Identification of a novel glycoside hydrolase family 8 xylanase from Deinococcus geothermalis and its application at low temperatures.

Xylanase is the most important hydrolase in the xylan hydrolase system, the main function of which is ß-1,4-endo-xylanase, which randomly cleaves xylans to xylo-oligosaccharides and xylose. Xylanase has wide ranging of applications, but there remains little research on the cold-adapted enzymes required in some low-temperature industries. Glycoside hydrolase family 8 (GH8) xylanases have been reported to have cold-adapted enzyme activity. In this study, the xylanase gene dgeoxyn was excavated from Deinococcus geothermalis through sequence alignment. The recombinant xylanase DgeoXyn encodes 403 amino acids with a theoretical molecular weight of 45.39 kDa. Structural analysis showed that DgeoXyn has a (α/α)6-barrel fold structure typical of GH8 xylanase. At the same time, it has strict substrate specificity, is only active against xylan, and its hydrolysis products include xylobiose, xylotrinose, xytetranose, xylenanose, and a small amount of xylose. DgeoXyn is most active at 70 â„ƒ and pH 6.0. It is very stable at 10, 20, and 30 â„ƒ, retaining more than 80% of its maximum enzyme activity. The enzyme activity of DgeoXyn increased by 10% after the addition of Mn2+ and decreased by 80% after the addition of Cu2+. The Km and Vmax of dgeox were 42 mg/ml and 20,000 U/mg, respectively, at a temperature of 70 â„ƒ and pH of 6.0 using 10 mg/ml beechwood xylan as the substrate. This research on DgeoXyn will provide a theoretical basis for the development and application of low-temperature xylanase.

A Review on Psychrophilic ß-D-Galactosidases and Their Potential Applications.

The majority of the Earth's ecosystem is frigid and frozen, which permits a vast range of microbial life forms to thrive by triggering physiological responses that allow them to survive in cold and frozen settings. The apparent biotechnology value of these cold-adapted enzymes has been targeted. Enzymes' market size was around USD 6.3 billion in 2017 and will witness growth at around 6.8% CAGR up to 2024 owing to shifting consumer preferences towards packaged and processed foods due to the rising awareness pertaining to food safety and security reported by Global Market Insights (Report ID-GMI 743). Various firms are looking for innovative psychrophilic enzymes in order to construct more effective biochemical pathways with shorter reaction times, use less energy, and are ecologically acceptable. D-Galactosidase catalyzes the hydrolysis of the glycosidic oxygen link between the terminal non-reducing D-galactoside unit and the glycoside molecule. At refrigerated temperature, the stable structure of psychrophile enzymes adjusts for the reduced kinetic energy. It may be beneficial in a wide variety of activities such as pasteurization of food, conversion of biomass, biological role of biomolecules, ambient biosensors, and phytoremediation. Recently, psychrophile enzymes are also used in claning the contact lens. ß-D-Galactosidases have been identified and extracted from yeasts, fungi, bacteria, and plants. Conventional (hydrolyzing activity) and nonconventional (non-hydrolytic activity) applications are available for these enzymes due to its transgalactosylation activity which produce high value-added oligosaccharides. This review content will offer new perspectives on cold-active ß-galactosidases, their source, structure, stability, and application.

Novel, acidic, and cold-adapted glycoside hydrolase family 8 endo-ß-1,4-glucanase from an Antarctic lichen-associated bacterium, Lichenicola cladoniae PAMC 26568.

Endo-ß-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted ß-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-ß-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-ß-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-ß-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley ß-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) and k cat/K m value (6.35 mg-1 s-1mL) of rGluL toward barley ß-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg-1) and k cat/K m value (2.83 mg-1 s-1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-ß-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization.

