High Substrate Specificity Research Articles

Improvement in the thermal stability of Mucor prainii-derived FAD-dependent glucose dehydrogenase via protein chimerization

FAD-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is an enzyme utilized industrially in glucose sensors. Previously, FAD-GDH isolated from Mucor prainii (MpGDH) was demonstrated to have high substrate specificity for glucose. However, MpGDH displays poor thermostability and is inactivated after incubation at 45 °C for only 15 min, which prevents its use in industrial applications, especially in continuous glucose monitoring (CGM) systems. Therefore, in this study, a chimeric MpGDH (Mr144–297) was engineered from the glucose-specific MpGDH and the highly thermostable FAD-GDH obtained from Mucor sp. RD056860 (MrdGDH). Mr144–297 demonstrated significantly higher heat resistance, with stability at even 55 °C. In addition, Mr144–297 maintained both high affinity and accurate substrate specificity for D-glucose. Furthermore, eight mutation sites that contributed to improved thermal stability and increased productivity in Escherichia coli were identified. Collectively, chimerization of FAD-GDHs can be an effective method for the construction of an FAD-GDH with greater stability, and the chimeric FAD-GDH described herein could be adapted for use in continuous glucose monitoring sensors.

OPTIMIZATION OF TOTAL SOLUBLE PROTEIN PRODUCTION BY Trametes sp. isolate B7 AND ENZYMATIC DEGRADATION OF SYNTHETIC DYES

Filamentous fungi as a source of bioproteins for biotechnological applications are preferred over bacteria and yeasts due to their excellent yield and total volume of production. Fungal laccases are useful because of high redox potentials and low substrate specificity to xenobiotics. However, challenges for large-scale utilizations are low enzyme yield and high cost of production. Saw-dust of Terminalia superba abounds locally and was utilized as alternative substrate for production using various optimization processes. Trametes sp. isolate B7 was isolated and identified using molecular techniques. Optimum pH and temperature for total soluble protein (TSP) and laccase activity were pH 5.0 (3.6 mg/mL, 2356 U/mL) and 25oC (2.3 mg/mL, 2395 U/mL), respectively. Enhanced production of TSA occurred in Cu2+ (2.70 mg/L) and Ca2+ (2.50 mg/L) at 3 mM. Laccase activity was recorded at 1 - 2 mM Cu2+ (2379 U/mL) and 3 - 4 mM Ca2+ (2385 U/mL). Productions of TSP and laccase activity were higher using ammonium sulphate and ammonium chloride respectively. Glucose induced the best TSP and laccase activity of 3.6 mg/mL and 2395 U/mL respectively. TSP and laccase activity were best on day 14 (3.6 mg/mL) and day 18 (2395 U/mL) respectively. Percentage degradation of synthetic dyes using crude laccase 1000 U/mL (and 2000 U/mL) were: Remazol Brilliant Blue Royal 100% (77%), Phenol red 28% (36%), Congo red 75% (64%), and Malachite green 62% (28%) respectively. The ability of laccase to degrade Phenol red and other synthetic dyes without using enzyme mediators made it a valuable tool for biotechnological applications.

바이오센서 응용을 위한 페록시다아제 모방 나노 소재

Biocatalysts, referred as enzymes, have been widely used in life, industrial, medical, and biological fields due to their high catalytic activity and substrate specificity for specific reaction. However, it has disadvantages in industrial applications due to the drawbacks in its characteristics as a catalyst, such as the high cost, the narrow operation conditions in terms of pH and temperature, and the low durability. Recently, many studies on nanozymes for replacing enzymes have been widely conducted to overcome these drawbacks. The nanozyme has a relatively wide pH and temperature operation range as compared with the enzyme, which is a kind of protein, and has a high durability over a long period of time. It is also expected to be able to be mass-produced at relatively low cost. However, the nanozymes still lacks of the substrate specificity, which is one of the most important features of enzymes. Furthermore, only few nanozymes have been reported which exhibits higher or comparable catalytic activity compared with enzymes. This review first discusses the classification of the nanozymes and the corresponding catalytic reactions, focusing on the most developed peroxidase -mimicking nanozymes. Then, it summarizes the most effective peroxidase nanozymes including iron oxides, nanocarbon, and iron- and carbon composite materials and their corresponding reaction mechanisms. After that, the nanotechnology- based approaches to improve the catalytic activity of nanozymes as well as the substrate specificity are covered. Furthermore, the current major application of nanozyme to the biosensor are introduced. Finally, the future directions for nanozyme researches and the applications are suggested.

