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Published: 9 March 2023
Last updated: 10 March 2023

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1

Kazuoka, Takayuki, Shouhei Takigawa, Noriaki Arakawa, Yoshiyuki Hizukuri, Ikuo Muraoka, Tadao Oikawa, and Kenji Soda. "Novel Psychrophilic and Thermolabile l-Threonine Dehydrogenase from Psychrophilic Cytophaga sp. Strain KUC-1." Journal of Bacteriology 185, no. 15 (August 1, 2003): 4483–89. http://dx.doi.org/10.1128/jb.185.15.4483-4489.2003.

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ABSTRACT A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD+-dependent l-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other l-threonine dehydrogenases that have thus far been studied. l-Threonine and dl-threo-3-hydroxynorvaline were the substrates, and NAD+ and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45°C. The kinetic parameters of the enzyme are highly influenced by temperatures. The Km for l-threonine was lowest at 20°C. Dead-end inhibition studies with pyruvate and adenosine-5′-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD+ binds to an enzyme prior to l-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, l-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. l-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.
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2

Jaafar, Nardiah Rizwana, Dene Littler, Travis Beddoe, Jamie Rossjohn, Rosli Md Illias, Nor Muhammad Mahadi, Mukram Mohamed Mackeen, Abdul Munir Abdul Murad, and Farah Diba Abu Bakar. "Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeastGlaciozyma antarcticaPI12." Acta Crystallographica Section F Structural Biology Communications 72, no. 11 (October 27, 2016): 831–39. http://dx.doi.org/10.1107/s2053230x16015612.

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Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA fromGlaciozyma antarcticaPI12 (GaFucA) was cloned and the enzyme was overexpressed inEscherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
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3

Rathour, Rashmi, Juhi Gupta, Bhawna Tyagi, and Indu Shekhar Thakur. "Production and characterization of psychrophilic α-amylase from a psychrophilic bacterium, Shewanella sp. ISTPL2." Amylase 4, no. 1 (November 18, 2020): 1–10. http://dx.doi.org/10.1515/amylase-2020-0001.

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AbstractA psychrophilic and halophilic bacterial isolate, Shewanella sp. ISTPL2, procured from the pristine Pangong Lake, Ladakh, Jammu and Kashmir, India, was used for the production and characterization of the psychrophilic and alkalophilic α-amylase enzyme. The α-amylase is a critical enzyme that catalyses the hydrolysis of α-1,4-glycosidic bonds of starch molecules and is predominately utilized in biotechnological applications. The highest enzyme activity of partially purified extracellular α-amylase was 10,064.20 U/mL after 12 h of incubation in a shake flask at pH 6.9 and 10 °C. Moreover, the maximum intracellular α-amylase enzyme activity (259.62 U/mL) was also observed at 6 h of incubation. The extracellular α-amylase was refined to the homogeneity with the specific enzyme activity of 36,690.47 U/mg protein corresponding to 6.87-fold purification. The optimized pH and temperature for the α-amylase were found to be pH 8 and 4 °C, respectively, suggesting its stability at alkaline conditions and low or higher temperatures. The amylase activity was highly activated by Cu2+, Fe2+ and Ca2+, while inhibited by Cd2+, Co2+ and Na2+. As per our knowledge, the current study reports the highest activity of a psychrophilic α-amylase enzyme providing prominent biotechnological potential.
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4

Mat-Sharani, Shuhaila, Reyad Al Moheer, Farah-Diba Abu-Bakar, Abdul-Munir Abdul-Murad, and Nor-Muhammad Mahadi. "Structure Flexibility of Alpha-galactosidase from a Marine Psychrophilic Yeast, Glaciozyma antarctica PI12." Squalen Bulletin of Marine and Fisheries Postharvest and Biotechnology 17, no. 3 (December 30, 2022): 121–30. http://dx.doi.org/10.15578/squalen.680.

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Factors that contribute to maintaining the flexibility or stability of an enzyme structure may depend on the composition of each amino acid with different characteristics, providing a purpose and bonding features within the structure. Based on this assumption, a study using homology modeling and a comparative study to observe different structure behaviors of an enzyme at an extremely low temperature (psychrophile) against temperate (mesophile) and high temperature (thermophile) was performed. The subject, a-galactosidase from Glaciozyma antarctica as a marine psychrophilic candidate was chosen against a-galactosidase from Trichoderma reesei (mesophile) and Ramsonia emersonii (thermophile). This enzyme catalyzes the hydrolysis of a-1-6 linked terminal galactosyl residues which can be found in a wide range of the organism. The ability of G. antarctica to grow in extremely cold temperatures rendered the question that the enzyme must have special characteristics to adapt to the cold condition. Based on the homology modeling and molecular dynamics study, a comparison of the structure of G. antarctica a-galactosidase enzymes with its homolog from the mesophilic and thermophilic fungi showed that G. antarctica a-galactosidase enzyme confers its flexibility by the increased number of small amino acids with reduced charges, more loops, a fewer number of hydrogen and disulfide bonds in its structure. Furthermore, a-galactosidase has potential for commercialization in bleach paper and the baking industry also a treatment for bloating and Fabry disease.
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5

Yang, Guang, Matteo Mozzicafreddo, Patrizia Ballarini, Sandra Pucciarelli, and Cristina Miceli. "An In-Silico Comparative Study of Lipases from the Antarctic Psychrophilic Ciliate Euplotes focardii and the Mesophilic Congeneric Species Euplotes crassus: Insight into Molecular Cold-Adaptation." Marine Drugs 19, no. 2 (January 27, 2021): 67. http://dx.doi.org/10.3390/md19020067.