We identified the raw-starch-digesting α-amylase genes a earthworm Eisenia fetid α amylase I and II (Ef-Amy I and Ef-Amy II). Each gene consists of 1,530 base pairs (bp) that encode proteins of 510 amino acids, as indicated by the corresponding mRNA sequences. Ef-Amy I and II showed an 89% amino acid identity. The amino acid sequences of Ef-Amy I and II were similar to those of the α-amylases from porcine pancreas, human pancreas, Tenebrio molitor, Oryctolagus cuniculus, and Xenopus (Silurana) tropicalis. Each gene encoding mature Ef-Amy I and II was expressed in the GS115 strain of Pichia pastoris. The molecular masses of the recombinant Ef-Amy I and II were 57 kDa each, and catalytically important residues of α-amylases of the GH family 13 were conserved in both proteins. These amylases exhibited raw-starch-digesting activity at 4 °C. The substrate specificities of rEf-Amy I and II were dissimilar. rEf-Amy I and II were shown to be active even in 40% ethanol, 4 M NaCl, and 4 M KCl.

Comparative characterization of the hemocyanin-derived phenol oxidase activity from spiders inhabiting different thermal habitats.

Enzymes adapted to cold temperatures are commonly characterized for having higher Michaelis-Menten constants (KM) values and lower optimum and denaturation temperature, when compared to other meso or thermophilic enzymes. Phenoloxidase (PO) enzymes are ubiquitous in nature, however, they have not been reported in spiders. It is the oxygen carrier protein hemocyanin (Hc), found at high concentrations in their hemolymph, which displays an inducible PO activity. Hence, we hypothesize that Hc-derived PO activity could show features of cold adaptation in alpine species. We analyzed the Hc from two species of Theraphosidae from different thermal environments: Euathlus condorito (2400 m a.s.l.) and Grammostola rosea (500 m a.s.l.). Hc was purified from the hemolymph of both spiders and was characterized by identifying subunit composition and measuring the sodium dodecyl sulfate (SDS)-induced PO activity. The high-altitude spider Hc showed higher PO activity under all conditions and higher apparent Michaelis-Menten constant. Moreover, the optimum temperature for PO activity was lower for E. condorito Hc. These findings suggest a potential adaptation at the level of Hc-derived PO activity in Euathlus condorito, giving insights on possible mechanisms used by this mygalomorph spider to occupy extremes and variable thermal environments.

Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

Study on a Novel Cold-Active and Halotolerant Monoacylglycerol Lipase Widespread in Marine Bacteria Reveals a New Group of Bacterial Monoacylglycerol Lipases Containing Unusual C(A/S)HSMG Catalytic Motifs.

Monoacylglycerol lipases (MGLs) are present in all domains of life. However, reports on bacterial MGLs are still limited. Until now, reported bacterial MGLs are all thermophilic/mesophilic enzymes from warm terrestrial environments or deep-sea hydrothermal vent, and none of them originates from marine environments vastly subject to low temperature, high salts, and oligotrophy. Here, we characterized a novel MGL, GnMgl, from the marine cold-adapted and halophilic bacterium Glaciecola nitratireducens FR1064T. GnMgl shares quite low sequence similarities with characterized MGLs (lower than 31%). GnMgl and most of its bacterial homologs harbor a catalytic Ser residue located in the conserved C(A/S)HSMG motif rather than in the typical GxSxG motif reported on other MGLs, suggesting that GnMgl-like enzymes might be different from reported MGLs in catalysis. Phylogenetic analysis suggested that GnMgl and its bacterial homologs are clustered as a separate group in the monoglyceridelipase_lysophospholipase family of the Hydrolase_4 superfamily. Recombinant GnMgl has no lysophospholipase activity but could hydrolyze saturated (C12:0-C16:0) and unsaturated (C18:1 and C18:2) MGs and short-chain triacylglycerols, displaying distinct substrate selectivity from those of reported bacterial MGLs. The substrate preference of GnMgl, predicted to be a membrane protein, correlates to the most abundant fatty acids within the strain FR1064T, suggesting the role of GnMgl in the lipid catabolism in this marine bacterium. In addition, different from known bacterial MGLs that are all thermostable enzymes, GnMgl is a cold-adapted enzyme, with the maximum activity at 30°C and retaining 30% activity at 0°C. GnMgl is also a halotolerant enzyme with full activity in 3.5M NaCl. The cold-adapted and salt-tolerant characteristics of GnMgl may help its source strain FR1064T adapt to the cold and saline marine environment. Moreover, homologs to GnMgl are found to be abundant in various marine bacteria, implying their important physiological role in these marine bacteria. Our results on GnMgl shed light on marine MGLs.