A comparative analysis of catalytic activity and stability of microbial transglutaminase in controlled denaturing conditions

Microbial transglutaminases (MTGs) catalyzes the formation of Gln-Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and in biotechnological applications for bioconjugation reactions. In view of its practical utility, a comparative study of the catalytic activity and stability of the enzyme in a wide range of denaturing conditions has been performed through Circular Dichroism (CD), fluorescence and activity assays performed with model substrates. In agreement with previous results, we show that MTG has a significant structural and functional tolerance to pH changes, whereas the enzyme stability and activity decrease in presence of increasing amounts of denaturing agents, such as urea and guanidinium chloride (GdnHCl). Noteworthy, the activity of MTG in denaturing conditions differs markedly from that in pseudo-physiological settings, shifting unexpectedly toward higher substrate specificity. Also, the use of controlled amounts of denaturing agents (1.0–1.5 M urea) largely improves yields and purity of the final products of 10–15% and 25–30%, respectively. These findings widen the range of applicability of the MTG-mediated biocatalysis for industrial and biotechnological purposes.

Cloning and characterization of a new endo-type polyG-specific alginate lyase from bacteria Vibrio sp. QD-5

Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV, was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about 62 kDa and a theoretical isoelectric point (pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na+, K+ and Mg2+ under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization (DP) and for determining the fine structure of alginate.

Production of Fungal Laccase under Solid State Bioprocessing of Agroindustrial Waste and Its Application in Decolourization of Synthetic Dyes

Fungal laccases are preferred due to high redox potentials and low substrate specificity to xenobiotics including synthetic dyes. For large-scale applications, low enzyme yield and high cost of production has remained the challenge. Agroindustrial waste such as saw-dust of Terminalia superba abounds locally and was utilized as low-cost alternative substrate for laccase production in Solid State Bioprocessing (SSB) using Trametes sp. isolate G31. The study optimized laccase production using various parameters. Optimal production of laccase was at pH 5.0 - 7.0 with 2356 U/mL - 2369 U/mL and 25°C (2336 U/mL). Among the sources of nitrogen and carbon tested, laccase production in ammonium sulphate and sucrose supplemented media were higher. The effect of activators on laccases production showed that Cu2+ and Ca2+induced high titres of laccase at 4 -5 mM and 2 - 4 mM respectively, while production of laccase by Mn2+ was significantly high at 40 mM. The effect of 2, 2-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), guaiacol and varatryl alcohol on laccase production was significantly different especially in glycerol. Optimum production for laccase was on day 14 with 2356 U/mL followed by steady declined up to day 34. The purified laccase had a specific activity of 5008 µmol/min/mg, purification factor of 3.85, and a molecular mass of ~40 kDa using N-PAGE. The potential of crude laccase to decolourize diverse dyes was tested. Phenol red achieved 40% decolourization for 1hr, while RBBR (93%), Crystal violet (60%), Methylene blue (53%) and Congo red (51%) for 24 hr, 72 hr, 48 hr and 120 hr respectively. Methyl red and Malachite green attained 42% (72 hr) and 32% (48 hr) decolourization. The enzyme ability to oxidize Phenol red and other synthetic dyes without mediators made it eco-friendly in treating dye wastewaters.

Characterization of a novel thermostable carboxylesterase from thermoalkaliphilic bacterium Bacillus thermocloaceae.

