Journal articles: 'Enzimi cold-active'

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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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Abd Latip, Muhammad Asyraf, Noor Faizul Hadry Nordin, Siti Aisyah Alias, Jerzy Smykla, Faridah Yusof, and Mohd Azrul Naim Mohamad. "The Optimization of Growth Condition of the Bacteria Producing Cold-Active Proteolytic Enzyme from the Antarctic Region." IIUM Engineering Journal 24, no. 1 (January 4, 2023): 27–39. http://dx.doi.org/10.31436/iiumej.v24i1.2447.

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The growth conditions of bacteria producing cold-active protease isolated from an Antarctic sample were screened using one-factor-at-time (OFAT). Then, crude protease of the strain was extracted during the late logarithmic phase for enzymatic assay. A strain that showed the highest enzyme activity was selected for optimization via response surface method (RSM). The parameters studied were incubation temperature (4 – 36 °C), pH media (4 – 10) and NaCl concentration (0 – 8%). Based on the OFAT results, all eight strains showed the highest growth rate at 20 °C, pH 7 and 4% (w/v) NaCl. The assay showed that the crude enzyme extracted from strain SC8 exhibited significantly higher activity (0.20 U and 0.37 U) than the positive control (0.11 U and 0.31 U) at -20 °C and 20 °C. RSM suggested that the optimized setting for growth of SC8 were at 20.5 °C, pH 6.83 and 2.05% (w/v) of NaCl with the results of the bacterial growth rate value was 3.70 ± 0.06 x 106 cells/hr. Optimal growth conditions of SC8 from this study are useful for the large-scale production of cold-active protease in future. ABSTRAK: Keadaan pertumbuhan bakteria yang menghasilkan enzim protease aktif sejuk daripada sampel Antartika disaring menggunakan satu faktor pada masa (OFAT). Kemudian, enzim protease ini diekstrak pada lewat fasa logaritma untuk ujian enzimatik. Strain yang menunjukkan aktiviti enzim tertinggi telah dipilih untuk tujuan pengoptimuman melalui kaedah permukaan tindak balas (RSM). Parameter yang dikaji ialah suhu pengeraman (4 – 36 °C), pH media (4 – 10) dan kepekatan NaCl (0 – 8%). Berdasarkan OFAT, kesemua lapan bakteria menunjukkan kadar pertumbuhan tertinggi pada 20 °C, pH 7 dan 4% NaCl. Hasil ujian enzimatik menunjukkan bahawa enzim protease yang diekstrak daripada SC8 mempamerkan aktiviti yang jauh lebih tinggi (0.20 U dan 0.37 U) daripada kawalan positif (0.11 U dan 0.31 U) pada -20 °C dan 20 °C. RSM mencadangkan tetapan optimum untuk pertumbuhan SC8 adalah pada 20.5 °C, pH 6.83 dan 2.05% NaCl dengan keputusan kadar pertumbuhan bakteria ialah 3.70 ± 0.06 x 106 sel/jam. Keadaan pertumbuhan optimum SC8 daripada kajian ini bermanfaat untuk menghasilkan produk protease aktif sejuk secara besar-besaran pada masa hadapan. The growth conditions of bacteria producing cold-active protease isolated from an Antarctic sample were screened using one-factor-at-time (OFAT). Then, crude protease of the strain was extracted during the late logarithmic phase for enzymatic assay. A strain that showed the highest enzyme activity was selected for optimization via response surface method (RSM). The parameters studied were incubation temperature (4 – 36 °C), pH media (4 – 10) and NaCl concentration (0 – 8%). Based on the OFAT results, all eight strains showed the highest growth rate at 20 °C, pH 7 and 4% (w/v) NaCl. The assay showed that the crude enzyme extracted from strain SC8 exhibited significantly higher activity (0.20 U and 0.37 U) than the positive control (0.11 U and 0.31 U) at -20 °C and 20 °C. RSM suggested that the optimized setting for growth of SC8 were at 20.5 °C, pH 6.83 and 2.05% (w/v) of NaCl with the results of the bacterial growth rate value was 3.70 ± 0.06 x 106 cells/hr. Optimal growth conditions of SC8 from this study are useful for the large-scale production of cold-active protease in future. ABSTRAK: Keadaan pertumbuhan bakteria yang menghasilkan enzim protease aktif sejuk daripada sampel Antartika disaring menggunakan satu faktor pada masa (OFAT). Kemudian, enzim protease ini diekstrak pada lewat fasa logaritma untuk ujian enzimatik. Strain yang menunjukkan aktiviti enzim tertinggi telah dipilih untuk tujuan pengoptimuman melalui kaedah permukaan tindak balas (RSM). Parameter yang dikaji ialah suhu pengeraman (4 – 36 °C), pH media (4 – 10) dan kepekatan NaCl (0 – 8%). Berdasarkan OFAT, kesemua lapan bakteria menunjukkan kadar pertumbuhan tertinggi pada 20 °C, pH 7 dan 4% NaCl. Hasil ujian enzimatik menunjukkan bahawa enzim protease yang diekstrak daripada SC8 mempamerkan aktiviti yang jauh lebih tinggi (0.20 U dan 0.37 U) daripada kawalan positif (0.11 U dan 0.31 U) pada -20 °C dan 20 °C. RSM mencadangkan tetapan optimum untuk pertumbuhan SC8 adalah pada 20.5 °C, pH 6.83 dan 2.05% NaCl dengan keputusan kadar pertumbuhan bakteria ialah 3.70 ± 0.06 x 106 sel/jam. Keadaan pertumbuhan optimum SC8 daripada kajian ini bermanfaat untuk menghasilkan produk protease aktif sejuk secara besar-besaran pada masa hadapan.