Analysis of amino acid residues involved in the thermal properties of isocitrate dehydrogenases from a psychrophilic bacterium, Colwellia maris, and a psychrotrophic bacterium, Pseudomonas psychrophila.

Monomeric NADP+-dependent isocitrate dehydrogenase (IDH) from a psychrophilic bacterium, Colwella maris, (CmIDH) is a cold-adapted enzyme, whereas that of a psychrotrophic bacterium, Pseudomonas psychrophila, (PpIDH) is mesophilic. However, the amino acid sequence identity of the two IDHs is high (67%). To identify the amino acid residues involved in the differences in their thermal properties, such as optimum temperature and thermostability for activity, six amino acid residues located in the corresponding positions of their regions 2 and 3 were substituted by site-directed mutagenesis, and several thermal properties of the mutated IDHs were examined. CmIDH mutants, CmE538L, CmE596L and CmA741S, substituted at Glu538, Glu596 and Ala741 by the corresponding PpIDH residues of Leu, Leu and Ser, respectively, exhibited higher thermostability than wild-type CmIDH (CmWT). Furthermore, the specific activity of CmE596L and CmA741S was higher than that of CmWT. On the other hand, the corresponding mutants of PpIDH PpL536E, PpL594E and PpS739A were more thermolabile than wild-type PpIDH, and PpL594E had a lower specific activity at temperatures over 45°C. These results suggested that these amino acid residues of CmIDH and PpIDH are involved in their thermal properties.

Molecular Cloning and Characterization of a Novel Cold-Adapted Alkaline 1,3-α-3,6-Anhydro-L-galactosidase, Ahg558, from Gayadomonas joobiniege G7.

Agar, a major polysaccharide of red algal cells, is degraded by ß-agarases into neoagarobiose, which is further hydrolyzed into the monomers, D-galactose and 3,6-anhydro-L-galactose, by 1,3-α-3,6-anhydro-L-galactosidases including α-1,3-L-neoagarooligasaccharide hydrolase (α-NAOSH). A novel cold-adapted alkaline α-NAOSH, Ahg558, consisting of 359 amino acids (40.8 kDa) was identified from Gayadomonas joobiniege G7. It was annotated as a glycosyl hydrolase family 43 based on genomic sequence analysis, showing 84% and 74% identities with the characterized α-NAOSHs from Agarivorans gilvus WH0801 and Saccharophagus degradans 2-40, respectively. The recombinant Ahg558 (rAhg558) purified from Escherichia coli formed dimers and cleaved α-1,3 glycosidic bonds at the non-reducing ends of the neoagarobiose, neoagarotetraose, and neoagarohexaose, which was confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for rAhg558 activity were 9.0 and 30 °C, respectively. Unusually, it retained over 93% activity in a broad range of temperatures between 0 and 40 °C and over 73% in a broad range of pH between pH 6.0 and pH 9.0, indicating it is a unique cold-adapted alkaline exo-acting α-NAOSH. Its enzymatic activity was dependent on Mn2+ ions. Km and Vmax values toward neoagarobiose were 2.6 mg/mL (8.01 mM) and 133.33 U/mg, respectively.

Cloning, Expression and Characterization of a Novel Cold-adapted ß-galactosidase from the Deep-sea Bacterium Alteromonas sp. ML52.

