillus cereus RGUJS2023 under submerged fermentation

Aim: To produce the highest amount of cold-active alpha-amylase within a short time using mesophilic bacteria with optimized media to save the energy consumption cost and obtain higher enzyme production. Methodology: Amylase producing twenty-three strains were isolated on starch peptone agar plates. Among them, one strain, A5 was selected on the basis of highest clear (12 mm) zone on starch peptone agar plates. It was characterized and identified following Bergey's Manual of Systematic Bacteriology. Enzyme was characterized as Cold alpha amylase. All physico-chemical parameters (temperature, pH, Inoculum size) including carbon, nitrogen, metal ion and amino acid sources were optimized for maximum production of the enzyme. The optimized media was used for enhancing the cold active amylase production. Results: The strain, A5 was identified as Bacillus cereus RGUJS2023 by 16S rRNA sequencing analysis for further experiments. This strain showed the highest activity (9.922± 0.143 U ml-1) on the basal starch peptone media. Though, crude enzyme showed its activity at 4°C to 48°C temperature, but the temperature was 28°C. The highest cold active enzyme was produced (18.87±0.06 U ml-1) at 16 hr of bacterial growth at 35 °C with a pH 6.5 in the optimized media containing 0.5% starch, 0.1% peptone and 0.03% MgSO4.7H2O as a carbon, nitrogen and metal ion sources, respectively, with addition of 0.03% arginine. Interpretation: The cold active alpha amylase could be used commercially for the benefit of pharmaceutical and starch processing industries. Key words: Bacillus cereus RGUJS2023, Cold-active amylase, Starch hydrolysis

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 → 3) sialyl linkages than those containing α(2 → 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.

Comparative genome analysis of the freshwater fungus Filosporella fistucella indicates potential for plant-litter degradation at cold temperatures.

Freshwater fungi play an important role in the decomposition of organic matter of leaf litter in rivers and streams. They also possess the necessary mechanisms to endure lower temperatures caused by habitat and weather variations. This includes the production of cold-active enzymes and antifreeze proteins. To better understand the physiological activities of freshwater fungi in their natural environment, different methods are being applied, and genome sequencing is one in the spotlight. In our study, we sequenced the first genome of the freshwater fungus Filosporella fistucella (45.7 Mb) and compared the genome with the evolutionary close-related species Tricladium varicosporioides (48.2 Mb). The genomes were annotated using the carbohydrate-active enzyme database where we then filtered for leaf-litter degradation-related enzymes (cellulase, hemicellulase, laccase, pectinase, cutinase, amylase, xylanase, and xyloglucanase). Those enzymes were analyzed for antifreeze properties using a machine-learning approach. We discovered that F. fistucella has more enzymes to participate in the breakdown of sugar, leaf, and wood than T. varicosporioides (855 and 719, respectively). Filosporella fistucella shows a larger set of enzymes capable of resisting cold temperatures than T. varicosporioides (75 and 66, respectively). Our findings indicate that in comparison with T. varicosporioides, F. fistucella has a greater capacity for aquatic growth, adaptability to freshwater environments, and resistance to low temperatures.

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.

Psychrophilic enzymes: strategies for cold-adaptation.

Psychrophilic organisms thriving at near-zero temperatures synthesize cold-adapted enzymes to sustain cell metabolism. These enzymes have overcome the reduced molecular kinetic energy and increased viscosity inherent to their environment and maintained high catalytic rates by development of a diverse range of structural solutions. Most commonly, they are characterized by a high flexibility coupled with an intrinsic structural instability and reduced substrate affinity. However, this paradigm for cold-adaptation is not universal as some cold-active enzymes with high stability and/or high substrate affinity and/or even an unaltered flexibility have been reported, pointing to alternative adaptation strategies. Indeed, cold-adaptation can involve any of a number of a diverse range of structural modifications, or combinations of modifications, depending on the enzyme involved, its function, structure, stability, and evolutionary history. This paper presents the challenges, properties, and adaptation strategies of these enzymes.

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.

