Journal articles: 'Cold-active enzyme'

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Iyo, Abiye H., and Cecil W. Forsberg. "A Cold-Active Glucanase from the Ruminal BacteriumFibrobacter succinogenes S85." Applied and Environmental Microbiology 65, no. 3 (March 1, 1999): 995–98. http://dx.doi.org/10.1128/aem.65.3.995-998.1999.

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ABSTRACT We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenesS85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25°C, and retains about 70% of its maximum activity at 0°C, while EGD has a temperature optimum of 35°C and retains only about 18% of its maximal activity at 0°C. When assayed at 4°C, CelG exhibits a 33-fold-higher kcat value and a 73-fold-higher physiological efficiency (kcat/Km ) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or thatF. succinogenes originated from the marine environment.

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Gatti-Lafranconi, Pietro, Serena Caldarazzo, Lilia Alberghina, and Marina Lotti. "Directed evolution of a cold-active lipolytic enzyme." Journal of Biotechnology 131, no. 2 (September 2007): S117. http://dx.doi.org/10.1016/j.jbiotec.2007.07.204.

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Čanak, Iva, Adrienn Berkics, Nikolett Bajcsi, Monika Kovacs, Agnes Belak, Renata Teparić, Anna Maraz, and Vladimir Mrša. "Purification and Characterization of a Novel Cold-Active Lipase from the Yeast Candida zeylanoides." Journal of Molecular Microbiology and Biotechnology 25, no. 6 (2015): 403–11. http://dx.doi.org/10.1159/000442818.

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Cold-active lipases have attracted attention in recent years due to their potential applications in reactions requiring lower temperatures. Both bacterial and fungal lipases have been investigated, each having distinct advantages for particular applications. Among yeasts, cold-active lipases from the genera Candida, Yarrowia, Rhodotorula, and Pichia have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from Candida zeylanoides isolated from refrigerated poultry meat are described. Heat-sterilized olive oil was found to be the best lipase biosynthesis inducer, while nonionic detergents were not effective. The enzyme was purified to homogeneity using hydrophobic chromatography and its enzymatic properties were tested. Pure enzyme activity at 7°C was about 60% of the maximal activity at 27°C. The enzyme had rather good activity at higher temperatures, as well. Optimal pH of pure lipase was between 7.3 and 8.2, while the enzyme from the crude extract had an optimum pH of about 9.0. The enzyme was sensitive to high ionic strength and lost most of its activity at high salt concentrations. Due to the described properties, cold-active C. zeylanoides lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.

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Isaksen, Geir Villy, Johan Åqvist, and Bjørn Olav Brandsdal. "Enzyme surface rigidity tunes the temperature dependence of catalytic rates." Proceedings of the National Academy of Sciences 113, no. 28 (June 27, 2016): 7822–27. http://dx.doi.org/10.1073/pnas.1605237113.

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The structural origin of enzyme adaptation to low temperature, allowing efficient catalysis of chemical reactions even near the freezing point of water, remains a fundamental puzzle in biocatalysis. A remarkable universal fingerprint shared by all cold-active enzymes is a reduction of the activation enthalpy accompanied by a more negative entropy, which alleviates the exponential decrease in chemical reaction rates caused by lowering of the temperature. Herein, we explore the role of protein surface mobility in determining this enthalpy–entropy balance. The effects of modifying surface rigidity in cold- and warm-active trypsins are demonstrated here by calculation of high-precision Arrhenius plots and thermodynamic activation parameters for the peptide hydrolysis reaction, using extensive computer simulations. The protein surface flexibility is systematically varied by applying positional restraints, causing the remarkable effect of turning the cold-active trypsin into a variant with mesophilic characteristics without changing the amino acid sequence. Furthermore, we show that just restraining a key surface loop causes the same effect as a point mutation in that loop between the cold- and warm-active trypsin. Importantly, changes in the activation enthalpy–entropy balance of up to 10 kcal/mol are almost perfectly balanced at room temperature, whereas they yield significantly higher rates at low temperatures for the cold-adapted enzyme.

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Liu, W. Y., Y. W. Shi, X. Q. Wang, and K. Lou. "Isolation and identification of a strain producing cold-adapted β galactosidase, and purification and characterisation of the enzyme." Czech Journal of Food Sciences 26, No. 4 (August 22, 2008): 284–90. http://dx.doi.org/10.17221/31/2008-cjfs.

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Enzymes with high specific activities at low temperatures have potential uses in the food industry. Cold-adapted microorganisms are potentially useful sources of cold-active enzyme. To find cold-adapted β-galactosidase, we isolated several cold-adapted microorganisms from glacier zone soil. One cold-adapted β-galactosidase producing strain was obtained. The biochemical characteristics and the results of 16S rDNA sequencing identified the strain as Rahnella aquatilis. The enzyme was purified by column chromatography after which a single protein band migrating near 60 kDa was observed by means of SDS-PAGE. The β-galactosidase was optimally active at 35°C and at pH 6.5 when assayed with o-nitrophenyl-β-D-galactopyrano-side as substrate. The enzyme activity was sensitive to temperatures above 40°C and was undetectable at 45°C. Metal ions Mn2+and K+ activated the enzyme while Cu2+, Zn2+, Fe3+, and Al3+ inhibited the activity. The enzyme was also assayed for lactose hydrolysis. When milk is treated with the enzyme at 30°C for 2 h, the degree of lactose hydrolysis can reach 80%. It has, thus, potential applications in the food industry.

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Siddiqui, Khawar S., Georges Feller, Salvino D'Amico, Charles Gerday, Laura Giaquinto, and Ricardo Cavicchioli. "The Active Site Is the Least Stable Structure in the Unfolding Pathway of a Multidomain Cold-Adapted α-Amylase." Journal of Bacteriology 187, no. 17 (September 1, 2005): 6197–205. http://dx.doi.org/10.1128/jb.187.17.6197-6205.2005.

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ABSTRACT The cold-active α-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30°C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3°C to 70°C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12°C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70°C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.

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Coombs, Jonna M., and Jean E. Brenchley. "Biochemical and Phylogenetic Analyses of a Cold-Active β-Galactosidase from the Lactic Acid Bacterium Carnobacterium piscicola BA." Applied and Environmental Microbiology 65, no. 12 (December 1, 1999): 5443–50. http://dx.doi.org/10.1128/aem.65.12.5443-5450.1999.