A novel thermostable carboxylesterase (Est5250) of thermoalkaliphilic bacterium Bacillus thermocloaceae was heterologously expressed in Escherichia coli and its biochemical properties were investigated. Est5250 showed optimum esterase activity at 60 °C and pH 8.0. The enzyme was highly thermostable at 60 °C, interestingly, the thermostability was enhanced in the presence of Ca2+, retaining more than 60% of its original activity after 12h of pre-incubation. Est5250 was active in the presence of 1% (v/v) of organic solvents and 0.1% (v/v) of non-ionic detergents. The enzyme activity was significantly enhanced up to 167% and 159% in the presence of 2-mercaptoethanol and dithiothreitol, respectively. Est5250 showed high substrate specificity for short-chain p-nitrophenyl-esters. Kinetic constants, Km and kcat, for p-nitrophenyl-acetate were 185.8μM and 186.6s-1, respectively. Est5250 showed outstanding thermostability and tolerance to various organic solvents under thermoalkaliphilic conditions, suggesting that it would be a highly suitable biocatalyst for various biotechnological applications. Abbreviations: B. thermocloaceae sp.: Bacillus thermocloaceae; E. coli: Escherichia coli; NP: nitrophenyl; DMSO: dimethyl sulfoxide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMF: dimethyl formamide; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; CTAB: cetrimonium bromide; PMSF: phenylmethylsulfonyl fluoride; DEPC: diethyl pyrocarbonate; 2-ME: 2-mercaptoethanol; DTT: dithiothreitol.

P38γ is essential for cell cycle progression and liver tumorigenesis.

The cell cycle is a tightly regulated process that is controlled by the conserved cyclin-dependent kinase (CDK)-cyclin protein complex1. However, control of the G0-to-G1 transition is not completely understood. Here we demonstrate that p38 MAPK gamma (p38γ) acts as a CDK-like kinase and thus cooperates with CDKs, regulating entry into the cell cycle. p38γ shares high sequence homology, inhibition sensitivity and substrate specificity with CDK family members. In mouse hepatocytes, p38γ induces proliferation after partial hepatectomy by promoting the phosphorylation of retinoblastoma tumour suppressor protein at known CDK target residues. Lack of p38γ or treatment with the p38γ inhibitor pirfenidone protects against the chemically induced formation of liver tumours. Furthermore, biopsies of human hepatocellular carcinoma show highexpression of p38γ, suggesting that p38γ could be a therapeutic target in the treatment of this disease.

Hoodwinking Cytochrome P450BM3 into Hydroxylating Non-Native Substrates by Exploiting Its Substrate Misrecognition.