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Cold-adapted enzymes produced by psychrophilic organisms have elevated catalytic activities at low temperatures compared to their mesophilic counterparts. This is largely due to amino acids changes in the protein sequence that often confer increased molecular flexibility in the cold. Comparison of structural changes between psychrophilic and mesophilic enzymes often reveal molecular cold adaptation. In the present study, we performed an in-silico comparative analysis of 104 hydrolytic enzymes belonging to the family of lipases from two evolutionary close marine ciliate species: The Antarctic psychrophilic Euplotes focardii and the mesophilic Euplotes crassus. By applying bioinformatics approaches, we compared amino acid composition and predicted secondary and tertiary structures of these lipases to extract relevant information relative to cold adaptation. Our results not only confirm the importance of several previous recognized amino acid substitutions for cold adaptation, as the preference for small amino acid, but also identify some new factors correlated with the secondary structure possibly responsible for enhanced enzyme activity at low temperatures. This study emphasizes the subtle sequence and structural modifications that may help to transform mesophilic into psychrophilic enzymes for industrial applications by protein engineering.
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6

Sulthana, Shaheen, Purusharth I. Rajyaguru, Pragya Mittal, and Malay K. Ray. "rnrGene from the Antarctic Bacterium Pseudomonas syringae Lz4W, Encoding a Psychrophilic RNase R." Applied and Environmental Microbiology 77, no. 22 (September 16, 2011): 7896–904. http://dx.doi.org/10.1128/aem.05683-11.

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ABSTRACTRNase R is a highly processive, hydrolytic 3′-5′ exoribonuclease belonging to the RNB/RNR superfamily which plays significant roles in RNA metabolism in bacteria. The enzyme was observed to be essential for growth of the psychrophilic Antarctic bacteriumPseudomonas syringaeLz4W at a low temperature. We present results here pertaining to the biochemical properties of RNase R and the RNase R-encoding gene (rnr) locus from this bacterium. By cloning and expressing a His6-tagged form of theP. syringaeRNase R (RNase RPs), we show that the enzyme is active at 0 to 4°C but exhibits optimum activity at ∼25°C. The enzyme is heat labile in nature, losing activity upon incubation at 37°C and above, a hallmark of many psychrophilic enzymes. The enzyme requires divalent cations (Mg2+and Mn2+) for activity, and the activity is higher in 50 to 150 mM KCl when it largely remains as a monomer. On synthetic substrates, RNase RPsexhibited maximum activity on poly(A) and poly(U) in preference over poly(G) and poly(C). The enzyme also degraded structuredmalE-malFRNA substrates. Analysis of the cleavage products shows that the enzyme, apart from releasing 5′-nucleotide monophosphates by the processive exoribonuclease activity, produces four-nucleotide end products, as opposed to two-nucleotide products, of RNA chain byEscherichia coliRNase R. Interestingly, three ribonucleotides (ATP, GTP, and CTP) inhibited the activity of RNase RPsin vitro. The ability of the nonhydrolyzable ATP-γS to inhibit RNase RPsactivity suggests that nucleotide hydrolysis is not required for inhibition. This is the first report on the biochemical property of a psychrophilic RNase R from any bacterium.
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7

Drienovska, Ivana, Eva Chovancova, Tana Koudelakova, Jiri Damborsky, and Radka Chaloupkova. "Biochemical Characterization of a Novel Haloalkane Dehalogenase from a Cold-Adapted Bacterium." Applied and Environmental Microbiology 78, no. 14 (May 11, 2012): 4995–98. http://dx.doi.org/10.1128/aem.00485-12.

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ABSTRACTA haloalkane dehalogenase, DpcA, fromPsychrobacter cryohalolentisK5, representing a novel psychrophilic member of the haloalkane dehalogenase family, was identified and biochemically characterized. DpcA exhibited a unique temperature profile with exceptionally high activities at low temperatures. The psychrophilic properties of DpcA make this enzyme promising for various environmental applications.
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8

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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9

Chessa, Jean-Pierre, Georges Feller, and Charles Gerday. "Purification and characterization of the heat-labile α-amylase secreted by the psychrophilic bacterium TAC 240B." Canadian Journal of Microbiology 45, no. 6 (July 15, 1999): 452–57. http://dx.doi.org/10.1139/w99-021.

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A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce α-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified α-amylase shows a molecular mass of about 50 000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other α-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic α-amylase requires both Cl-and Ca2+for its amylolytic activity. Br-is also quite effecient as an allosteric effector. The comparison of the amino acid composition with those of other α-amylases from various organisms shows that the cold α-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment. Key words: α-amylase, psychrophilic microorganisms, Antarctic.
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10

Benrezkallah, Djamila, Hayat Sediki, and Abdelghani Mohammed Krallafa. "The Structural Flexibility of Cold- and Warm-Adapted Enzymes (Endonucleases I) by Molecular Dynamics Simulation." Chemistry Proceedings 3, no. 1 (November 14, 2020): 128. http://dx.doi.org/10.3390/ecsoc-24-08387.