Production and characterization of a novel cold-active ß-glucosidase and its influence on aromatic precursors of Muscat wine

ß-Glucosidases (ßGL)—widely used in the enological field—are enzymes that catalyze the liberation of aromatic volatiles from their glycosidic precursors during winemaking. In the present study, a ßGL obtained from the Antarctic yeast Mrakia sp. LP 7.1.2016 was produced on a bioreactor scale, purified, characterized, and the enzyme's properties studied for a potential enological application. Sodium-dodecylsulfate–polyacrylamide-gel electrophoresis indicated a high molecular weight for this enzyme, it having at least two subunits of 134 and 14 kDa. ßGL exhibited a pH optimum of 5.0 and retained substantial activity and stability at pH 4.0. The temperature range for optimal activity was 50–55 °C, with the enzyme demonstrating thermostability up to 50 °C and retaining 87% of residual activity at that temperature after 3 h. The respective kinetic constants determined with p-nitrophenyl-ß-d-glucopyranoside and cellobiose were 0.38 and 1.79 mM for Km and 20.1 and 5.65 μmol−1 mg−1 min.1 for Vmax, ß-Glucosidase manifested high activity in 10–25% (v/v) ethanol, 30.0 mg L−1 sulfur dioxide, and 10–200 g L−1 fructose; but it was strongly inhibited by glucose, retaining only 6% of residual activity in the presence of 20 g L−1. Upon investigating the influence of the enzyme on Muscat-wine glycosidic precursors, we found significant differences in terpene content after 14 days of ßGL treatment at an eightfold increase over control-wine levels. The enzyme was more active toward the precursors of the monoterpenes nerol and geraniol and oxides of trans- and cis-linalool. These findings contribute to our understanding of the potential of cold-active enzymes in advantageous biotechnological applications to enology.

A Cold-Active Flavin-Dependent Monooxygenase from Janthinobacterium svalbardensis Unlocks Applications of Baeyer-Villiger Monooxygenases at Low Temperature.

Cold-active enzymes maintain a large part of their optimal activity at low temperatures. Therefore, they can be used to avoid side reactions and preserve heat-sensitive compounds. Baeyer-Villiger monooxygenases (BVMO) utilize molecular oxygen as a co-substrate to catalyze reactions widely employed for steroid, agrochemical, antibiotic, and pheromone production. Oxygen has been described as the rate-limiting factor for some BVMO applications, thereby hindering their efficient utilization. Considering that oxygen solubility in water increases by 40% when the temperature is decreased from 30 to 10 °C, we set out to identify and characterize a cold-active BVMO. Using genome mining in the Antarctic organism Janthinobacterium svalbardensis, a cold-active type II flavin-dependent monooxygenase (FMO) was discovered. The enzyme shows promiscuity toward NADH and NADPH and high activity between 5 and 25 °C. The enzyme catalyzes the monooxygenation and sulfoxidation of a wide range of ketones and thioesters. The high enantioselectivity in the oxidation of norcamphor (eeS = 56%, eeP > 99%, E > 200) demonstrates that the generally higher flexibility observed in the active sites of cold-active enzymes, which compensates for the lower motion at cold temperatures, does not necessarily reduce the selectivity of these enzymes. To gain a better understanding of the unique mechanistic features of type II FMOs, we determined the structure of the dimeric enzyme at 2.5 Å resolution. While the unusual N-terminal domain has been related to the catalytic properties of type II FMOs, the structure shows a SnoaL-like N-terminal domain that is not interacting directly with the active site. The active site of the enzyme is accessible only through a tunnel, with Tyr-458, Asp-217, and His-216 as catalytic residues, a combination not observed before in FMOs and BVMOs.

Evaluation of scale-up effect on cold-active enzyme production and biodegradation tests using pilot-scale bioreactors and a 3D soil tank

Despite recent attention being paid to the biodegradation of petroleum hydrocarbons in cold environments, scale-up studies of biodegradation are lacking. Herein, the effect of scale-up on the enzymatic biodegradation of highly contaminated soil at low temperatures was studied. A novel cold-adapted bacteria (Arthrobacter sp. S2TR-06) was isolated that could produce cold-active degradative enzymes (xylene monooxygenase (XMO) and catechol 2,3-dioxygenase (C2,3D)). Enzyme production was investigated on 4 different scales (lab to pilot scale). The results showed a shorter fermentation time, and the highest production of enzymes and biomass (107 g/L for biomass, 109 U/mL, and 203 U/mL for XMO and C2,3D after 24 h) was achieved in the 150-L bioreactor due to enhanced oxygenation. Multi-pulse injection of p-xylene into the production medium was needed every 6 h. The stability of membrane-bound enzymes can be increased up to 3-fold by adding FeSO4 at 0.1% (w/v) before extraction. Soil tests also showed that biodegradation is scale-dependent. The maximum biodegradation rate decreased from 100% at lab-scale to 36% in the 300-L sand tank tests due to limited access of enzymes to trapped p-xylene in soil pores, low dissolved oxygen in the water-saturated zone, soil heterogeneity, and the presence of the free phase of p-xylene. The result demonstrated that formulation of enzyme mixture with FeSO4 and direct injection of enzyme mixture (third scenario) can increase the efficiency of bioremediation in heterogeneous soil. In this study, it was demonstrated that cold-active degradative enzyme production can be scaled up to an industrial scale and enzymatic treatment can be used to effectively bioremediate p-xylene contaminated sites. This study could provide key scale-up guidance for the enzymatic bioremediation of mono-aromatic pollutants in water-saturated soil under cold conditions.