Bacterial cytochrome P450s (P450s) are at the focus of attention as potential biocatalysts for applications in green synthetic chemistry, as they possess high activity for the hydroxylation of inert substrate C-H bonds. The high activity of bacterial P450s, such as P450BM3, is chiefly due to their high substrate specificity, and consequently, the catalytic activity of P450BM3 toward non-native substrates is very low, limiting the utility of bacterial P450s as biocatalysts. To enable oxidation of non-native substrates by P450BM3 without any mutagenesis, we have developed a series of "decoy molecules", inert dummy substrates, with structures that resemble those of the native substrates. Decoy molecules fool P450BM3 into generating the active species, so-called Compound I, enabling the catalytic oxidation of non-native substrates other than fatty acids. Perfluorinated carboxylic acids (PFCs) serve as decoy molecules to initiate the activation of molecular oxygen in the same manner as long-alkyl-chain fatty acids, due to their structural similarity, and induce the generation of Compound I, but, unlike the native substrates, PFCs are not oxidizable by Compound I, allowing the hydroxylation of non-native substrates, such as gaseous alkanes and benzene. The catalytic activity for non-native substrate hydroxylation was significantly enhanced by employing second generation decoy molecules, PFCs modified with amino acids (PFC-amino acids). Cocrystals of P450BM3 with PFC9-Trp revealed clear electron density in the fatty-acid-binding channel that was readily assigned to PFC9-Trp. The alkyl chain terminus of PFC9-Trp does not reach the active site owing to multiple hydrogen bonding interactions between the carboxyl and carbonyl groups of PFC9-Trp and amino acids located at the entrance of the substrate binding channel of P450BM3 that fix it in place. The remaining space above the heme after binding of PFC9-Trp can be utilized to accommodate non-native substrates. Further developments revealed that third generation decoy molecules, N-acyl amino acids, such as pelargonoyl-l-phenylalanine (C9-Phe), can serve as decoy molecules, indicating that the rationale "fluorination is required for decoy molecule function" can be safely discarded. Diverse carboxylic acids including dipeptides could now be exploited as building blocks, and a library of decoy molecules possessing diverse structures was prepared. Among the third-generation decoy molecules examined N-enanthyl-l-proline modified with l-phenylalanine (C7-Pro-Phe) afforded the maximum turnover rate for benzene hydroxylation. The structural diversity of third-generation decoy molecules was also utilized to control the stereoselectivity of hydroxylation for the benzylic hydroxylation of Indane, showing that decoy molecules can alter stereoselectivity. As both the catalytic activity and enantioselectivity are dependent upon the structure of the decoy molecules, their design allows us to regulate reactions catalyzed by wild-type enzymes. Furthermore, decoy molecules can also activate intracellular P450BM3, allowing the use of E. coli expressing wild-type P450BM3 as an efficient whole-cell bioreactor. It should be noted that Mn-substituted full-length P450BM3 (Mn-P450BM3) is also active for the hydroxylation of propane in which the regioselectivity diverged from that of Fe-P450BM3. The results summarized in this Account represent good examples of how the reactive properties of P450BM3 can be controlled for the monooxygenation of non-native substrates in vitro as well as in vivo to expand the potential of P450BM3.

Polyfunctional Bis-Lewis-Acid-/Bis-Triazolium Catalysts for Stereoselective 1,4-Additions of 2-Oxindoles to Maleimides.

Achieving enzyme-like catalytic activity and stereoselectivity without the typically high substrate specificity of enzymes is a challenge in the development of artificial catalysts for asymmetric synthesis. Polyfunctional catalysts are considered to be a promising tool for achieving excellent catalytic efficiency. A polyfunctional catalyst system was developed, which incorporates two Lewis acidic/Brønsted basic cobalt centers in combination with triazolium moieties that are crucial for high reactivity and excellent stereoselectivity in the direct 1,4-addition of oxindoles to maleimides. The catalyst is assembled through click chemistry and is readily recyclable through precipitation by making use of its charges. Kinetic studies support a cooperative mode of action. Diastereodivergency is achievable with either Boc-protected or unprotected maleimide.

Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens

Clostridium perfringens is a gram-positive, anaerobic, pathogenic bacterium that can cause a wide range of diseases in humans, poultry and agriculturally important livestock. A pyridoxal-5-phosphate-dependent alanine racemase with a function in the racemization of d- and l-alanine is an attractive drug target for C.perfringens and other pathogens due to its absence in animals and humans. In this study alanine racemase from C.perfringens (CPAlr) was successfully expressed and purified in Escherichia coli and biochemically characterized. The purified CPAlr protein was a dimeric PLP-dependent enzyme with high substrate specificity. The optimal racemization temperature and pH were 40°C and 8.0, respectively. The kinetic parameters Km and kcat of CPAlr, determined by HPLC at 40°C were 19.1mM and 17.2s-1 for l-alanine, and 10.5mM and 8.7s-1 for d-alanine, respectively. Gel filtration chromatographic analysis showed that the molecular weight of mutant Y359A was close to monomeric form, suggesting that the inner layer residue Tyr359 might play an essential role in dimer-formation. Furthermore, the mutation at residues Asp171 and Tyr359 resulted in a dramatic increase in Km value and/or decreased in kcat value, indicating that the middle and inner layer residues Asp171 and Tyr359 of CPAlr might have the key role in substrate binding, catalytic activity or oligomerization state through the hydrogen-bonding interaction with the pentagonal ring waters and/or PLP cofactor.

