Littérature scientifique sur le sujet « Cold-active enzyme »

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.

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 <i>Candida, Yarrowia, Rhodotorula</i>, and <i>Pichia </i>have been reported. In this paper, biosynthesis and properties of a novel cold-active lipase from <i>Candida zeylanoides</i> 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 <i>C. zeylanoides</i> lipase has comparative advantages to most similar enzymes with technological applications and may have potential to become an industrially important enzyme.

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.

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 &beta;-galactosidase, we isolated several cold-adapted microorganisms from glacier zone soil. One cold-adapted &beta;-galactosidase producing strain was obtained. The biochemical characteristics and the results of 16S rDNA sequencing identified the strain as <I>Rahnella aquatilis</I>. 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 &beta;-galactosidase was optimally active at 35°C and at pH 6.5 when assayed with <I>o</I>-nitrophenyl-&beta;-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.

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.

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.

<p><strong>Objective: </strong>The objective of the present study was on <em>Penicillium pinophilum </em>strain F2 from soil samples of Jammu city having the potentiality to produce alkaline cold active endoglucanase and pigment.</p><p><strong>Methods: </strong><em>Penicillium pinophilum </em>strain F2,<em> </em>a<em> </em>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.</p><p><strong>Results: </strong>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%.</p><p><strong>Conclusion: </strong>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.</p>

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.

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.

Biodiversity of organisms and their genomic content is a valuable source of enzymes, some of which can be isolated and turned into biocatalysts, useful for more sustainable and efficient industrial processes. Organisms thriving in constantly cold environments produce enzymes that may be more efficient in the cold and more thermolabile than enzymes from other organisms, and that display interesting features for the catalysis of several processes that require or are better at low temperature. In the first part of this thesis, two glycoside hydrolases of family 19 (GH19), named LYS177 and LYS188, were identified in the genome of an Antarctic Pseudomonas strain and characterized. Even though most of the characterized GH19 are chitinases, LYS177 and LYS188 showed no chitinolytic activity, but were active as lysozymes with an optimum temperature of 25-35°C, and retained 40% of their highest activity at 5°C. The temperatures of midpoint unfolding transition were estimated to be 20°C higher than their optimum of activity. Based on these features and sequence analysis, LYS177 and LYS188 can be considered cold-active phage endolysins integrated in prophagic regions of the bacterial host. Moreover, the best performing of the two, LYS177, was active and structurally stable over several days only at 4°C, indicating it as a candidate for potential application on the preservation of food and beverages during cold storage. In protein families, enzymes can rapidly acquire new specializations. Therefore, best practices should be implemented to select optimal candidates with the activity of interest and new, potentially promising, features. Characterized GH19 enzymes showed an enhanced in vivo crop defence against chitin containing pathogens and antimicrobial potentialities. In the second part of this thesis, the sequence space of the GH19 family was explored and a database was created to highlight non-described sequences potentially endowed with interesting variants. Based on global pairwise sequence identity of all proteins available in public databases, GH19s were assigned to two subfamilies, the chitinases and the endolysins. Subfamilies were further split into homologous families, which differ in the n° of characterized enzymes they harbour, in the taxonomical distribution, in the presence of accessory domains and loop insertions. Despite this heterogeneity, a core consisting of 27 amino acids around the active site, including important substrate binding residues, was inferred to be conserved between GH19 subfamilies. Thus, this shared core is suggested to be associated to the GH19 capacity to bind sugars containing N-acetyl-glucosamine. Moreover, specifically conserved positions in each subfamily alignment were identified to be a “signature” useful for predicting the substrate specialization of chitinases and endolysins, and to indicate possible outliers with different features. The GH19 evolution was also investigated through molecular phylogeny to explain the observed sequence and structural plasticity: despite endolysins were divided in an higher number of homologous families, they remained in phages and their bacterial hosts, contrary to chitinases, which spread to both prokaryotic and eukaryotic taxa, and acquired at least four loop insertions; moreover, the GH19 chitinase catalytic domain passed from plants to bacteria by horizontal gene transfer in at least two cases. In conclusion, the second part of this thesis shows how bioinformatic tools can be used to analyse the sequence space of a glycoside hydrolase family and extract information to help both experts and non-experts to optimize the discovery of new biocatalysts potentially applied in the field of human health and nutrition.
Biodiversity of organisms and their genomic content is a valuable source of enzymes, some of which can be isolated and turned into biocatalysts, useful for more sustainable and efficient industrial processes. Organisms thriving in constantly cold environments produce enzymes that may be more efficient in the cold and more thermolabile than enzymes from other organisms, and that display interesting features for the catalysis of several processes that require or are better at low temperature. In the first part of this thesis, two glycoside hydrolases of family 19 (GH19), named LYS177 and LYS188, were identified in the genome of an Antarctic Pseudomonas strain and characterized. Even though most of the characterized GH19 are chitinases, LYS177 and LYS188 showed no chitinolytic activity, but were active as lysozymes with an optimum temperature of 25-35°C, and retained 40% of their highest activity at 5°C. The temperatures of midpoint unfolding transition were estimated to be 20°C higher than their optimum of activity. Based on these features and sequence analysis, LYS177 and LYS188 can be considered cold-active phage endolysins integrated in prophagic regions of the bacterial host. Moreover, the best performing of the two, LYS177, was active and structurally stable over several days only at 4°C, indicating it as a candidate for potential application on the preservation of food and beverages during cold storage. In protein families, enzymes can rapidly acquire new specializations. Therefore, best practices should be implemented to select optimal candidates with the activity of interest and new, potentially promising, features. Characterized GH19 enzymes showed an enhanced in vivo crop defence against chitin containing pathogens and antimicrobial potentialities. In the second part of this thesis, the sequence space of the GH19 family was explored and a database was created to highlight non-described sequences potentially endowed with interesting variants. Based on global pairwise sequence identity of all proteins available in public databases, GH19s were assigned to two subfamilies, the chitinases and the endolysins. Subfamilies were further split into homologous families, which differ in the n° of characterized enzymes they harbour, in the taxonomical distribution, in the presence of accessory domains and loop insertions. Despite this heterogeneity, a core consisting of 27 amino acids around the active site, including important substrate binding residues, was inferred to be conserved between GH19 subfamilies. Thus, this shared core is suggested to be associated to the GH19 capacity to bind sugars containing N-acetyl-glucosamine. Moreover, specifically conserved positions in each subfamily alignment were identified to be a “signature” useful for predicting the substrate specialization of chitinases and endolysins, and to indicate possible outliers with different features. The GH19 evolution was also investigated through molecular phylogeny to explain the observed sequence and structural plasticity: despite endolysins were divided in an higher number of homologous families, they remained in phages and their bacterial hosts, contrary to chitinases, which spread to both prokaryotic and eukaryotic taxa, and acquired at least four loop insertions; moreover, the GH19 chitinase catalytic domain passed from plants to bacteria by horizontal gene transfer in at least two cases. In conclusion, the second part of this thesis shows how bioinformatic tools can be used to analyse the sequence space of a glycoside hydrolase family and extract information to help both experts and non-experts to optimize the discovery of new biocatalysts potentially applied in the field of human health and nutrition.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第8424号
農博第1108号
新制||農||799(附属図書館)
学位論文||H12||N3381(農学部図書室)
UT51-2000-F328
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 江﨑 信芳, 教授 清水 昌, 教授 加藤 暢夫
学位規則第4条第1項該当