The bacterium Alteromonas sp. ML52, isolated from deep-sea water, was found to synthesize an intracellular cold-adapted ß-galactosidase. A novel ß-galactosidase gene from strain ML52, encoding 1058 amino acids residues, was cloned and expressed in Escherichia coli. The enzyme belongs to glycoside hydrolase family 2 and is active as a homotetrameric protein. The recombinant enzyme had maximum activity at 35 °C and pH 8 with a low thermal stability over 30 °C. The enzyme also exhibited a Km of 0.14 mM, a Vmax of 464.7 U/mg and a kcat of 3688.1 S-1 at 35 °C with 2-nitrophenyl-ß-d-galactopyranoside as a substrate. Hydrolysis of lactose assay, performed using milk, indicated that over 90% lactose in milk was hydrolyzed after incubation for 5 h at 25 °C or 24 h at 4 °C and 10 °C, respectively. These properties suggest that recombinant Alteromonas sp. ML52 ß-galactosidase is a potential biocatalyst for the lactose-reduced dairy industry.

Organic Solvent-Tolerant Esterase from Sphingomonas glacialis Based on Amino Acid CompositionAnalysis: Cloning and Characterization of EstSP2.

Organic solvent-tolerant (OST) enzymes are widely applied in various industries due to their activity and stability in organic solvents, higher substrate solubility, and increased stereo-selectivity. However, the criteria for identifying OST enzymes largely remain unresolved. In this study, we compared the amino acid composition of 19 OST esterases and 19 non-OST esterases. OST esterases have increased ratio of Ala and Arg residues and decreased ratio of Asn, Ile, Tyr, and Ser residues. Based on the amino acid composition analysis, we cloned acarboxylesterase (EstSP2) from a psychrophilic bacterium, Sphingomonas glacialis PAMC 26605, and characterized its recombinant protein. EstSP2 is substrate specific to p-nitrophenyl acetate and hydrolyzed aspirin, with optimal activityat 40°C; at 4°C, the activity is approximately 50% of its maximum. As expected, EstSP2showstolerance in up to 40% concentration of polar organic solvents, including dimethyl sulfoxide, methanol, and ethanol. The results of this study suggest that selection of OST esterases based on their amino acid composition analysis could be a novel approach to identify OST esterases produced from bacterial genomes.

Identification and biochemical characterization of a novel cold-adapted 1,3-α-3,6-anhydro-L-galactosidase, Ahg786, from Gayadomonas joobiniege G7.

Agar is a major polysaccharide of red algal cells and is mainly decomposed into neoagarobiose by the co-operative effort of ß-agarases. Neoagarobiose is hydrolyzed into monomers, D-galactose and 3,6-anhydro-L-galactose, via a microbial oxidative process. Therefore, the enzyme, 1,3-α-3,6-anhydro-L-galactosidase (α-neoagarobiose/neoagarooligosaccharide hydrolase) involved in the final step of the agarolytic pathway is crucial for bioindustrial application of agar. A novel cold-adapted α-neoagarooligosaccharide hydrolase, Ahg786, was identified and characterized from an agarolytic marine bacterium Gayadomonas joobiniege G7. Ahg786 comprises 400 amino acid residues (45.3 kDa), including a 25 amino acid signal peptide. Although it was annotated as a hypothetical protein from the genomic sequencing analysis, NCBI BLAST search showed 57, 58, and 59% identities with the characterized α-neoagarooligosaccharide hydrolases from Saccharophagus degradans 2-40, Zobellia galactanivorans, and Bacteroides plebeius, respectively. The signal peptide-deleted recombinant Ahg786 expressed and purified from Escherichia coli showed dimeric forms and hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose into 3,6-anhydro-L-galactose and other compounds by cleaving α-1,3-glycosidic bonds from the non-reducing ends of neoagarooligosaccharides, as confirmed by thin-layer chromatography and mass spectrometry. The optimum pH and temperature for Ahg786 activity were 7.0 and 15 °C, respectively, indicative of its unique cold-adapted features. The enzymatic activity severely inhibited with 0.5 mM ethylenediaminetetraacetic acid was completely restored or remarkably enhanced by Mn2+ in a concentration-dependent manner, suggestive of the dependence of the enzyme on Mn2+ ions. Km and Vmax values for neoagarobiose were 4.5 mM and 1.33 U/mg, respectively.