Nanozymes: Classification, Catalytic Mechanisms, Activity Regulation, and Applications.

Because of the high catalytic activities and substrate specificity, natural enzymes have been widely used in industrial, medical, and biological fields, etc. Although promising, they often suffer from intrinsic shortcomings such as high cost, low operational stability, and difficulties of recycling. To overcome these shortcomings, researchers have been devoted to the exploration of artificial enzyme mimics for a long time. Since the discovery of ferromagnetic nanoparticles with intrinsic horseradish peroxidase-like activity in 2007, a large amount of studies on nanozymes have been constantly emerging in the next decade. Nanozymes are one kind of nanomaterials with enzymatic catalytic properties. Compared with natural enzymes, nanozymes have the advantages such as low cost, high stability and durability, which have been widely used in industrial, medical, and biological fields. A thorough understanding of the possible catalytic mechanisms will contribute to the development of novel and high-efficient nanozymes, and the rational regulations of the activities of nanozymes are of great significance. In this review, we systematically introduce the classification, catalytic mechanism, activity regulation as well as recent research progress of nanozymes in the field of biosensing, environmental protection, and disease treatments, etc. in the past years. We also propose the current challenges of nanozymes as well as their future research focus. We anticipate this review may be of significance for the field to understand the properties of nanozymes and the development of novel nanomaterials with enzyme mimicking activities.

A thermostable laccase from Thermus sp. 2.9 and its potential for delignification of Eucalyptus biomass

Laccases are multicopper oxidases that are being studied for their potential application in pretreatment strategies of lignocellulosic feedstocks for bioethanol production. Here, we report the expression and characterization of a predicted laccase (LAC_2.9) from the thermophilic bacterial strain Thermus sp. 2.9 and investigate its capacity to delignify lignocellulosic biomass. The purified enzyme displayed a blue color typical of laccases, showed strict copper dependence and retained 80% of its activity after 16 h at 70 °C. At 60 °C, the enzyme oxidized 2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,6-dimethoxyphenol (DMP) at optimal pH of 5 and 6, respectively. LAC_2.9 had higher substrate specificity (kcat/KM) for DMP with a calculated value that accounts for one of the highest reported for laccases. Further, the enzyme oxidized a phenolic lignin model dimer. The incubation of steam-exploded eucalyptus biomass with LAC_2.9 and 1-hydroxybenzotriazole (HBT) as mediator changed the structural properties of the lignocellulose as evidenced by Fourier transform infrared (FTIR) spectroscopy and thermo-gravimetric analysis (TGA). However, this did not increase the yield of sugars released by enzymatic saccharification. In conclusion, LAC_2.9 is a thermostable laccase with potential application in the delignification of lignocellulosic biomass.

Purification and biochemical characterization of extracellular glucoamylase from Paenibacillus amylolyticus strain.

In the present study, glucoamylase produced from a soil bacterium Paenibacillus amylolyticus NEO03 was cultured under submerged fermentation conditions. The extracellular enzyme was purified by starch adsorption chromatography and further by gel filtration, with 2.73-fold and recovery of 40.02%. The protein exhibited molecular mass of ∼66,000 Da as estimated by SDS-PAGE and depicted to be a monomer. The enzyme demonstrated optimum activity at pH range 6.0-7.0 and temperature range 30-40 °C. Glucoamylase was mostly activated by Mn2+ metal ions and depicted no dependency on Ca2+ ions. The enzyme preferentially hydrolyzed all the starch substrates. High substrate specificity was demonstrated towards soluble starch and kinetic values Km and Vmax were 2.84 mg/ml and 239.2 U/ml, respectively. The products of hydrolysis of soluble starch were detected by thin layer chromatography which showed only D -glucose, indicating a true glucoamylase. The secreted glucoamylase from P. amylolyticus strain possesses properties suitable for saccharification processes such as biofuel production.