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The constraints exerted on the molecular edifices by different environmental parameters are not the same, which is translated by different adaptive strategies. Thus, for the extracellular and periplasmic enzymes of marine organisms which are directly exposed to environments in which large variations in temperature and salinity can occur, it is not an easy task to separate the adaptation of the enzyme to one of the two environmental parameters without the involvement of the other. In such a scenario, a comparative study of a marine psychrophilic and an estuarine mesophilic endonuclease I wasundertaken. The different salt optima of the enzymes were taken into consideration when the temperature-dependent enzymatic properties were characterized, but the results did not show an adaptive strategy at the molecular edifice. For that purpose, we employed multiple all-atom explicit solvent and ions molecular dynamics simulations, in conjunction with different temperatures at the nanosecond time scale to analyzethe structural flexibility of the cold adapted enzyme, endonuclease I psychrophile marine (VsEndA), and its mesophile homologous enzyme, endonuclease I mesophile estuarine (VcEndA). The Root Mean Square Fluctuation (RMSF) profiles of the two enzymes are almost similar with the most flexible residues located at the loop regions for both enzymes. We underlined a different trend against temperature for the two enzymes. The cold adapted enzyme was dominated by the lowest temperatures of T = 300 and T = 318 K, compared to its warm adapted homologous counterpart for which the highest temperature studiedwas T = 326 K as the dominantone. The lifetimes of the hydrogen bonding network of the most flexible residues of both enzymes correlate well with the RMSF of the considered enzymes.
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11

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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12

Takasawa, Toshihide, Keiko Sagisaka, Koichi Yagi, Kyoko Uchiyama, Atsushi Aoki, Kyo Takaoka, and Katsuhiro Yamamato. "Polygalacturonase isolated from the culture of the psychrophilic fungus Sclerotinia borealis." Canadian Journal of Microbiology 43, no. 5 (May 1, 1997): 417–24. http://dx.doi.org/10.1139/m97-059.

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A polygalacturonase was isolated from the culture medium of Sclerotinia borealis, a psychrophilic fungus that grows on lawn and wheat seedling under the snow in winter and induces the snow mold disease. Pectic acid was a better substrate of this enzyme than pectin when the activity was determined by measuring the reducing sugar produced. However, when the activity was measured by viscosity change, the viscosity of pectin decreased more rapidly than that of pectic acid. The results of viscosity change apparently indicate that the polygalacturonase catalyzes pectin hydrolysis as an endo-type enzyme. Highly methyl-esterified pectin was a poor substrate, as determined by measurements of reducing sugar production and viscosity change. It is suggested from the results that the methoxy group of pectin affects the polygalacturonase reaction. A reaction mechanism was proposed for the polygalacturonase reaction. Molecular mass of this enzyme was 40 kDa and its isoelectric point was pH 7.5. Optimum pH of the enzyme reaction was 4.5 and its optimum temperature was 40–50 °C. Thirty percent of the maximum activity was observed at 5 °C, but it was only slightly active above 60 °C. The activity was preserved for more than 2 years at 5 °C and pH 4.5, but it was lost when kept at room temperature overnight or heated at 50 °C for 30 min. The amino acid sequence of the N-terminal region of the psychrophilic polygalacturonase of Sclerotinia borealis is compared with those of polygalacturonases of mesophilic fungi. The function of this enzyme against the target plants is discussed with reference to the reaction of polygalacturonases of mesophilic fungi.Key words: polygalacturonase, pectin-hydrolyzing enzyme, psychrophilic fungi, snow mold disease, Sclerotinia borealis.
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13

Xu, Bi-Lin, Meihong Dai, Yuanhao Chen, Dongheng Meng, Yasi Wang, Nan Fang, Xiao-Feng Tang, and Bing Tang. "Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart." Applied and Environmental Microbiology 81, no. 18 (July 6, 2015): 6302–13. http://dx.doi.org/10.1128/aem.01478-15.

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ABSTRACTThe incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The half-life of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (Tm) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values ofKmandkcat(25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications.
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14

Sheridan, Peter P., and Jean E. Brenchley. "Characterization of a Salt-Tolerant Family 42 β-Galactosidase from a Psychrophilic AntarcticPlanococcus Isolate." Applied and Environmental Microbiology 66, no. 6 (June 1, 2000): 2438–44. http://dx.doi.org/10.1128/aem.66.6.2438-2444.2000.

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ABSTRACT We isolated a gram-positive, halotolerant psychrophile from a hypersaline pond located on the McMurdo Ice Shelf in Antarctica. A phylogenetic analysis of the 16S rRNA gene sequence of this organism showed that it is a member of the genus Planococcus. This assignment is consistent with the morphology and physiological characteristics of the organism. A gene encoding a β-galactosidase in this isolate was cloned in an Escherichia coli host. Sequence analysis of this gene placed it in glycosidase family 42 most closely related to an enzyme from Bacillus circulans. Even though an increasing number of family 42 glycosidase sequences are appearing in databases, little information about the biochemical features of these enzymes is available. Therefore, we purified and characterized this enzyme. The purified enzyme did not appear to have any metal requirement, had an optimum pH of 6.5 and an optimum temperature of activity at 42°C, and was irreversibly inactivated within 10 min when it was incubated at 55°C. The enzyme had an apparent Km of 4.9 μmol ofo-nitrophenyl-β-d-galactopyranoside, and theV max was 467 μmol ofo-nitrophenol produced/min/mg of protein at 39°C. Of special interest was the finding that the enzyme remained active at high salt concentrations, which makes it a possible reporter enzyme for halotolerant and halophilic organisms.
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15

Arcus, Vickery L., and Adrian J. Mulholland. "Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates." Annual Review of Biophysics 49, no. 1 (May 6, 2020): 163–80. http://dx.doi.org/10.1146/annurev-biophys-121219-081520.