Accidental hypothermia is defined as a core temperature of <35°C and is uncommon. It may present in any age group at any time of the year. Hypothermia may be primary, where the cold injury is the major pathology, or secondary where patients develop hypothermia incidental to another illness. Since the severely cold patient may be in cardiac arrest, areflexic, and in coma, decision making regarding treatment, its initiation, and continuation, may be difficult. Hypothermia is classified into mild (33–35°C), moderate (28–33°C) and severe (<28°C), but these are not distinct clinical syndromes. A more recent classification into stages has emerged from alpine medicine along with a treatment algorithm based on it. Many pathophysiogical changes are due to reduced enzyme action. Clinical features include changes in higher cerebral functions with bizarre behaviour progressing to coma. In the circulation initial tachycardia and hypertension (‘cold stress’) are replaced, as the patient cools, with worsening hypotension and bradycardia and, eventually, ventricular fibrillation and asystole. Rewarming methods are classified as passive or active and the latter subdivided into external, core, and extracorporeal. Active warming should be considered for patients with a temperature of 32°C or lower. Peritoneal lavage has the advantage of warming the liver directly and also the heart through the diaphragm. Cardiopulmonary bypass is the extracorporeal method with most experience, but the advent of extracorporeal membrane oxygenation has the advantage of portability.

Piperine is known for its versatile therapeutic activity. It has been used for various disease conditions (e.g., cold, cough, etc.). Piperine is an alkaloid found in black pepper. It possesses various pharmacological actions like anti-inflammatory, anti-oxidant, anti-cholinergic, and anti-cancerous. The above-mentioned properties will be studied by selecting target proteins COX-2 protein, angiotensin converting enzyme, acetylcholineesterases, and survivin using computational docking study. This chapter explains the inhibition property of piperine against selected target protein from the results of docking studies. Based on the docking scores and protein-ligand interactions, piperine was found to bind well in the active site of the selected target proteins. It ensures the binding efficacy of piperine against selected target proteins. Docking scores and protein-ligand interactions plays an important role in its therapeutic activity.