Characterization of a cold-active transglutaminase from a crayfish, Pacifastacus leniusculus.

Transglutaminase (TGase) from signal crayfish (Pacifastacus leniusculus) and its activity at low temperatures was studied. TGase is an abundant protein in the hematopoietic (HPT) cells and this tissue was used for TGase enzyme preparation. The optimal temperature and pH for the activity of crayfish TGase were determined. We found that TGase activity at 4 °C showed nearly the same activity as at a temperature of 22 °C. TGase activity from crayfish was compared with guinea pig liver TGase activity at 4 °C and the crayfish TGase displayed a higher activity while guinea pig liver TGase had a very low activity at this low temperature. By comparing kinetic parameters to guinea pig liver TGase, the results showed that a high activity of crayfish TGase was due to a decreasing Km value for pentylamine as a substrate, while it did not affect the kcat value (at 22 °C). The amino acid sequences of a krill and a crayfish TGase, which both are cold adapted, do not give any clue to why these two enzymes are cold-adapted. These results demonstrate that crayfish TGase is adapted to have significant activity at low temperatures and since crayfish are living in quite cold waters this is an interesting adaptation of this enzyme.

Crystal structure of 5-enolpyruvylshikimate-3-phosphate synthase from a psychrophilic bacterium, Colwellia psychrerythraea 34H.

To survive at low temperatures, psychrophiles seem to produce cold-adapted enzymes with a high flexibility around active sites for high catalytic efficiency. To gain insights into the cold-adaptation of psychrophilic enzymes in atomic detail, we determined the crystal structure of 5-enolpyruvylshikimate-3-phosphate synthase (CpsEPSPS) from Colwellia psychrerythraea, a psychrophilic bacterium. EPSPS is the primary target for the broad-spectrum herbicide, glyphosate, and a promising target for the development of antimicrobial and antiparasitic agents since it is absent in animals. The crystal structure of unliganded, open CpsEPSPS was determined at 2.2 Å resolution in space group P21 with two protomers per asymmetric unit. Superposition of separate domain I and II of CpsEPSPS structure with those of Escherichia coli EPSPS (EcoEPSPS) structure showed relatively small differences of RMSD values of 0.423 Å and 0.693 Å for domains I and II, respectively, implying the residues in ligand binding and catalysis of cold-adapted CpsEPSPS showed no significant flexibility. This result is conflicting to other cases of cold-adapted proteins. We also observed that hydrogen-bond forming residues in the surface of EcoEPSPS was mutated to non- or lesser hydrogen-bond forming one in CpsEPSPS, which makes the protein surface softer and eventually makes the protein more active at low temperature. In addition, domain rotation angle between open and closed states of CpsEPSPS was smaller than those of any EPSPSs whose structures are known. The restriction of the domain closure, which reduces the entropy cost of ligand binding and catalysis, may be a novel molecular adaptations of cold-adapted enzymes.

A new cold-adapted serine peptidase from Antarctic Lysobacter sp. A03: Insights about enzyme activity at low temperatures.

Currently, there is a great interest for customized biocatalysts that can supply the ongoing demand of industrial processes, but also deal with the growing concern about the environment. In this scenario, cold-adapted enzymes have features that make them very attractive for industrial and biotechnological purposes. Here, we describe A03Pep1, a new cold-adapted serine peptidase isolated from Lysobacter sp. A03 by screening a genomic library. The enzyme is synthesized as a large inactive prepropeptidase that, after intramolecular processing, gives rise to the active form, of 35kDa. The heterologous expression of A03Pep1 was carried out in E. coli cells harboring the vector pGEX-4T-2-a0301. Its activity was optimal at pH 9.0 and 40°C, in the presence of 25mM Ca2+, which may contribute to the thermal stability of the enzyme. The 3D structure modelling predicted a less deep and more open binding pocket in A03Pep1 than that observed in the crystal structure of its mesophilic homologous AprV2, presumably as a way to enhance the probability of substrate binding at low temperatures. These results provide possible approaches in developing new biotechnologically relevant peptidases active at low to moderate temperatures.