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We review the adaptations of enzyme activity to different temperatures. Psychrophilic (cold-adapted) enzymes show significantly different activation parameters (lower activation enthalpies and entropies) from their mesophilic counterparts. Furthermore, there is increasing evidence that the temperature dependence of many enzyme-catalyzed reactions is more complex than is widely believed. Many enzymes show curvature in plots of activity versus temperature that is not accounted for by denaturation or unfolding. This is explained by macromolecular rate theory: A negative activation heat capacity for the rate-limiting chemical step leads directly to predictions of temperature optima; both entropy and enthalpy are temperature dependent. Fluctuations in the transition state ensemble are reduced compared to the ground state. We show how investigations combining experiment with molecular simulation are revealing fundamental details of enzyme thermoadaptation that are relevant for understanding aspects of enzyme evolution. Simulations can calculate relevant thermodynamic properties (such as activation enthalpies, entropies, and heat capacities) and reveal the molecular mechanisms underlying experimentally observed behavior.
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16

Chrast, Lukas, Katsiaryna Tratsiak, Joan Planas-Iglesias, Lukas Daniel, Tatyana Prudnikova, Jan Brezovsky, David Bednar, Ivana Kuta Smatanova, Radka Chaloupkova, and Jiri Damborsky. "Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17." Microorganisms 7, no. 11 (October 28, 2019): 498. http://dx.doi.org/10.3390/microorganisms7110498.

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Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.
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17

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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18

Secades, P., B. Alvarez, and J. A. Guijarro. "Purification and Characterization of a Psychrophilic, Calcium-Induced, Growth-Phase-Dependent Metalloprotease from the Fish Pathogen Flavobacterium psychrophilum." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2436–44. http://dx.doi.org/10.1128/aem.67.6.2436-2444.2001.

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ABSTRACT Flavobacterium psychrophilum is a fish pathogen that commonly affects salmonids. This bacterium produced an extracellular protease with an estimated molecular mass of 55 kDa. This enzyme, designated Fpp1 ( F . p sychrophilum p rotease 1), was purified to electrophoretic homogeneity from the culture supernatant by using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography. On the basis of its biochemical characteristics, Fpp1 can be included in the group of metalloproteases that have an optimum pH for activity of 6.5 and are inhibited by 1,10-phenanthroline, EDTA, or EGTA but not by phenylmethylsulfonyl fluoride. Fpp1 activity was dependent on calcium ions not only for its activity but also for its thermal stability. In addition to calcium, strontium and barium can activate the protein. The enzyme showed typical psychrophilic behavior; it had an activation energy of 5.58 kcal/mol and was more active at temperatures between 25 and 40°C, and its activity decreased rapidly at 45°C. Fpp1 cleaved gelatin, laminin, fibronectin, fibrinogen, collagen type IV, and, to a lesser extent, collagen types I and II. Fpp1 also degraded actin and myosin, basic elements of the fish muscular system. The presence of this enzyme in culture media was specifically dependent on the calcium concentration. Fpp1 production started early in the exponential growth phase and reached a maximum during this period. Addition of calcium during the stationary phase did not induce Fpp1 production at all. Besides calcium and the growth phase, temperature also seems to play a role in production of Fpp1. In this study we found that production of Fpp1 depends on factors such as calcium concentration, growth phase of the culture, and temperature. The combination of these parameters corresponds to the combination in the natural host during outbreaks of disease caused by F. psychrophilum. Consequently, we suggest that environmental host factors govern Fpp1 production.
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Wang, Wei, Mi Sun, Wanshun Liu, and Bin Zhang. "Purification and characterization of a psychrophilic catalase from Antarctic Bacillus." Canadian Journal of Microbiology 54, no. 10 (October 2008): 823–28. http://dx.doi.org/10.1139/w08-066.

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Catalase from Bacillus sp. N2a (BNC) isolated from Antarctic seawater was purified to homogeneity. BNC has a molecular mass of about 230 kDa and is composed of four identical subunits of 56 kDa. The catalase showed optimal activity at 25 °C and at a pH range of 6–11. The enzyme could be inhibited by azide, hydroxylamine, and mercaptoethanol. These characteristics suggested that BNC is a small-subunit monofunctional catalase. The activation energy of BNC was 13 kJ/mol and the apparent kcat/Km values were 3.6 × 106 and 4 × 106 L·mol–1·s–1 at 4 and 25 °C, respectively. High catalytic efficiency of BNC at low temperatures enables this bacterium to scavenge H2O2 efficiently. BNC exhibited activation energy, catalytic efficiency, and thermostability comparable with some mesophilic homologues. Such similarity of enzymatic characteristics to mesophilic homologues, although uncommon among the cold-adapted enzymes in general, has also been observed in other psychrophilic small-subunit monofunctional catalases.
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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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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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Da-qing, Wang, Jin Wen-ran, Sun Tai-peng, Meng Yu-tian, Zhao Wei, and Wang Hong-yan. "Screening Psychrophilic Fungi of Cellulose Degradation and Characteristic of Enzyme Production." Journal of Northeast Agricultural University (English Edition) 23, no. 2 (June 2016): 20–27. http://dx.doi.org/10.1016/s1006-8104(16)30044-7.

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23

TranNgoc, Kiet, Nhung Pham, ChangWoo Lee, and Sei-Heon Jang. "Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621." International Journal of Molecular Sciences 20, no. 6 (March 18, 2019): 1362. http://dx.doi.org/10.3390/ijms20061362.

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Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5–8.5 and 30 °C for NADP+ and 20 °C for NAD+. SpG6PD1 utilized both NADP+ and NAD+, with the preferential utilization of NADP+. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4–40 °C, its activity and tertiary structure were better preserved between 4–20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.
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MAKARIOS-LAHAM, IBRAHIM K., and RICHARD W. TRAXLER. "Kinetics of Extracellular Protease From the Obligately Psychrophilic Vibrio OP7 of Fish Origin." Journal of Food Protection 54, no. 8 (August 1, 1991): 578–81. http://dx.doi.org/10.4315/0362-028x-54.8.578.