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ABSTRACT We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A β-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal) at 4°C and possessed higher activity in crude cell lysates at 25 than at 37°C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an α-galactosidase and two β-galactosidases. The larger of the two β-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30°C, and it was inactivated at 40°C in 10 min. TheKm of freshly purified enzyme at 30°C was 1.7 mM, and the V max was 450 μmol · min−1 · mg−1 with o-nitrophenyl β-d-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme fromBacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.

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Maharana, Abhas Kumar. "EXTRACELLULAR COLD ACTIVE ENDOGLUCANASE AND PIGMENT PRODUCING PSYCHROTOLERANT PENICILLIUM PINOPHILUM." International Journal of Pharmacy and Pharmaceutical Sciences 8, no. 10 (August 12, 2016): 164. http://dx.doi.org/10.22159/ijpps.2016v8i10.13441.

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Objective: The objective of the present study was on Penicillium pinophilum strain F2 from soil samples of Jammu city having the potentiality to produce alkaline cold active endoglucanase and pigment.Methods: Penicillium pinophilum strain F2, a psychrotolerant micro-fungus was isolated from soil of Jammu city, India by taking Czapek’s Dox agar incubated at 15 °C. The strain was screened for production of cold active enzymes by taking various substrates at 15 °C. Final production was done for cold active endoglucanase by using sugarcane bagasse and ground nut shell as substrates. Besides, the strain was also able to produce red color pigment at a low temperature which was further studied to optimize its production by changing pH and growth medium. The produced pigment was used for dyeing of wool and silk, and absorption percentages were also calculated.Results: Screening for the production of cold active enzymes revealed it as a good producer of cellulose followed by lipase and amylase. Endoglucanase production revealed the total enzyme titer (total enzyme activity) was found to be 5.032 folds higher in sugarcane bagasse (38.91 units) than groundnut shell (7.732 units). Endoglucanase activity was maximum 9.82±0.33 units/ml and 2.29±0.31 units/ml after 120 h of incubation at 15 °C by sugarcane bagasse and groundnut shells, respectively. Red color pigment production was maxima at pH 5 in Czapek’s Dox broth. Maximum absorption percentage was seen by the treatment soaked with mordant, i.e. 5% CuSO4 (51.52%) and without a mordant, it showed about 45.54%.Conclusion: Due to the above unique features and capability to produce cold active endoglucanase and pigment by strain F2, can be used significantly in various industries.

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Mohamad Ali, Mohd Shukuri, Siti Farhanie Mohd Fuzi, Menega Ganasen, Raja Noor Zaliha Raja Abdul Rahman, Mahiran Basri, and Abu Bakar Salleh. "Structural Adaptation of Cold-Active RTX Lipase fromPseudomonassp. Strain AMS8 Revealed via Homology and Molecular Dynamics Simulation Approaches." BioMed Research International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/925373.

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The psychrophilic enzyme is an interesting subject to study due to its special ability to adapt to extreme temperatures, unlike typical enzymes. Utilizing computer-aided software, the predicted structure and function of the enzyme lipase AMS8 (LipAMS8) (isolated from the psychrophilicPseudomonassp., obtained from the Antarctic soil) are studied. The enzyme shows significant sequence similarities with lipases fromPseudomonassp. MIS38 andSerratia marcescens. These similarities aid in the prediction of the 3D molecular structure of the enzyme. In this study, 12 ns MD simulation is performed at different temperatures for structural flexibility and stability analysis. The results show that the enzyme is most stable at 0°C and 5°C. In terms of stability and flexibility, the catalytic domain (N-terminus) maintained its stability more than the noncatalytic domain (C-terminus), but the non-catalytic domain showed higher flexibility than the catalytic domain. The analysis of the structure and function of LipAMS8 provides new insights into the structural adaptation of this protein at low temperatures. The information obtained could be a useful tool for low temperature industrial applications and molecular engineering purposes, in the near future.

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Necula-Petrareanu, Georgiana, Paris Lavin, Victoria Ioana Paun, Giulia Roxana Gheorghita, Alina Vasilescu, and Cristina Purcarea. "Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002." Fermentation 8, no. 1 (December 27, 2021): 7. http://dx.doi.org/10.3390/fermentation8010007.

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Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 °C for 18 days. F-ALDH used both NAD+ and NADP+ and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory KM and kcat values and the highest catalytic efficiency (0.50 µM−1 s−1) for isovaleraldehyde. The enzyme was active in the 4–60 °C-temperature interval, with an optimal pH of 9.5, and a preference for NAD+-dependent reactions. Arrhenius plots of both NAD(P)+-dependent reactions indicated conformational changes occurring at 30 °C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.

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Tsigos, Iason, Konstantinos Mavromatis, Maria Tzanodaskalaki, Charalambos Pozidis, Michael Kokkinidis, and Vassilis Bouriotis. "Engineering the properties of a cold active enzyme through rational redesign of the active site." European Journal of Biochemistry 268, no. 19 (October 1, 2001): 5074–80. http://dx.doi.org/10.1046/j.0014-2956.2001.02432.x.

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Guo, Chenchen, Rikuan Zheng, Ruining Cai, Chaomin Sun, and Shimei Wu. "Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium." Microorganisms 9, no. 4 (April 10, 2021): 802. http://dx.doi.org/10.3390/microorganisms9040802.

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The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes.

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Laye, Victoria J., Ram Karan, Jong-Myoung Kim, Wolf T. Pecher, Priya DasSarma, and Shiladitya DasSarma. "Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic β-galactosidase." Proceedings of the National Academy of Sciences 114, no. 47 (November 6, 2017): 12530–35. http://dx.doi.org/10.1073/pnas.1711542114.

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The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic β-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or β-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

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Lee, Charles C., Michael Smith, Rena E. Kibblewhite-Accinelli, Tina G. Williams, Kurt Wagschal, George H. Robertson, and Dominic W. S. Wong. "Isolation and Characterization of a Cold-Active Xylanase Enzyme from Flavobacterium sp." Current Microbiology 52, no. 2 (January 31, 2006): 112–16. http://dx.doi.org/10.1007/s00284-005-4583-9.

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Xu, Ying, Georges Feller, Charles Gerday, and Nicolas Glansdorff. "Moritella Cold-Active Dihydrofolate Reductase: Are There Natural Limits to Optimization of Catalytic Efficiency at Low Temperature?" Journal of Bacteriology 185, no. 18 (September 15, 2003): 5519–26. http://dx.doi.org/10.1128/jb.185.18.5519-5526.2003.

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ABSTRACT Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2°C or less. The enzyme is monomeric (M r = 18,291), 55% identical to its Escherichia coli counterpart, and displays Tm and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0°C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5°C these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (k cat /Km ) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.