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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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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Joseph, Babu, Supriya Upadhyaya, and Pramod Ramteke. "Production of Cold-Active Bacterial Lipases through Semisolid State Fermentation Using Oil Cakes." Enzyme Research 2011 (April 26, 2011): 1–6. http://dx.doi.org/10.4061/2011/796407.

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Production of cold active lipase by semisolid state fermentation involves the use of agroindustrial residues. In the present study, semisolid state fermentation was carried out for the production of cold active lipase using Micrococcus roseus, isolated from soil samples of Gangotri glaciers, Western Himalayas. Among various substrate tested, groundnut oil cake (GOC) favored maximal yield of lipases at 15 ± 1°C within 48 h. Supplementation of glucose 1% (w/v) as additional carbon source and ammonium nitrate 2% (w/v) as additional nitrogen source enhanced production of lipase. Addition of triglycerides 0.5% (v/v) tends to repress the lipase production. Further mixed preparation of groundnut oil cake (GOC) along with mustard oil cake (MOC) in the ratio of 1 : 1, and its optimization resulted in improved production of cold active lipase. The enzyme exhibited maximum activity at 10–15°C and was stable at temperatures lower than 30°C. The lipase exhibited optimum activity at pH 8 and showed more than 60% stability at pH 9. Semisolid state fermentation process by utilizing agroindustrial wastes will direct to large-scale commercialization of lipase catalyzed process in cost-effective systems.

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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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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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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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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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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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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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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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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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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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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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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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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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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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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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Kryukova, M. V., L. E. Petrovskaya, E. A. Kryukova, G. Yu Lomakina, S. A. Yakimov, E. G. Maksimov, K. M. Boyko, V. O. Popov, D. A. Dolgikh, and M. P. Kirpichnikov. "Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library." Biomolecules 9, no. 12 (December 16, 2019): 880. http://dx.doi.org/10.3390/biom9120880.

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PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.

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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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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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Huston, Adrienne L., Barbara Methe, and Jody W. Deming. "Purification, Characterization, and Sequencing of an Extracellular Cold-Active Aminopeptidase Produced by Marine Psychrophile Colwellia psychrerythraea Strain 34H." Applied and Environmental Microbiology 70, no. 6 (June 2004): 3321–28. http://dx.doi.org/10.1128/aem.70.6.3321-3328.2004.

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ABSTRACT The limited database on cold-active extracellular proteases from marine bacteria was expanded by successful purification and initial biochemical and structural characterization of a family M1 aminopeptidase (designated ColAP) produced by the marine psychrophile Colwellia psychrerythraea strain 34H. The 71-kDa enzyme displayed a low optimum temperature (19Ã¯Â¿Â½C) and narrow pH range (pH 6 to 8.5) for activity and greater thermolability than other extracellular proteases. Sequencing of the gene encoding ColAP revealed a predicted amino acid sequence with the highest levels of identity (45 to 55%) to M1 aminopeptidases from mesophilic members of the γ subclass of the Proteobacteria and the next highest levels of identity (35 to 36%) to leukotriene A4 hydrolases from mammalian sources. Compared to mesophilic homologs, ColAP had structural differences thought to increase the flexibility for activity in the cold; for example, it had fewer proline residues, fewer ion pairs, and a lower hydrophobic residue content. In addition to intrinsic properties that determine enzyme activity and stability, we also investigated effects of extracellular polymeric substances (EPS) from spent culture medium of strain 34H on ColAP activity at an environmentally relevant temperature (0Ã¯Â¿Â½C) and at 45Ã¯Â¿Â½C (the maximum temperature for activity). In both cases, ColAP stability increased significantly in the presence of EPS, indicating the importance of considering environmentally relevant extrinsic factors when enzyme structure and function are investigated.

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Noby, Nehad, Rachel L. Johnson, Jonathan D. Tyzack, Amira M. Embaby, Hesham Saeed, Ahmed Hussein, Sherine N. Khattab, Pierre J. Rizkallah, and D. Dafydd Jones. "Structure-Guided Engineering of a Family IV Cold-Adapted Esterase Expands Its Substrate Range." International Journal of Molecular Sciences 23, no. 9 (April 24, 2022): 4703. http://dx.doi.org/10.3390/ijms23094703.

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Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining suggested that triacetin was a potential commercially valuable substrate for EstN7, which was subsequently proved experimentally with the final product being a single isomeric product, 1,2-glyceryl diacetate. Enzyme kinetics revealed that EstN7’s activity is restricted to C2 and C4 substrates due to a plug at the end of the acyl binding pocket that blocks access to a buried water-filled cavity. Residues M187, N211 and W206 were identified as key plug forming residues. N211A stabilised EstN7 allowing incorporation of the destabilising M187A mutation. The M187A-N211A double mutant had the broadest substrate range, capable of hydrolysing a C8 substrate. W206A did not appear to have any significant effect on substrate range either alone or when combined with the double mutant. Thus, the enzyme kinetics and engineering together with a recently determined structure of EstN7 provide new insights into substrate specificity and the role of acyl binding pocket plug residues in determining family IV esterase stability and substrate range.

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Wang, Yatong, Yanhua Hou, Ping Nie, Yifan Wang, Xiulian Ren, Qifeng Wei, and Quanfu Wang. "A Novel Cold-Adapted and Salt-Tolerant RNase R from Antarctic Sea-Ice Bacterium Psychrobacter sp. ANT206." Molecules 24, no. 12 (June 14, 2019): 2229. http://dx.doi.org/10.3390/molecules24122229.

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A novel RNase R, psrnr, was cloned from the Antarctic bacterium Psychrobacter sp. ANT206 and expressed in Escherichia coli (E. coli). A bioinformatics analysis of the psrnr gene revealed that it contained an open reading frame of 2313 bp and encoded a protein (PsRNR) of 770 amino acids. Homology modeling indicated that PsRNR had reduced hydrogen bonds and salt bridges, which might be the main reason for the catalytic efficiency at low temperatures. A site directed mutation exhibited that His 667 in the active site was absolutely crucial for the enzyme catalysis. The recombinant PsRNR (rPsRNR) showed maximum activity at 30 °C and had thermal instability, suggesting that rPsRNR was a cold-adapted enzyme. Interestingly, rPsRNR displayed remarkable salt tolerance, remaining stable at 0.5–3.0 M NaCl. Furthermore, rPsRNR had a higher kcat value, contributing to its efficient catalytic activity at a low temperature. Overall, cold-adapted RNase R in this study was an excellent candidate for antimicrobial treatment.

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MURAKAWA, Takeshi, Hiroshi YAMAGATA, Hiroki TSURUTA, and Yasuo AIZONO. "Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile,Shewanellasp., and Expression of the Recombinant Enzyme." Bioscience, Biotechnology, and Biochemistry 66, no. 4 (January 2002): 754–61. http://dx.doi.org/10.1271/bbb.66.754.

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Roohi and Mohammed Kuddus. "Statistical optimization of cold-active chitinase production by mutagenized cells of multi-enzyme producing Bacillus cereus GA6." Rendiconti Lincei 26, no. 3 (July 14, 2015): 271–80. http://dx.doi.org/10.1007/s12210-015-0447-9.

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Zeng, Runying, Rui Zhang, Jing Zhao, and Nianwei Lin. "Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: enzyme purification and characterization." Extremophiles 7, no. 4 (August 1, 2003): 335–37. http://dx.doi.org/10.1007/s00792-003-0323-x.

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50

Dolhaji, N. H., I. D. Muhammad, H. Yaakob, and A. Mohd Marsin. "Chilling injury in pineapple fruits: physical quality attributes and antioxidant enzyme activity." Food Research 4, S5 (December 20, 2020): 86–95. http://dx.doi.org/10.26656/fr.2017.4(s5).004.

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Harvested fruit have high metabolic and moisture content which leads to an active biochemical reaction that contributes to decrement of nutritional value such as vitamin, proteins and lipids. The application of low temperature as a single-effective management to prolong shelf-life of fruits is a common practice which applied to keep agriculture commodities at high quality. A matured stage pineapple is very perishable and cold storage chain is crucial in maintaining the chemical and physical quality attributes in order to assure its commercial value for market. The main objective of this study is to evaluate the effect of sub-optimum cold storage scenario on changes of pineapple fruits physical quality attributes, the activity of browning enzyme and antioxidant related enzyme in 2 pineapple cultivars (cv.), Morris (Queen-type) and Josapine (hybrid of Spanish and Smooth Cayeen). Malaysian pineapple fruit cv. with different sensitivity toward CI, Morris (Queen-type) and Josapine (Smooth-Cayenne-type) were stored at sub-optimal storage temperatures (4±2°C) for 28 days to investigate the effects of CI towards physical quality attributes and antioxidant enzyme activity. The result indicated both cv. was affected with CI towards the 28 days of sub-optimum cold storage. Overall physical quality attributes indicated CI was found positively correlated with the increase of EL and TTA and on the contrary decrease its firmness, weight (% w/w), brightness (L*) and redness (A*). Similarly, a positive correlation was also deduced between CI symptoms and the activities of PPO and APX which reflect the incident of oxidative stress. The results derived from this study may serve as a basis for evaluation of better postharvest strategies to control CI during cold chain storage of pineapple fruits and thus assure the quality and nutritional value till it reaches to consumer.

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Journal articles on the topic 'Enzimi cold-active'

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