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The extracellular protease recovered from the obligately psychrophilic marine Vibrio OP7 of fish origin was able to degrade fish actomyosin at 5°C. The Vmax and the apparent Km values for the enzyme were 33.3 μg/ml/min and 2.5 mg/ml, respectively. Ethylenediamine tetracetic acid (EDTA) and sodium tripolyphosphate (TPP) were found to inhibit the enzyme's activity at concentrations of 0.075 mg/ml and 0.25 mg/ml, respectively. EDTA produced a noncompetitive inhibition, whereas TPP produced a mixed of noncompetitive and uncompetitive inhibitions of the enzyme.
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25

Mazur, Andrii, Tatyana Prudnikova, Pavel Grinkevich, Jeroen R. Mesters, Daria Mrazova, Radka Chaloupkova, Jiri Damborsky, Michal Kuty, Petr Kolenko, and Ivana Kuta Smatanova. "The tetrameric structure of the novel haloalkane dehalogenase DpaA from Paraglaciecola agarilytica NO2." Acta Crystallographica Section D Structural Biology 77, no. 3 (February 17, 2021): 347–56. http://dx.doi.org/10.1107/s2059798321000486.

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Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane dehalogenase DpaA described here was isolated from the psychrophilic and halophilic bacterium Paraglaciecola agarilytica NO2, which was found in marine sediment collected from the East Sea near Korea. Gel-filtration experiments and size-exclusion chromatography provided information about the dimeric composition of the enzyme in solution. The DpaA enzyme was crystallized using the sitting-drop vapour-diffusion method, yielding rod-like crystals that diffracted X-rays to 2.0 Å resolution. Diffraction data analysis revealed a case of merohedral twinning, and subsequent structure modelling and refinement resulted in a tetrameric model of DpaA, highlighting an uncommon multimeric nature for a protein belonging to haloalkane dehalogenase subfamily I.
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Zhang, Wen Jing, Qiong Fang Li, Xin He, Qun Wei Dai, Wei Zhang, and Jian Ping Wang. "Psychrophilic Carbonic Anhydrase Producing Bacteria for Screening and Optimizing the Enzyme Production." Advanced Materials Research 1092-1093 (March 2015): 617–20. http://dx.doi.org/10.4028/www.scientific.net/amr.1092-1093.617.

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Carbonic anhydrase(CA)has a catalytic effect on calcium carbonate (CaCO3) deposition, and can also control the deposition of CaCO3. Screening strains that can produce carbonic anhydrase can solve the microbial CA from the natural environment in the role of CaCO3 deposition. Through the single and orthogonal test, the present paper focused on temperature, pH value, culture time and inoculation of 4 factors to optimally culture the psychrotrophic strains, which could produce CA by independent screening from Huanglong of cold calcified water, in order to get high yield of CA psychrophilic strains. The results showed that the optimal condition of the 15-33 strains producing enzyme was temperature 15 °C?, incubation time 12 h, pH 6.0, and inoculation 2.0 mL. The 18-10 strains, was temperature 15 °C?, incubation time 24 h, pH 6.5, and qinoculation 2.0 mL.
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27

Arpigny, J. L., Z. Zekhnini, J. Swings, C. Gerday, E. Narinx, and G. Feller. "Temperature dependence of growth, enzyme secretion and activity of psychrophilic Antarctic bacteria." Applied Microbiology and Biotechnology 41, no. 4 (June 1, 1994): 477–79. http://dx.doi.org/10.1007/s002530050176.

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28

Feller, G., E. Narinx, J. L. Arpigny, Z. Zekhnini, J. Swings, and C. Gerday. "Temperature dependence of growth, enzyme secretion and activity of psychrophilic Antarctic bacteria." Applied Microbiology and Biotechnology 41, no. 4 (June 1994): 477–79. http://dx.doi.org/10.1007/bf00939039.

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29

Feller, G., E. Narinx, J. L. Arpigny, Z. Zekhnini, J. Swings, and C. Gerday. "Temperature dependence of growth, enzyme secretion and activity of psychrophilic Antarctic bacteria." Applied Microbiology and Biotechnology 41, no. 4 (June 1994): 477–79. http://dx.doi.org/10.1007/bf00212261.

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30

Lund, Bjarte Aarmo, and Bjørn Olav Brandsdal. "ThermoSlope: A Software for Determining Thermodynamic Parameters from Single Steady-State Experiments." Molecules 26, no. 23 (November 26, 2021): 7155. http://dx.doi.org/10.3390/molecules26237155.

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The determination of the temperature dependence of enzyme catalysis has traditionally been a labourious undertaking. We have developed a new approach to the classical Arrhenius parameter estimation by fitting the change in velocity under a gradual change in temperature. The evaluation with a simulated dataset shows that the approach is valid. The approach is demonstrated as a useful tool by characterizing the Bacillus pumilus LipA enzyme. Our results for the lipase show that the enzyme is psychrotolerant, with an activation energy of 15.3 kcal/mol for the chromogenic substrate para-nitrophenyl butyrate. Our results demonstrate that this can produce equivalent curves to the traditional approach while requiring significantly less sample, labour and time. Our method is further validated by characterizing three α-amylases from different species and habitats. The experiments with the α-amylases show that the approach works over a wide range of temperatures and clearly differentiates between psychrophilic, mesophilic and thermophilic enzymes. The methodology is released as an open-source implementation in Python, available online or used locally. This method of determining the activation parameters can make studies of the temperature dependence of enzyme catalysis more widely adapted to understand how enzymes have evolved to function in extreme environments. Moreover, the thermodynamic parameters that are estimated serve as functional validations of the empirical valence bond calculations of enzyme catalysis.
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31

Tutino, Maria Luisa, Ermenegilda Parrilli, Laura Giaquinto, Angela Duilio, Giovanni Sannia, Georges Feller, and Gennaro Marino. "Secretion of α-Amylase from Pseudoalteromonas haloplanktis TAB23: Two Different Pathways in Different Hosts." Journal of Bacteriology 184, no. 20 (October 15, 2002): 5814–17. http://dx.doi.org/10.1128/jb.184.20.5814-5817.2002.

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ABSTRACT Secretion of cold-adapted α-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria. We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for α-amylase secretion in Escherichia coli. The occurrence of two different secretion pathways in different hosts is proposed.
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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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33

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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34

Malecki, Piotr H., Magdalena Bejger, Wojciech Rypniewski, and Constantinos E. Vorgias. "The Crystal Structure of a Streptomyces thermoviolaceus Thermophilic Chitinase Known for Its Refolding Efficiency." International Journal of Molecular Sciences 21, no. 8 (April 21, 2020): 2892. http://dx.doi.org/10.3390/ijms21082892.

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Analyzing the structure of proteins from extremophiles is a promising way to study the rules governing the protein structure, because such proteins are results of structural and functional optimization under well-defined conditions. Studying the structure of chitinases addresses an interesting aspect of enzymology, because chitin, while being the world’s second most abundant biopolymer, is also a recalcitrant substrate. The crystal structure of a thermostable chitinase from Streptomyces thermoviolaceus (StChi40) has been solved revealing a β/α-barrel (TIM-barrel) fold with an α+β insertion domain. This is the first chitinase structure of the multi-chitinase system of S. thermoviolaceus. The protein is also known to refold efficiently after thermal or chemical denaturation. StChi40 is structurally close to the catalytic domain of psychrophilic chitinase B from Arthrobacter TAD20. Differences are noted in comparison to the previously examined chitinases, particularly in the substrate-binding cleft. A comparison of the thermophilic enzyme with its psychrophilic homologue revealed structural features that could be attributed to StChi40’s thermal stability: compactness of the structure with trimmed surface loops and unique disulfide bridges, one of which is additionally stabilized by S–π interactions with aromatic rings. Uncharacteristically for thermophilic proteins, StChi40 has fewer salt bridges than its mesophilic and psychrophilic homologues.
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Yumoto, Isao, Daisen Ichihashi, Hideaki Iwata, Anita Istokovics, Nobutoshi Ichise, Hidetoshi Matsuyama, Hidetoshi Okuyama, and Kosei Kawasaki. "Purification and Characterization of a Catalase from the Facultatively Psychrophilic Bacterium Vibrio rumoiensis S-1T Exhibiting High Catalase Activity." Journal of Bacteriology 182, no. 7 (April 1, 2000): 1903–9. http://dx.doi.org/10.1128/jb.182.7.1903-1909.2000.

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ABSTRACT Catalase from the facultatively psychrophilic bacteriumVibrio rumoiensis S-1T, which was isolated from an environment exposed to H2O2 and exhibited high catalase activity, was purified and characterized, and its localization in the cell was determined. Its molecular mass was 230 kDa, and the molecule consisted of four identical subunits. The enzyme, which was not apparently reduced by dithionite, showed a Soret peak at 406 nm in a resting state. The catalytic activity was 527,500 U · mg of protein−1 under standard reaction conditions at 40°C, 1.5 and 4.3 times faster, respectively, than those of theMicrococcus luteus and bovine catalases examined under the same reaction conditions, and showed a broad optimum pH range (pH 6 to 10). The catalase from strain S-1T is located not only in the cytoplasmic space but also in the periplasmic space. There is little difference in the activation energy for the activity between strain S-1T catalase and M. luteus and bovine liver catalases. The thermoinstability of the activity of the former catalase were significantly higher than those of the latter catalases. The thermoinstability suggests that the catalase from strain S-1T should be categorized as a psychrophilic enzyme. Although the catalase from strain S-1T is classified as a mammal type catalase, it exhibits the unique enzymatic properties of high intensity of enzymatic activity and thermoinstability. The results obtained suggest that these unique properties of the enzyme are in accordance with the environmental conditions under which the microorganism lives.
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36

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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37

Xu, Ying, Georges Feller, Charles Gerday, and Nicolas Glansdorff. "Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritellaabyssi." Journal of Bacteriology 185, no. 7 (April 1, 2003): 2161–68. http://dx.doi.org/10.1128/jb.185.7.2161-2168.2003.

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ABSTRACT The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCaseMab), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCaseMab displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCaseMab displays higher Km values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue δ-N-phosphonoacetyl-l-ornithine (PALO). OTCaseMab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high Km values and the lower sensitivity to PALO. The Km for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k cat/Km ) of OTCases adapted to different temperatures showed that OTCaseMab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the Km value for ornithine observed when the temperature is brought from 20 to 5°C. OTCaseMab adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.
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Roovers, Martine, Rony Sanchez, Christianne Legrain, and Nicolas Glansdorff. "Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase." Journal of Bacteriology 183, no. 3 (February 1, 2001): 1101–5. http://dx.doi.org/10.1128/jb.183.3.1101-1105.2001.

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ABSTRACT We have obtained mutants of Pyrococcus furiosusornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15°C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.
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Chen, Li, Hao Yu, Shengping Yang, Yunfang Qian, and Jing Xie. "Study on the Mechanism of Cold Tolerance of the Strain Shewanella putrefaciens WS13 Through Fatty Acid Metabolism." Nanoscience and Nanotechnology Letters 11, no. 12 (December 1, 2019): 1718–23. http://dx.doi.org/10.1166/nnl.2019.3055.