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Núñez-Montero, Kattia, Rodrigo Salazar, Andrés Santos, Olman Gómez-Espinoza, Scandar Farah, Claudia Troncoso, Catalina Hoffmann, Damaris Melivilu, Felipe Scott, and Leticia Barrientos Díaz. "Antarctic Rahnella inusitata: A Producer of Cold-Stable β-Galactosidase Enzymes." International Journal of Molecular Sciences 22, no. 8 (April 16, 2021): 4144. http://dx.doi.org/10.3390/ijms22084144.

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There has been a recent increase in the exploration of cold-active β-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active β-galactosidases. A screening revealed 81 out of 304 strains with β-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41–62% of its β-galactosidase activity in the cold (4 °C–15 °C). Three β-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzymes exhibited higher lactose predicted affinity than the commercial control enzyme from Aspergillus oryzae. Hence, this work reports a new Rahnella inusitata strain from the Antarctic continent as a prominent cold-active β-galactosidase producer.

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Orlando, Marco, Sandra Pucciarelli, and Marina Lotti. "Endolysins from Antarctic Pseudomonas Display Lysozyme Activity at Low Temperature." Marine Drugs 18, no. 11 (November 20, 2020): 579. http://dx.doi.org/10.3390/md18110579.

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Organisms specialized to thrive in cold environments (so-called psychrophiles) produce enzymes with the remarkable ability to catalyze chemical reactions at low temperature. Cold activity relies on adaptive changes in the proteins’ sequence and structural organization that result in high conformational flexibility. As a consequence of flexibility, several such enzymes are inherently heat sensitive. Cold-active enzymes are of interest for application in a number of bioprocesses, where cold activity coupled with easy thermal inactivation can be of advantage. We describe the biochemical and functional properties of two glycosyl hydrolases (named LYS177 and LYS188) of family 19 (GH19), identified in the genome of an Antarctic marine Pseudomonas. Molecular evolutionary analysis placed them in a group of characterized GH19 endolysins active on lysozyme substrates, such as peptidoglycan. Enzyme activity peaks at about 25–35 °C and 40% residual activity is retained at 5 °C. LYS177 and LYS188 are thermolabile, with Tm of 52 and 45 °C and half-lives of 48 and 12 h at 37 °C, respectively. Bioinformatics analyses suggest that low heat stability may be associated to temperature-driven increases in local flexibility occurring mainly in a specific region of the polypeptide that is predicted to contain hot spots for aggregation.

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Zhang, Zhi-Bi, Yuan-Ling Xia, Guang-Heng Dong, Yun-Xin Fu, and Shu-Qun Liu. "Exploring the Cold-Adaptation Mechanism of Serine Hydroxymethyltransferase by Comparative Molecular Dynamics Simulations." International Journal of Molecular Sciences 22, no. 4 (February 11, 2021): 1781. http://dx.doi.org/10.3390/ijms22041781.

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Cold-adapted enzymes feature a lower thermostability and higher catalytic activity compared to their warm-active homologues, which are considered as a consequence of increased flexibility of their molecular structures. The complexity of the (thermo)stability-flexibility-activity relationship makes it difficult to define the strategies and formulate a general theory for enzyme cold adaptation. Here, the psychrophilic serine hydroxymethyltransferase (pSHMT) from Psychromonas ingrahamii and its mesophilic counterpart, mSHMT from Escherichia coli, were subjected to μs-scale multiple-replica molecular dynamics (MD) simulations to explore the cold-adaptation mechanism of the dimeric SHMT. The comparative analyses of MD trajectories reveal that pSHMT exhibits larger structural fluctuations and inter-monomer positional movements, a higher global flexibility, and considerably enhanced local flexibility involving the surface loops and active sites. The largest-amplitude motion mode of pSHMT describes the trends of inter-monomer dissociation and enlargement of the active-site cavity, whereas that of mSHMT characterizes the opposite trends. Based on the comparison of the calculated structural parameters and constructed free energy landscapes (FELs) between the two enzymes, we discuss in-depth the physicochemical principles underlying the stability-flexibility-activity relationships and conclude that (i) pSHMT adopts the global-flexibility mechanism to adapt to the cold environment and, (ii) optimizing the protein-solvent interactions and loosening the inter-monomer association are the main strategies for pSHMT to enhance its flexibility.

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Siddiqui, Khawar Sohail, Anne Poljak, Michael Guilhaus, Georges Feller, Salvino D'Amico, Charles Gerday, and Ricardo Cavicchioli. "Role of Disulfide Bridges in the Activity and Stability of a Cold-Active α-Amylase." Journal of Bacteriology 187, no. 17 (September 1, 2005): 6206–12. http://dx.doi.org/10.1128/jb.187.17.6206-6212.2005.

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ABSTRACT The cold-adapted α-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30°C and unfolds reversibly and sequentially with two transitions at temperatures below 12°C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with β-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.

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Krishnan, Abiramy, Zazali Alias, Peter Convey, Marcelo González-Aravena, Jerzy Smykla, Mohammed Rizman-Idid, and Siti Aisyah Alias. "Temperature and pH Profiling of Extracellular Amylase from Antarctic and Arctic Soil Microfungi." Fermentation 8, no. 11 (November 3, 2022): 601. http://dx.doi.org/10.3390/fermentation8110601.

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While diversity studies and screening for enzyme activities are important elements of understanding fungal roles in the soil ecosystem, extracting and purifying the target enzyme from the fungal cellular system is also required to characterize the enzyme. This is, in particular, necessary before developing the enzyme for industrial-scale production. In the present study, partially purified α-amylase was obtained from strains of Pseudogymnoascus sp. obtained from Antarctic and Arctic locations. Partially purified α-amylases from these polar fungi exhibited very similar characteristics, including being active at 15 °C, although having a small difference in optimum pH. Both fungal taxa are good candidates for the potential application of cold-active enzymes in biotechnological industries, and further purification and characterization steps are now required. The α-amylases from polar fungi are attractive in terms of industrial development because they are active at lower temperatures and acidic pH, thus potentially creating energy and cost savings. Furthermore, they prevent the production of maltulose, which is an undesirable by-product often formed under alkaline conditions. Psychrophilic amylases from the polar Pseudogymnoascus sp. investigated in the present study could provide a valuable future contribution to biotechnological applications.

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Rutkiewicz, Maria, Anna Bujacz, Marta Wanarska, Anna Wierzbicka-Wos, and Hubert Cieslinski. "Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB." International Journal of Molecular Sciences 20, no. 17 (September 3, 2019): 4301. http://dx.doi.org/10.3390/ijms20174301.

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ArthβDG is a dimeric, cold-adapted β-d-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthβDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthβDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The resulting three-dimensional structures of complexes: ArthβDG_E441Q/LACs and ArthβDG/IPTG (ligand bound in shallow mode) and structures of complexes ArthβDG_E441Q/LACd, ArthβDG/ONPG (ligands bound in deep mode), and galactose ArthβDG/GAL and their analysis enabled structural characterization of the hydrolysis reaction mechanism. Furthermore, comparative analysis with mesophilic analogs revealed the most striking differences in catalysis mechanisms. The key role in substrate transfer from shallow to deep binding mode involves rotation of the F581 side chain. It is worth noting that the 10-aa loop restricting access to the active site in mesophilic GH2 βDGs, in ArthβDG is moved outward. This facilitates access of substrate to active site. Such a permanent exposure of the entrance to the active site may be a key factor for improved turnover rate of the cold adapted enzyme and thus a structural feature related to its cold adaptation.

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Taguchi, Seiichi, Shingo Komada, and Haruo Momose. "The Complete Amino Acid Substitutions at Position 131 That Are Positively Involved in Cold Adaptation of Subtilisin BPN′." Applied and Environmental Microbiology 66, no. 4 (April 1, 2000): 1410–15. http://dx.doi.org/10.1128/aem.66.4.1410-1415.2000.

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ABSTRACT To ascertain whether position 131 of a mesophilic protease, subtilisin BPN′, is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492–495, 1998), a full set of subtilisin BPN′ mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10°C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN′ and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k cat/Km ) at 10°C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k cat and a decrease inKm . All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10°C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60°C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.

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Gheorghita, Giulia Roxana, Victoria Ioana Paun, Simona Neagu, Gabriel-Mihai Maria, Madalin Enache, Cristina Purcarea, Vasile I. Parvulescu, and Madalina Tudorache. "Cold-Active Lipase-Based Biocatalysts for Silymarin Valorization through Biocatalytic Acylation of Silybin." Catalysts 11, no. 11 (November 17, 2021): 1390. http://dx.doi.org/10.3390/catal11111390.

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Extremophilic biocatalysts represent an enhanced solution in various industrial applications. Integrating enzymes with high catalytic potential at low temperatures into production schemes such as cold-pressed silymarin processing not only brings value to the silymarin recovery from biomass residues, but also improves its solubility properties for biocatalytic modification. Therefore, a cold-active lipase-mediated biocatalytic system has been developed for silybin acylation with methyl fatty acid esters based on the extracellular protein fractions produced by the psychrophilic bacterial strain Psychrobacter SC65A.3 isolated from Scarisoara Ice Cave (Romania). The extracellular production of the lipase fraction was enhanced by 1% olive-oil-enriched culture media. Through multiple immobilization approaches of the cold-active putative lipases (using carbodiimide, aldehyde-hydrazine, or glutaraldehyde coupling), bio-composites (S1–5) with similar or even higher catalytic activity under cold-active conditions (25 °C) have been synthesized by covalent attachment to nano-/micro-sized magnetic or polymeric resin beads. Characterization methods (e.g., FTIR DRIFT, SEM, enzyme activity) strengthen the biocatalysts’ settlement and potential. Thus, the developed immobilized biocatalysts exhibited between 80 and 128% recovery of the catalytic activity for protein loading in the range 90–99% and this led to an immobilization yield up to 89%. The biocatalytic acylation performance reached a maximum of 67% silybin conversion with methyl decanoate acylating agent and nano-support immobilized lipase biocatalyst.

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Baltulionis, Gediminas, Maeve O'Neill, Denise Gallagher, Andrew Ellis, Dimitrios Charalampapolous, and Kimberly Watson. "Exploration of strategies to altering thermal properties of industrial enzymes." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C435. http://dx.doi.org/10.1107/s2053273314095643.

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Endoproteases and exopeptidases occupy a pivotal position with respect to their commercial applications in food (e.g. as additives in whey protein processing) and, as additives in detergent, textile and a number of other industries. Food processing at low temperatures by cold-active enzymes has many advantages as it minimises undesirable chemical reactions as well as the risk of microbial contamination. Cold-active enzymes were found to display higher specific activity and catalytic efficiency resulting in lower quantities of enzyme required and significantly shortened processing times. On the other hand, industrial hydrolysis typically occur at elevated temperatures due to the faster reaction rates, increased substrate solubility and thermophilic biocatalysts are required to maintain reactions at very high temperatures. The aim of our work is to exploit structure-function relationships of extremophilic enzymes that give rise to novel industrially useful proteases. We are using the high-throughput capability of the Oxford Protein Purification Facility (OPPF) to study a number of structural modifications leading to protein extremophilic functional behaviour. Several strategies to effectively alter the thermal properties of commercial serine endoproteases and aminopeptidases are being tested including; i) site directed mutagenesis targeted to reduce quantity of prolines, salt bridges, S-S bridges, and hydrophobic clusters, and ii) iterative saturation mutagenesis relying on residues with low B-factors (local rigidity) according to available 3D structures are currently being implemented. Our recent results reveal the potential for an emerging universal mechanism to modify the thermostability of any given enzyme.

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Kamaruddin, Shazilah, Rohaiza Ahmad Redzuan, Nurulermila Minor, Wan Mohd Khairulikhsan Wan Seman, Mahzan Md Tab, Nardiah Rizwana Jaafar, Nazahiyah Ahmad Rodzli, et al. "Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12." Catalysts 12, no. 7 (June 30, 2022): 722. http://dx.doi.org/10.3390/catal12070722.

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Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.

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ABD LATIP, MUHAMMAD ASYRAF, SITI AISYAH ALIAS, SMYKLA JERZY, FARIDAH YUSOF, MOHD AZRUL NAIM MOHAMAD, and NOOR FAIZUL HADRY NORDIN. "DISCOVERY OF COLD-ACTIVE PROTEASE FROM PSYCHROPHILIC BACTERIA ISOLATED FROM ANTARCTIC REGION FOR BIO-PROSPECTING." Malaysian Applied Biology 49, no. 1 (June 30, 2020): 55–60. http://dx.doi.org/10.55230/mabjournal.v49i1.1654.

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The Antarctic region is a new frontier as natural sources for bio-prospecting purposes. Its extreme cold temperature may provide unique enzyme characteristics that have valuable potential for industrial and biotechnological applications. This study was designed to discover proteases that are activate and can work at very low temperatures. Soil samples from the Antarctic region were screened for protease activity on skim milk agar at 4°C. Bacteria that showed clear halo zone around the colonies were selected and identified through 16S rDNA sequencing. Out of 35 bacteria, 10 bacteria that showed rapid halo zone formation were selected and further analyzed by enzymatic assay. By using azocasein as a substrate, the reaction was measured using spectrophotometer at OD340 nm. Based on the 16S rDNA sequence, phylogenetic analysis showed that 88% of the bacteria producing protease were from Pseudomonas sp., 9% from Arthrobacter sp. and 3% from Paenibacillus sp. For enzymatic assay analysis, sample SC8 showed the highest protease activity compared to other 10 samples. This preliminary study successfully demonstrated cold active protease producers that can be further investigated for bioprospecting. In future, purification and characterization of this enzyme is required in order to optimize the enzyme activity.

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Ji, Xiu Ling, Muhammad Kamran Taj, Xiao Bo Lu, Lian Bing Lin, Qi Zhang, and Yun Lin Wei. "Purification and Characterization of an Extracellular Cold-Active Protease Produced by the Psychrotrophic Bacterium serratia Sp. WJ39." Applied Mechanics and Materials 618 (August 2014): 330–34. http://dx.doi.org/10.4028/www.scientific.net/amm.618.330.

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Proteases have diverse applications in a wide variety of industries, such as in detergent, leather, food, pharmaceutical and silk. The extracellular cold-active protease was purified from the psychrotrophic bacteriumSerratiasp. WJ39 from a meat factory. The protease was cold-active with a molecular mass of 47.6 kDa estimated on SDS-PAGE. It showed an optimal activity at pH of 8 and was stable at pH 6 to 10, while its optimal temperature was 37°C and it was stable at 0-25°C, even remained 35% residual activity at 0°C. The protease was totally inhibited by PMSF which was telling that the purified enzyme was a serine protease. The properties like moderate thermostability, activity in a broad pH range and resistance to metal ions make this enzyme a suitable candidate for the possible use in food and leather industry.

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Ramya, L. N., and K. K. Pulicherla. "Molecular insights into cold active polygalacturonase enzyme for its potential application in food processing." Journal of Food Science and Technology 52, no. 9 (November 26, 2014): 5484–96. http://dx.doi.org/10.1007/s13197-014-1654-6.

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29

Kuddus, Mohammed, and Pramod W. Ramteke. "A cold-active extracellular metalloprotease from Curtobacterium luteum (MTCC 7529): Enzyme production and characterization." Journal of General and Applied Microbiology 54, no. 6 (2008): 385–92. http://dx.doi.org/10.2323/jgam.54.385.

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30

Lee, Charles C., Rena E. Kibblewhite-Accinelli, Kurt Wagschal, George H. Robertson, and Dominic W. S. Wong. "Cloning and characterization of a cold-active xylanase enzyme from an environmental DNA library." Extremophiles 10, no. 4 (March 11, 2006): 295–300. http://dx.doi.org/10.1007/s00792-005-0499-3.

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31

Kuddus, Mohammed, and Pramod W. Ramteke. "Cold-active extracellular alkaline protease from an alkaliphilicStenotrophomonas maltophilia: production of enzyme and its industrial applications." Canadian Journal of Microbiology 55, no. 11 (November 2009): 1294–301. http://dx.doi.org/10.1139/w09-089.

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A novel psychro-tolerant bacterium Stenotrophomonas maltophilia (MTCC 7528) with an ability to produce extracellular, cold-active, alkaline, and detergent-stable protease was isolated from soil samples obtained from Gangotri glacier, Western Himalaya, India. The culture conditions for higher protease production were optimized with respect to incubation time, agitation, substrate, pH, and temperature. Maximum protease production of 56.2 U·mL–1was achieved in the medium at 20 °C and pH 9.0 after 120 h incubation. The protease was partially purified by ion-exchange chromatography and approximately 55-fold purification was achieved. The purified enzyme was a 75 kDa protease with maximum activity and stability at pH 10 and 20 °C. The activity of enzyme is stimulated by Mn2+and inhibited completely by metalloprotease inhibitors, indicating that it is a metalloprotease. The protease showed excellent stability and compatibility with commercial detergents and exhibited high efficiency for the removal of different types of protein-containing stains at low temperature. The wash performance analysis of blood and grass stains on cotton fabric showed an increase in reflectance by 26% and 23%, respectively, after treatment with enzyme in comparison to detergent only. These results indicate that it may be a potential component to use as a detergent additive for cold washing and in environmental bioremediation in cold regions.

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Miao, Li-Li, Yan-Jie Hou, Hong-Xia Fan, Jie Qu, Chao Qi, Ying Liu, De-Feng Li, and Zhi-Pei Liu. "Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus." Applied and Environmental Microbiology 82, no. 7 (January 22, 2016): 2021–30. http://dx.doi.org/10.1128/aem.03158-15.

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ABSTRACTPsychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacteriumMicrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, theTm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

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Le, Ly Thi Huong Luu, Wanki Yoo, Changwoo Lee, Ying Wang, Sangeun Jeon, Kyeong Kyu Kim, Jun Hyuck Lee, and T. Doohun Kim. "Molecular Characterization of a Novel Cold-Active Hormone-Sensitive Lipase (HaHSL) from Halocynthiibacter Arcticus." Biomolecules 9, no. 11 (November 5, 2019): 704. http://dx.doi.org/10.3390/biom9110704.

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Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from Halocynthiibacter arcticus (HaHSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HaHSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HaHSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp23, Tyr74, Phe78, Trp83, and Phe245) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr74 played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HaHSL from H. arcticus, which can be efficiently and sustainably used in a wide range of biotechnological applications.

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Monsalves, María T., Gabriela P. Ollivet-Besson, Maximiliano J. Amenabar, and Jenny M. Blamey. "Isolation of a Psychrotolerant and UV-C-Resistant Bacterium from Elephant Island, Antarctica with a Highly Thermoactive and Thermostable Catalase." Microorganisms 8, no. 1 (January 10, 2020): 95. http://dx.doi.org/10.3390/microorganisms8010095.

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Microorganisms present in Antarctica have to deal not only with cold temperatures but also with other environmental conditions, such as high UV radiation, that trigger the generation of reactive oxygen species. Therefore, Antarctic microorganisms must have an important antioxidant defense system to prevent oxidative damage. One of these defenses are antioxidant enzymes, such as catalase, which is involved in the detoxification of hydrogen peroxide produced under oxidative conditions. Here, we reported the isolation and partial characterization of an Antarctic bacterium belonging to the Serratia genus that was resistant to UV-C radiation and well-adapted to cold temperatures. This microorganism, denominated strain I1P, was efficient at decreasing reactive oxygen species levels produced after UV-C irradiation. Genomic and activity assays suggested that the enzymatic antioxidant defense mechanisms of strain I1P, especially its catalase enzyme, may confer UV resistance. This catalase was active in a wide range of temperatures (20–70 °C), showing optimal activity at 50 °C (at pH 7.0), a remarkable finding considering its psychrotolerant origin. In addition, this enzyme was thermostable, retaining around 60% of its activity after 6 h of incubation at 50 °C. The antioxidant defense systems of strain I1P, including its surprisingly thermoactive and thermostable catalase enzyme, make this microorganism a good source of biocompounds with potential biotechnological applications.

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35

Xu, Ying, Ziyuan Liang, Christianne Legrain, Hans J. Rüger, and Nicolas Glansdorff. "Evolution of Arginine Biosynthesis in the Bacterial Domain: Novel Gene-Enzyme Relationships from Psychrophilic Moritella Strains (Vibrionaceae) and Evolutionary Significance of N-α-Acetyl Ornithinase." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1609–15. http://dx.doi.org/10.1128/jb.182.6.1609-1615.2000.

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ABSTRACT In the arginine biosynthetic pathway of the vast majority of prokaryotes, the formation of ornithine is catalyzed by an enzyme transferring the acetyl group of N-α-acetylornithine to glutamate (ornithine acetyltransferase [OATase]) (argJencoded). Only two exceptions had been reported—theEnterobacteriaceae and Myxococcus xanthus(members of the γ and δ groups of the classProteobacteria, respectively)—in which ornithine is produced from N-α-acetylornithine by a deacylase, acetylornithinase (AOase) (argE encoded). We have investigated the gene-enzyme relationship in the arginine regulons of two psychrophilic Moritella strains belonging to theVibrionaceae, a family phylogenetically related to theEnterobacteriaceae. Most of the arg genes were found to be clustered in one continuous sequence divergently transcribed in two wings, argE and argCBFGH(A)[“H(A)” indicates that the argininosuccinase gene consists of a part homologous to known argH sequences and of a 3′ extension able to complement an Escherichia colimutant deficient in the argA gene, encodingN-α-acetylglutamate synthetase, the first enzyme committed to the pathway]. Phylogenetic evidence suggests that this new clustering pattern arose in an ancestor common toVibrionaceae and Enterobacteriaceae, where OATase was lost and replaced by a deacylase. The AOase and ornithine carbamoyltransferase of these psychrophilic strains both display distinctly cold-adapted activity profiles, providing the first cold-active examples of such enzymes.

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Bakunina, Irina, Lubov Slepchenko, Stanislav Anastyuk, Vladimir Isakov, Galina Likhatskaya, Natalya Kim, Liudmila Tekutyeva, Oksana Son, and Larissa Balabanova. "Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants." Marine Drugs 16, no. 10 (September 24, 2018): 349. http://dx.doi.org/10.3390/md16100349.

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A novel wild-type recombinant cold-active α-d-galactosidase (α-PsGal) from the cold-adapted marine bacterium Pseudoalteromonas sp. KMM 701, and its mutants D451A and C494N, were studied in terms of their structural, physicochemical, and catalytic properties. Homology models of the three-dimensional α-PsGal structure, its active center, and complexes with D-galactose were constructed for identification of functionally important amino acid residues in the active site of the enzyme, using the crystal structure of the α-galactosidase from Lactobacillus acidophilus as a template. The circular dichroism spectra of the wild α-PsGal and mutant C494N were approximately identical. The C494N mutation decreased the efficiency of retaining the affinity of the enzyme to standard p-nitrophenyl-α-galactopiranoside (pNP-α-Gal). Thin-layer chromatography, matrix-assisted laser desorption/ionization mass spectrometry, and nuclear magnetic resonance spectroscopy methods were used to identify transglycosylation products in reaction mixtures. α-PsGal possessed a narrow acceptor specificity. Fructose, xylose, fucose, and glucose were inactive as acceptors in the transglycosylation reaction. α-PsGal synthesized -α(1→6)- and -α(1→4)-linked galactobiosides from melibiose as well as -α(1→6)- and -α(1→3)-linked p-nitrophenyl-digalactosides (Gal2-pNP) from pNP-α-Gal. The D451A mutation in the active center completely inactivated the enzyme. However, the substitution of C494N discontinued the Gal-α(1→3)-Gal-pNP synthesis and increased the Gal-α(1→4)-Gal yield compared to Gal-α(1→6)-Gal-pNP.

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Soror, Sameh H., V. Verma, Ren Rao, Shafaq Rasool, S. Koul, G. N. Qazi, and John Cullum. "A cold-active esterase of Streptomyces coelicolor A3(2): from genome sequence to enzyme activity." Journal of Industrial Microbiology & Biotechnology 34, no. 8 (June 7, 2007): 525–31. http://dx.doi.org/10.1007/s10295-007-0224-6.

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38

Bujacz, Anna, Maria Rutkiewicz-Krotewicz, Karolina Nowakowska-Sapota, and Marianna Turkiewicz. "Crystal structure and enzymatic properties of a broad substrate-specificity psychrophilic aminotransferase from the Antarctic soil bacteriumPsychrobactersp. B6." Acta Crystallographica Section D Biological Crystallography 71, no. 3 (February 26, 2015): 632–45. http://dx.doi.org/10.1107/s1399004714028016.

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Aminotransferases (ATs) are enzymes that are commonly used in the chemical and pharmaceutical industries for the synthesis of natural and non-natural amino acids by transamination reactions. Currently, the easily accessible enzymes from mesophilic organisms are most commonly used; however, for economical and ecological reasons the utilization of aminotransferases from psychrophiles would be more advantageous, as their optimum reaction temperature is usually significantly lower than for the mesophilic ATs. Here, gene isolation, protein expression, purification, enzymatic properties and structural studies are reported for the cold-active aromatic amino-acid aminotransferase (PsyArAT) fromPsychrobactersp. B6, a psychrotrophic, Gram-negative strain from Antarctic soil. Preliminary computational analysis indicated dual functionality of the enzyme through the ability to utilize both aromatic amino acids and aspartate as substrates. This postulation was confirmed by enzymatic activity tests, which showed that it belonged to the class EC 2.6.1.57. The first crystal structures of a psychrophilic aromatic amino-acid aminotransferase have been determined at resolutions of 2.19 Å for the native enzyme (PsyArAT) and 2.76 Å for its complex with aspartic acid (PsyArAT/D). Both types of crystals grew in the monoclinic space groupP21under slightly different crystallization conditions. ThePsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of thePsyArAT/D complex included four dimers showing different stages of the transamination reaction.

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Nespolo, Roberto F., Leonardo D. Bacigalupe, Pablo Sabat, and Francisco Bozinovic. "Interplay among energy metabolism, organ mass and digestive enzyme activity in the mouse-opossum Thylamys elegans: the role of thermal acclimation." Journal of Experimental Biology 205, no. 17 (September 1, 2002): 2697–703. http://dx.doi.org/10.1242/jeb.205.17.2697.

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SUMMARY The potential for thermal acclimation in marsupials is controversial. Initial studies suggest that the thermoregulatory maximum metabolic rate (MMR)in metatherians cannot be changed by thermal acclimation. Nevertheless, recent studies reported conspicuous seasonality in both MMR and in basal metabolic rate (BMR). We studied the role of thermal acclimation in the Chilean mouse-opossum, Thylamys elegans, by measuring MMR and BMR before and after acclimation to cold or warm conditions. Following acclimation we also measured the mass of metabolically active organs, and the activity of a key digestive enzyme, aminopeptidase-N. No significant effect of thermal acclimation (i.e. between cold- and warm-acclimated animals) was observed for body mass, MMR, body temperature or factorial aerobic scope. However, the BMR of cold-acclimated animals was 30 % higher than for warm-acclimated individuals. For organ mass, acclimation had a significant effect on the dry mass of caecum, liver and kidneys only. Stepwise multiple regression using pooled data showed that 71 % of the variation in BMR is explained by the digestive organs. Overall, these results suggest that MMR is a rather rigid variable, while BMR shows plasticity. It seems that T. elegans cannot respond to thermal acclimation by adjusting its processes of energy expenditure (i.e. thermogenic capacity and mass of metabolically active organs). The lack of any significant difference in aminopeptidase-N specific activity between warm- and cold-acclimated animals suggests that this response is mainly quantitative (i.e. cell proliferation) rather than qualitative (i.e. differential enzyme expression). Finally, as far as we know, this study is the first to report the effects of thermal acclimation on energy metabolism, organ mass and digestive enzyme activity in a marsupial.

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Rutkiewicz-Krotewicz, Maria, Agnieszka J. Pietrzyk-Brzezinska, Bartosz Sekula, Hubert Cieśliński, Anna Wierzbicka-Woś, Józef Kur, and Anna Bujacz. "Structural studies of a cold-adapted dimeric β-D-galactosidase fromParacoccussp. 32d." Acta Crystallographica Section D Structural Biology 72, no. 9 (August 31, 2016): 1049–61. http://dx.doi.org/10.1107/s2059798316012535.

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The crystal structure of a novel dimeric β-D-galactosidase fromParacoccussp. 32d (ParβDG) was solved in space groupP212121at a resolution of 2.4 Å by molecular replacement with multiple models using theBALBESsoftware. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric β-D-galactosidases fromEscherichia coliandArthrobactersp. C2-2, respectively. It is the second known structure of a cold-active GH2 β-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted β-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical β-D-galactosidase, the crystal structure in complex with galactose (ParβDG–Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric β-D-galactosidase reveals significant differences in comparison to other β-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 inParβDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

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Journal, Baghdad Science. "Study the Effects of Olive Leaves Extracts in the activity of the enzyme GOT and their Biological Activities." Baghdad Science Journal 14, no. 1 (March 5, 2017): 48–59. http://dx.doi.org/10.21123/bsj.14.1.48-59.

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The olive tree, has been used it is important plant for the time being some of their parts on a large scale in the treatment of gastrointestinal disorders and stimulate circulation . Moreover, it is used as antibacterial material and also to address some of the respiratory system, diabetes, food preservation osteoporosis. This study involved the collection of olive leaves from different areas in Baghdad / Iraq. These leaves have been harvested, wash it, then dried and crushed, where the study aimed to identify the active ingredients and chemical elements in the olive leaf as well as its effect on the action of GOT enzyme .The study showed that the aqueous extracts (cold and hot) of the olive leaves powder are acidic in nature pH values are of (5.74 and 5.40) for the aqueous extracts hot and cold respectively. Study revealed the extract contain the a collection of Glycosides, tannins, phenolic compounds, resins, flavonoids, alkaloids, terpenes and compound Alaolurobin. The study also showed ability to activate the enzyme GOT in cold aqueous extract (8.36%). and the percentage (27.35%) of hot aqueous extract. That can be analyzed to the presence of higher concentrations of the active compounds in hot aqueous extract compared with cold aqueous extract especially tannins working to activate enzymes carrier in the cell membrane in the body. The study showed that the analysis of thin layer chromatography, liquid high-performance, ( TLC, HPLC) in extract compounds of phenols olive leaf using a mixture (chloroform, acetic acid) and a 2:5 was more impact in the Gram-positive Staphylococcus aureus, while less impact in a negative Gram Salmonella typhimurium. As proved accurate analysis of the chemical elements in powder of plant leaf olive tree and at different concentrations contain of chemical elements Major estimated by (g /kg) and trace estimated by (mg/kg). Since the existence of these elements led to increasing of enzymatic effectiveness through an increase process of activity the enzyme (GOT), which plays role of important in our bodies being gives indication of the nature of the work and effectiveness of the activity of some members of the body (liver, kidney, pancreas, etc.).

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42

Kleczkowski, Leszek A., and Gerald E. Edwards. "Hysteresis and Reversible Cold Inactivation of Maize Phosphoenolpyruvate Carboxylase." Zeitschrift für Naturforschung C 45, no. 1-2 (February 1, 1990): 42–46. http://dx.doi.org/10.1515/znc-1990-1-209.

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Abstract Maize (Zea mays L.) leaf phosphoenolpyruvate (PEP) carboxylase (PEPCase) (EC 4.1.1.31) showed a lag in activity when assayed after storage at 0-4 °C. The lag was promoted by high pH on storage (7.8 -8.5) and was observed over a range of assay pH (7.1 -8.5). Thermal reactivation of the cold-stored enzyme by assay temperature (18 °C) accounted for most of the hysteretic effect, but presence of PEP in the reaction mixture was required to completely eliminate the lag. Based on steady-state rates after the lag, stability of PEPCase in the cold was independent of protein concentration . It is suggested that low temperature and high pH induce a change in the oligomerization state of PEPCase, resulting in a less active but relatively stable form of the enzyme. The lag probably reflects a reversal of this process, promoted by assay temperature and presence of PEP.