Preparation of malto-oligosaccharides with specific degree of polymerization by a novel cyclodextrinase from Palaeococcus pacificus

An open reading frame from Palaeococcus pacificus was discovered by genomic analysis. The recombinant PpCD was proved as novel extremely thermal-stable cyclodextrinase with potential on production of malto-oligosaccharides with specific degree of polymerization. This enzyme has extremely high substrate specificity toward cyclodextrins, while the rates of hydrolysis toward macromolecular or linear substrates such as starch, pullulan and amylose were pretty slow. The optimal pH and temperature of PpCD were determined as 6.0 and 95 °C respectively. It was confirmed that PpCD could produce malto-oligosaccharides with a certain degree of polymerization in consistent with that of cyclodextrin substrates. In this study, the preparation of high purity maltoheptaose was achieved and the suitable β-cyclodextrin concentration was about 8–10%. Therefore, PpCD may have a potential application in the production of specific degree of polymerization malto-oligosaccharides.

Cloning and characterization of a Flavin-free oxygen-insensitive azoreductase from Klebsiella oxytoca GS-4-08.

This study reports the identification of a new bacterial azoreductase (AzoR) from Klebsiella oxytoca GS-4-08, its heterologous production in Escherichia coli and the decolorization of azo dyes. The AzoR, as a flavin-free, oxygen-insensitive enzyme, has a molecular mass of 22kDa and a high substrate specificity for methyl red (MR). In the presence of 5mM of NADH, the enzyme activity for decolorization of 50mgl-1 of MR reached 5.61μmolmin-1 mg protein-1. Double-reciprocal plots indicated that the NADH and MR reductions proceed by ping-pong mechanism. The calculated Vm was 8.17μMmin-1 mg protein-1, and the AzoR retained over 75% of relative activity under temperature range from 20 to 50°C. This study for the first time identified an AzoR from Klebsiella oxytoca strain, which enabled efficient degradation of azo dyes by strain GS-4-08.

An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking, binding free energy calculation, and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔGvdW, −57.30 kcal/mol) and electrostatic solvation (ΔGsolv, −36.86 kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.

Biochemical and structural insights into PLP fold type IV transaminase from Thermobaculum terrenum

The high catalytic efficiency of enzymes under reaction conditions is one of the main goals in biocatalysis. Despite the dramatic progress in the development of more efficient biocatalysts by protein design, the search for natural enzymes with useful properties remains a promising strategy. The pyridoxal 5'-phosphate (PLP)-dependent transaminases represent a group of industrially important enzymes due to their ability to stereoselectively transfer amino groups between diverse substrates; however, the complex mechanism of substrate recognition and conversion makes the design of transaminases a challenging task. Here we report a detailed structural and kinetic study of thermostable transaminase from the bacterium Thermobaculum terrenum (TaTT) using the methods of enzyme kinetics, X-ray crystallography and molecular modeling. TaTT can convert L-branched-chain and L-aromatic amino acids as well as (R)-(+)-1-phenylethylamine at a high rate and with high enantioselectivity. The structures of TaTT in complex with the cofactor pyridoxal 5′-phosphate covalently bound to enzyme and in complex with its reduced form, pyridoxamine 5′-phosphate, were determined at resolutions of 2.19 Å and 1.5 Å, and deposited in the Protein Data Bank as entries 6GKR and 6Q8E, respectively. TaTT is a fold type IV PLP-dependent enzyme. In terms of structural similarity, the enzyme is close to known branched-chain amino acid aminotransferases, but differences in characteristic sequence motifs in the active site were observed in TaTT compared to canonical branched-chain amino acid aminotransferases, which can explain the improved binding of aromatic amino acids and (R)-(+)-1-phenylethylamine. This study has shown for the first time that high substrate specificity towards both various l-amino acids and (R)-primary amines can be implemented within one pyridoxal 5′-phosphate-dependent active site of fold type IV. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further biochemical and bioinformatic studies to understand the sequence-structure-function relationship in these enzymes.