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In order to investigate the cold tolerance mechanism of Shewanella, the whole genome of strain Shewanella putrefaciens WS13 was used to study the comparative genome related to cold tolerance of Shewanella . By comparing and analyzing the key enzymes involved in the process of lipid synthesis with those of other psychrophilic and non-psychrophilic bacteria, the results showed that in S. putrefaciens WS13, the genes fabA, fabB, fabD, fabF, fabG, fabH and fabZ, as the key enzymes of fatty acid synthesis, were found in the target strain, but the gene fabI did not exist in the type II fatty acid synthesis pathway. However, due to the absence of the key enzyme fabI gene, the synthesis process of saturated fatty acids will be blocked, and the pathway of unsaturated fatty acid synthesis still exists, which leads to the bacteria Shewanella start to synthesize a large number of unsaturated fatty acids, thus increasing the synthesis of unsaturated fatty acids and reducing the synthesis of saturated fatty acids. It is precisely because unsaturated fatty acids have lower phase transition temperature than that saturated fatty acids have, which can increase the fluidity of biofilm, so that Shewanella has better cold adaptability than that other bacteria have. It is a complex biological process for microorganisms to adapt to the environment, and the biosynthesis of fatty acids is only one aspect. However, the mechanism of cold adaptation of Shewanella in other aspects remains to be further discussed.
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Mandelman, David, Mostafa Bentahir, Georges Feller, Charles Gerday, and Richard Haser. "Crystallization and preliminary X-ray analysis of a bacterial psychrophilic enzyme, phosphoglycerate kinase." Acta Crystallographica Section D Biological Crystallography 57, no. 11 (October 25, 2001): 1666–68. http://dx.doi.org/10.1107/s0907444901012069.

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Hou, Yanhua, Chenhui Qiao, Yifan Wang, Yatong Wang, Xiulian Ren, Qifeng Wei, and Quanfu Wang. "Cold-Adapted Glutathione S-Transferases from Antarctic Psychrophilic Bacterium Halomonas sp. ANT108: Heterologous Expression, Characterization, and Oxidative Resistance." Marine Drugs 17, no. 3 (March 1, 2019): 147. http://dx.doi.org/10.3390/md17030147.

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Glutathione S-transferases are one of the most important antioxidant enzymes to protect against oxidative damage induced by reactive oxygen species. In this study, a novel gst gene, designated as hsgst, was derived from Antarctic sea ice bacterium Halomonas sp. ANT108 and expressed in Escherichia coli (E. coli) BL21. The hsgst gene was 603 bp in length and encoded a protein of 200 amino acids. Compared with the mesophilic EcGST, homology modeling indicated HsGST had some structural characteristics of cold-adapted enzymes, such as higher frequency of glycine residues, lower frequency of proline and arginine residues, and reduced electrostatic interactions, which might be in relation to the high catalytic efficiency at low temperature. The recombinant HsGST (rHsGST) was purified to apparent homogeneity with Ni-affinity chromatography and its biochemical properties were investigated. The specific activity of the purified rHsGST was 254.20 nmol/min/mg. The optimum temperature and pH of enzyme were 25 °C and 7.5, respectively. Most importantly, rHsGST retained 41.67% of its maximal activity at 0 °C. 2.0 M NaCl and 0.2% H2O2 had no effect on the enzyme activity. Moreover, rHsGST exhibited its protective effects against oxidative stresses in E. coli cells. Due to its high catalytic efficiency and oxidative resistance at low temperature, rHsGST may be a potential candidate as antioxidant in low temperature health foods.
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42

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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43

Alam, Syed Imteyaz, Smita Dube, Mukesh Kumar Agarwal, and Lokendra Singh. "Purification and characterization of an extracellular protease produced by psychrotolerant Clostridium sp. LP3 from lake sediment of Leh, India." Canadian Journal of Microbiology 52, no. 12 (December 1, 2006): 1238–46. http://dx.doi.org/10.1139/w06-089.

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An anaerobic, proteolytic bacterium isolated from lake sediments of Leh, India, was characterized with respect to morphology, biochemical characteristics, and 16S rRNA sequence and was identified as Clostridium species, with closest similarity to Clostridium subterminale. Isolate LP3 was psychrophilic, forming maximum cell mass between 10 and 20 °C, and produced extracellular protease. Growth was observed in the pH range of 7.0–8.5, with optimum at pH 7.5. Protease was purified 62.4-fold with a total yield of 17.5%. The effects of temperature, pH, and salt concentration on enzyme activity were studied. Protease was found to be a serine-type metallo-enzyme, active in a broad range of pHs. It was thermolabile and resistant to sodium dodecyl sulfate. Enzyme kinetics showed a tendency to increase Km with an increase in temperature for casein substrate.Key words: Clostridium sp., psychrotolerant, protease, anaerobe.
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44

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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45

de Wijn, Raphaël, Oliver Hennig, Felix G. M. Ernst, Bernard Lorber, Heike Betat, Mario Mörl, and Claude Sauter. "Combining crystallogenesis methods to produce diffraction-quality crystals of a psychrophilic tRNA-maturation enzyme." Acta Crystallographica Section F Structural Biology Communications 74, no. 11 (October 31, 2018): 747–53. http://dx.doi.org/10.1107/s2053230x18014590.