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43

GARSOUX, Geneviève, Josette LAMOTTE, Charles GERDAY, and Georges FELLER. "Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis." Biochemical Journal 384, no. 2 (November 23, 2004): 247–53. http://dx.doi.org/10.1042/bj20040325.

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The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli. This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the surface of cellulose available to a bound enzyme molecule. The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi. Both kcat and kcat/Km values at 4 °C for the psychrophilic cellulase are similar to the values for Cel5 at 30–35 °C, suggesting temperature adaptation of the kinetic parameters. The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data. The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site. No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures.

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44

Dachuri, VinayKumar, Sei-Heon Jang, and ChangWoo Lee. "Different Effects of Salt Bridges near the Active Site of Cold-Adapted Proteus mirabilis Lipase on Thermal and Organic Solvent Stabilities." Catalysts 12, no. 7 (July 9, 2022): 761. http://dx.doi.org/10.3390/catal12070761.

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Organic solvent-tolerant (OST) enzymes have been discovered in psychrophiles. Cold-adapted OST enzymes exhibit increased conformational flexibility in polar organic solvents resulting from their intrinsically flexible structures. Proteus mirabilis lipase (PML), a cold-adapted OST lipase, was used to assess the contribution of salt bridges near the active site involving two arginine residues (R237 and R241) on the helix η1 and an aspartate residue (D248) on the connecting loop to the thermal and organic solvent stabilities of PML. Alanine substitutions for the ion pairs (R237A, R241A, D248A, and R237A/D248A) increased the conformational flexibility of PML mutants compared to that of the wild-type PML in an aqueous buffer. The PML mutants became more susceptible to denaturation after increasing the dimethyl sulfoxide or methanol concentration than after a temperature increase. Methanol was more detrimental to the structural stability of PML compared to dimethyl sulfoxide. These results suggest that direct interactions of dimethyl sulfoxide and methanol with the residues near the active site can have a destructive effect on the structure of PML compared with the global effect of heat on the protein structure. This study provides insight into the conformational changes within an OST enzyme with different effects on its thermal and organic solvent stabilities.

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45

Kim, Min Ju, Ha Ju Park, Pilsung Kang, Il Chan Kim, Joung Han Yim, and Se Jong Han. "Purification and characterization of a new cold-active cellulolytic enzyme produced by Pseudoalteromonas sp. ArcC09 from the Arctic Beaufort Sea." BioResources 17, no. 2 (April 20, 2022): 3163–77. http://dx.doi.org/10.15376/biores.17.2.3163-3177.

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A cold-active endoglucanase-producing bacterium was isolated from the Beaufort Sea of the Arctic Ocean and identified as Pseudoalteromonas sp. ArcC09. Cellulolytic activity of ArcC09 reached a maximum of 60 U/mg when cultivated in ZoBell medium for 72 h at 15 °C. This purified endoglucanase, with a molecular mass of 28 kDa, exhibited maximum activity at pH 7.0 and 55 °C. The ArcC09 endoglucanase exhibited 10% and 36% of its maximal activity even at low temperatures of 5 °C and 15 °C, respectively. However, it showed lower thermal stability than a mesophilic cellulase, which is characteristic of a psychrophilic enzyme. The activity was inhibited by CuSO4, and linear alkylbenzene sulfonate (LAS). These findings supplement the understanding of cold-active endoglucanases and may have commercial applications in enzymatic digestion of cellulosic biomass to fermentable sugars.

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Lan, Dong-Ming, Ning Yang, Wen-Kai Wang, Yan-Fei Shen, Bo Yang, and Yong-Hua Wang. "A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme." International Journal of Molecular Sciences 12, no. 6 (June 14, 2011): 3950–65. http://dx.doi.org/10.3390/ijms12063950.

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47

Maharana, Abhas Kumar, and Shiv Mohan Singh. "Extracellular Cold Active Alkaline Proteolytic Enzyme Produced By Pseudomonas gessardii Strain N-86 Isolated from Antarctica." International Journal of Current Microbiology and Applied Sciences 9, no. 6 (June 10, 2020): 3119–34. http://dx.doi.org/10.20546/ijcmas.2020.906.374.

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48

Nilsen, Inge W., Kersti Øverbø, Erling Sandsdalen, Elin Sandaker, Knut Sletten, and Bjørnar Myrnes. "Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity." FEBS Letters 464, no. 3 (December 27, 1999): 153–58. http://dx.doi.org/10.1016/s0014-5793(99)01693-2.

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49

Coker, James A., Peter P. Sheridan, Jennifer Loveland-Curtze, Kevin R. Gutshall, Ann J. Auman, and Jean E. Brenchley. "Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate." Journal of Bacteriology 185, no. 18 (September 15, 2003): 5473–82. http://dx.doi.org/10.1128/jb.185.18.5473-5482.2003.

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ABSTRACT A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 β-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both β-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-β-d-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ β-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 β-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.

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Liu, Xiaoyu, Mingyang Zhou, Shu Xing, Tao Wu, Hailun He, John Kevin Bielicki, and Jianbin Chen. "Identification and Biochemical Characterization of a Novel Hormone-Sensitive Lipase Family Esterase Est19 from the Antarctic Bacterium Pseudomonas sp. E2-15." Biomolecules 11, no. 11 (October 20, 2021): 1552. http://dx.doi.org/10.3390/biom11111552.

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Esterases represent an important class of enzymes with a wide variety of industrial applications. A novel hormone-sensitive lipase (HSL) family esterase, Est19, from the Antarctic bacterium Pseudomonas sp. E2-15 is identified, cloned, and expressed. The enzyme possesses a GESAG motif containing an active serine (S) located within a highly conserved catalytic triad of Ser155, Asp253, and His282 residues. The catalytic efficiency (kcat/Km) of Est19 for the pNPC6 substrate is 148.68 s−1mM−1 at 40 °C. Replacing Glu154 juxtaposed to the critical catalytic serine with Asp (E154→D substitution) reduced the activity and catalytic efficiency of the enzyme two-fold, with little change in the substrate affinity. The wild-type enzyme retained near complete activity over a temperature range of 10–60 °C, while ~50% of its activity was retained at 0 °C. A phylogenetic analysis suggested that Est19 and its homologs may represent a new subfamily of HSL. The thermal stability and stereo-specificity suggest that the Est19 esterase may be useful for cold and chiral catalyses.

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Journal articles on the topic 'Cold-active enzyme'

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