Discrimination of psychrophilic enzymes using machine learning algorithms with amino acid composition descriptor.

Psychrophilic enzymes are a class of macromolecules with high catalytic activity at low temperatures. Cold-active enzymes possessing eco-friendly and cost-effective properties, are of huge potential application in detergent, textiles, environmental remediation, pharmaceutical as well as food industry. Compared with the time-consuming and labor-intensive experiments, computational modeling especially the machine learning (ML) algorithm is a high-throughput screening tool to identify psychrophilic enzymes efficiently. In this study, the influence of 4 ML methods (support vector machines, K-nearest neighbor, random forest, and naïve Bayes), and three descriptors, i.e., amino acid composition (AAC), dipeptide combinations (DPC), and AAC + DPC on the model performance were systematically analyzed. Among the 4 ML methods, the support vector machine model based on the AAC descriptor using 5-fold cross-validation achieved the best prediction accuracy with 80.6%. The AAC outperformed than the DPC and AAC + DPC descriptors regardless of the ML methods used. In addition, amino acid frequencies between psychrophilic and non-psychrophilic proteins revealed that higher frequencies of Ala, Gly, Ser, and Thr, and lower frequencies of Glu, Lys, Arg, Ile,Val, and Leu could be related to the protein psychrophilicity. Further, ternary models were also developed that could classify psychrophilic, mesophilic, and thermophilic proteins effectively. The predictive accuracy of the ternary classification model using AAC descriptor via the support vector machine algorithm was 75.8%. These findings would enhance our insight into the cold-adaption mechanisms of psychrophilic proteins and aid in the design of engineered cold-active enzymes. Moreover, the proposed model could be used as a screening tool to identify novel cold-adapted proteins.

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

Cold environments characterised by diverse temperatures close to or below the water freezing point dominate about 80% of the Earth's biosphere. One of the survival strategies adopted by microorganisms living in cold environments is their expression of cold-active enzymes that enable them to perform an efficient metabolic flux at low temperatures necessary to thrive and reproduce under those constraints. Cold-active enzymes are ideal biocatalysts that can reduce the need for heating procedures and improve industrial processes' quality, sustainability, and cost-effectiveness. Despite their wide applications, their industrial usage is still limited, and the major contributing factor is the lack of complete understanding of their structure and cold adaptation mechanisms. The current review looked at the recombinant overexpression, purification, and recent mechanism of cold adaptation, various approaches for purification, and three-dimensional (3D) crystal structure elucidation of cold-active lipases and esterase.

Identification of Fungal Communities Isolated from Himalayan Glacier Cryoconites

The current study focuses on fungi that were isolated from cryoconite holes of the Hamtah glacier in the Himalayas. Cryoconite holes have ecological and biotechnological importance. A total of seven cryoconite samples were collected from different locations and subjected to the isolation of psychotropic fungi at 1, 4, 15 and 22 °C. Isolates were identified by ITS and D1/D2 region sequences. The result showed culturable yeasts (45) and filamentous fungi (10) belonging to four ascomycetous classes (Dothideomycetes, Eurotiomycetes, Saccharomycetes and Sordariomycetes) and two basidiomycetes’ classes (Microbotryomycetes and Tremellomycetes). Physiological characteristics such as the pH, temperature, salt tolerance, carbon source utilization and antibiotics sensitivity of the isolates were studied. All the isolates were grown from acidic to alkaline pH and were able to grow at 1 to 22 °C. The fungal cultures isolated were screened to produce cold active enzymes such as amylase, cellulase, lipase, protease and catalase. Cellulase activity was detected at its maximum at both 4 and 15 °C. Himalayan cryoconites fungi showed immense potential for biotechnological and industrial applications. To the best of our knowledge, this is the first record of the characterization of fungal communities present in the glacier cryoconites of the Himalayas.