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The determination of conditions for the reproducible growth of well diffracting crystals is a critical step in every biocrystallographic study. On the occasion of a new structural biology project, several advanced crystallogenesis approaches were tested in order to increase the success rate of crystallization. These methods included screening by microseed matrix screening, optimization by counter-diffusion and crystal detection by trace fluorescent labeling, and are easily accessible to any laboratory. Their combination proved to be particularly efficient in the case of the target, a 48 kDa CCA-adding enzyme from the psychrophilic bacterium Planococcus halocryophilus. A workflow summarizes the overall strategy, which led to the production of crystals that diffracted to better than 2 Å resolution and may be of general interest for a variety of applications.
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46

Mohamad Tahir, Hiryahafira, Raja Noor Zaliha Raja Abd Rahman, Adam Thean Chor Leow, and Mohd Shukuri Mohamad Ali. "Expression, Characterisation and Homology Modelling of a Novel Hormone-Sensitive Lipase (HSL)-Like Esterase from Glaciozyma antarctica." Catalysts 10, no. 1 (January 1, 2020): 58. http://dx.doi.org/10.3390/catal10010058.

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Microorganisms, especially those that survive in extremely cold places such as Antarctica, have gained research attention since they produce a unique feature of the protein, such as being able to withstand at extreme temperature, salinity, and pressure, that make them desired for biotechnological application. Here, we report the first hormone-sensitive lipase (HSL)-like esterase from a Glaciozyma species, a psychrophilic yeast designated as GlaEst12-like esterase. In this study, the putative lipolytic enzyme was cloned, expressed in E. coli, purified, and characterised for its biochemical properties. Protein sequences analysis showed that GlaEst12 shared about 30% sequence identity with chain A of the bacterial hormone-sensitive lipase of E40. It belongs to the H group since it has the conserved motifs of Histidine-Glycine-Glycine-Glycine (HGGG)and Glycine-Aspartate-Serine-Alanine-Glycine (GDSAG) at the amino acid sequences. The recombinant GlaEst12 was successfully purified via one-step Ni-Sepharose affinity chromatography. Interestingly, GlaEst12 showed unusual properties with other enzymes from psychrophilic origin since it showed an optimal temperature ranged between 50–60 °C and was stable at alkaline pH conditions. Unlike other HSL-like esterase, this esterase showed higher activity towards medium-chain ester substrates rather than shorter chain ester. The 3D structure of GlaEst12, predicted by homology modelling using Robetta software, showed a secondary structure composed of mainly α/β hydrolase fold, with the catalytic residues being found at Ser232, Glu341, and His371.
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47

Karasová, P., V. Spiwok, Š. Malá, B. Králová, and N. J. Russell. "Beta-galactosidase activity in psychrotrophic microorganisms and their potential use in food industry." Czech Journal of Food Sciences 20, No. 2 (November 18, 2011): 43–47. http://dx.doi.org/10.17221/3508-cjfs.

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Twenty-one psychrotrophic resp. psychrophilic bacterial strains were screened for presence of b-galactosidase activity which showed 8 of them. b-Galactosidase activity of these strains was determined for 2 substrates – synthetic substrate (ONPG) and lactose – and also temperature profile of this enzyme was measured. b-Galactosidase from Arthrobacter sp. C2-2 not only proved the typical properties of cold-active enzyme, but it also preferred lactose as a substrate. Therefore, it was chosen for further isolation and purification and was found that it contains two b-galactosidase isoenzymes. One of them had strong preference for lactose and was able to catalyse transglycosylation reactions at low temperature. It has, thus, potential use in food technology.  
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48

Sonan, Guillaume K., Véronique Receveur-Brechot, Colette Duez, Nushin Aghajari, Mirjam Czjzek, Richard Haser, and Charles Gerday. "The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium." Biochemical Journal 407, no. 2 (September 25, 2007): 293–302. http://dx.doi.org/10.1042/bj20070640.

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The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.
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49

MANCO, Giuseppe, Elena ADINOLFI, Francesca M. PISANI, Gianluca OTTOLINA, Giacomo CARREA, and Mosè ROSSI. "Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily." Biochemical Journal 332, no. 1 (May 15, 1998): 203–12. http://dx.doi.org/10.1042/bj3320203.

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We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B´-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11±2 µM (mean±S.D., n = 3) and 6610±880 s-1 (mean±S.D., n = 3) respectively at 70 °C and pH 7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic MoraxellaTA144lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (±)-3-bromo-5-(hydroxymethyl)-Δ2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity–stability–temperature relationship is discussed in relation to those of the homologous members of the HSL group.
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50

Devi, Sarita, Savitri, Tilak Raj, Nikhil Sharma, and Wamik Azmi. "In silicoAnalysis of L-Glutaminase from Extremophiles." Current Proteomics 16, no. 3 (February 18, 2019): 210–21. http://dx.doi.org/10.2174/1570164615666180911110606.

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Background:L-glutaminase enzyme belongs to the family of hydrolases, those acting on carbon-nitrogen bonds other than peptide bonds, specifically in linear amides. Protein L-glutaminase, which converts amino acid glutamine to a glutamate residue, is useful as antileukemic agent, antiretroviral agent and a new food-processing enzyme.Objective:The sequences representing L-glutaminase from extremophiles were analyzed for different physico-chemical properties and to relate these observed differences to their extremophilic properties, phylogenetic tree construction and the evolutionary relationship among them.Methods:In this work, in silico analysis of amino acid sequences of extremophilic (thermophile, halophile and psychrophiles) proteins has been done. The physiochemical properties of these four groups of proteins for L-glutaminase also differ in number of amino acids, aliphatic index and grand average of hydropathicity (GRAVY).Result:The GRAVY was found to be significantly high in thermophilic (2.29 fold) and psychrophilic bacteria (3.3 fold) as compare to mesophilic bacteria. The amino acid Cys (C) was found to be statistically significant in mesophilic bacteria (approximately or more than 3 fold) as compared to the abundance of this amino acid in extremophilic bacteria.Conclusion:Multiple sequence alignment revealed the domain/motif for glutaminase that consists of Ser-74, Lys-77, Asn-126, Lys-268, and Ser-269, which is highly conserved in all microorganisms.
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