Production and characterization of β-cyclodextrin glucanotransferase from Bacillus sp. ND1.

A potent β-CGTase producing bacterium ND1 has been isolated from sugarcane field soil in India. The biochemical, physiologicaland phylogenetic analyses based on 16S rRNA gene suggest that the isolate belongs to Bacillus cereus group. The enzyme β-CGTase produced from isolate ND1 catalyzes production of β-cyclodextrin utilizing starch as a substrate which has diverse applications in various fields. The enzyme production parameters pH, temperature, and substrate concentration were optimized using Central Composite Design (CCD) of Response Surface Methodology (RSM) and were found to be 8.9, 30.55 °C, and 1.88%, respectively for optimal enzyme activity. The crude enzyme was partially purified (29-fold) using ammonium sulphate precipitation followed by ion exchange chromatography. The specific activity of the purified enzyme was found to be 63.53 U mg-1 . The enzyme is monomeric in nature with a molecular weight of 97.4 kD as determined by SDS-PAGE. It is stable in a wide range of pH (6-10) and temperature (40-60 °C) values. The maximum CGTase activity was observed at pH 9 and temperature 50 °C. The Km value was found to be 2.613 ± 0.5 and Vmax was 0.309 ± 0.05 µg min-1 indicating high substrate specificity. Together; these results suggest that the enzyme may be of wide commercial value in various industrial processes.

NEW DATA ON APHYLLO-PHOROID FUNGI AND SOME OTHER GROUPS OF MACROMYCETES OF THE SOLOVETSKY ARCHIPALAGO

This paper provides additions to the biota of aphyllophoroid fungi of the Solovetsky Archipelago. To date, the list includes 307 species of aphyllophoroid fungi, 22 of them are found only in the territory in question. A previously published list was supplemented with 31 species of aphyllophoroid fungi, including 9 species new for the Arkhangelsk Region (Craterellus tubaeformis, Hypochnicium erikssonii, Phellodon melaleucus, Pterula gracilis, Sistotrema alboluteum, Trechispora invisitata, T. kavinioides, T. stevensonii, Tubulicrinis accedens). In addition, new data on 13 species of ascomycetes, including 6 new for the Arkhangelsk Region (Gyromitra infula, Humaria hemisphaerica, Hypoxylon fragiforme, Peziza badia, Trichoderma pulvinatum, T. viride), 3 species of dacrymyce-tes and 39 species of agaricomycetes, including 19 new for the region (Amanita fulva, A. muscaria, Boletus edulis, B. paluster, Clitocybe gibba, Coprinus comatus, Cortinarius armillatus, C. caperatus, C. sanguineus, C. traganus, Laccaria laccata, Lachnella albo-violascens, Lycoperdon perlatum, L. utriforme, Mycena haematopus, Pholiota squar-rosa, Pleurotus ostreatus, Russula aeruginea, Sebacina grisea) are provided. The state of knowledge about aphyllophoroid fungi on different substrates was analyzed. The correlation between the number of species and the habitat was discussed. The number of species increased especially notably along with stand density. Quite high substrate specificity was demonstrated. Almost every fifth of the species that we found only on the main forest-forming tree species was encountered sporadically. The Turing coefficient for the Solovetsky Archipelago is 67.3 %. The territory has been found to harbor 17 species listed in Red Data Books of adjacent areas and 33 indicator species, including 24 indicators of old-growth and 9 of very old spruce and pine forests. The forests of the archipelago can therefore be considered particularly valuable. The group of aphyllophoroid fungi has been studied quite thoroughly, while data on other groups of macromycetes are still scarce.