NADP+-dependent isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina.

The gene encoding NADP+-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity.

Biochemical characterization of a novel cold-adapted GH39 ß-agarase, AgaJ9, from an agar-degrading marine bacterium Gayadomonas joobiniege G7.

Gayadomonas joobiniege G7 is an agar-degrading marine bacterium belonging to a novel genus. Genomic sequencing of G. joobiniege revealed that AgaJ9 (formerly YjdB) belonging to the glycoside hydrolase (GH) 39 family. It showed the highest similarity (47% identity) to a putative ß-agarase from Catenovulum agarivorans DS-2, an agar-degrading marine bacterium sharing the highest similarity in the nucleotide sequence of 16s rRNA gene with G. joobiniege G7. The agaJ9 gene encodes a protein (134 kDa) of 1205 amino acids, including a 23-amino acid signal peptide. The agarase activity of purified AgaJ9 was confirmed by zymogram analysis. The optimum pH and temperature for AgaJ9 activity were determined as 5 and 25 °C, respectively. Notably, AgaJ9 is a cold-adapted ß-agarase retaining more than 80% of its activity even at a temperature of 5 °C. In addition, gel filtration chromatography revealed that AgaJ9 exists as two forms, dimer and monomer. Although the two forms had similar enzymatic properties, their kinetic parameters were different. The K m and V max of dimeric AgaJ9 for agarose was 0.68 mg/ml (5.7 × 10-6 M) and 17.2 U/mg, respectively, whereas the monomeric form had a K m of 1.43 mg/ml (1.2 × 10-5 M) and V max of 10.7 U/mg. Thin-layer chromatography and agarose-liquefying analyses revealed that AgaJ9 is an endo-type ß-agarase that hydrolyzes agarose into neoagarotetraose and neoagarobiose. This study is the first report of a GH39 ß-agarase with a cold-adapted enzymatic feature, a unique attribute, which may be useful for industrial applications.

Characterization of a New Cold-Adapted and Salt-Activated Polysaccharide Lyase Family 7 Alginate Lyase from Pseudoalteromonas sp. SM0524.

Marine bacterial alginate lyases play a role in marine alginate degradation and carbon cycling. Although a large number of alginate lyases have been characterized, reports on alginate lyases with special characteristics are still rather less. Here, a gene alyPM encoding an alginate lyase of polysaccharide lyase family 7 (PL7) was cloned from marine Pseudoalteromonas sp. SM0524 and expressed in Escherichia coli. AlyPM shows 41% sequence identity to characterized alginate lyases, indicating that AlyPM is a new PL7 enzyme. The optimal pH for AlyPM activity was 8.5. AlyPM showed the highest activity at 30°C and remained 19% of the highest activity at 5°C. AlyPM was unstable at temperatures above 30°C and had a low T m of 37°C. These data indicate that AlyPM is a cold-adapted enzyme. Moreover, AlyPM is a salt-activated enzyme. AlyPM activity in 0.5-1.2 M NaCl was sixfolds higher than that in 0 M NaCl, probably caused by a significant increase in substrate affinity, because the K m of AlyPM in 0.5 M NaCl decreased more than 20-folds than that in 0 M NaCl. AlyPM preferably degraded polymannuronate and mainly released dimers and trimers. These data indicate that AlyPM is a new PL7 endo-alginate lyase with special characteristics.

Characterization of NADP(+)-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium, Colwellia psychrerythraea strain 34H.

NADP(+)-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.

Isolation and Characterization of a Novel Cold-Adapted Esterase, MtEst45, from Microbulbifer thermotolerans DAU221.

A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23-24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2-C18) was p-nitrophenyl butyrate, and the K m and V max values were 0.0998 mM and 550 µmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg(2+), Zn(2+), and Cu(2+) ions; by phenylmethanesulfonyl fluoride; and by ß-mercaptoethanol. Ca(2+) did not affect the enzyme's activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications.