When Synthesis Gets It Wrong: Unexpected Epimerization Using PyBOP in the Synthesis of the Cyclic Peptide Thermoactinoamide A.

Chemical synthesis is commonly seen as the final proof of the structure of complex natural products, but even a seemingly easy and well-established synthetic procedure may lead to an unexpected result. This is what happened with the synthesis of thermoactinoamide A (1a), an antimicrobial and antitumor nonribosomal cyclic hexapeptide produced by the thermophilic bacterium Thermoactinomyces vulgaris. The synthetic thermoactinoamide A outsourced to a company and the one described in a synthetic paper showed spectroscopic data identical to each other but different from those of the natural product. After a detailed spectroscopic, degradative, and synthetic study, the synthetic compound was shown to be an epimer (1b) of the intended target compound, originating during the cyclization reaction by extensive epimerization at the activated C-terminal amino acid. This allowed confirmation of the structure of the natural product.

Proposal of Thermoactinomyces mirandus sp. nov., a filamentous, anaerobic bacterium isolated from a biogas plant.

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1T, from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0-2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1T (= DSM 110094T = LMG 31503T). The description of the genus Thermoactinomyces is emended accordingly.

Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis.

Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.

Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes.

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.

Mobile Loop in the Active Site of Metallocarboxypeptidases as an Underestimated Determinant of Substrate Specificity.

It is generally accepted that the primary specificity of metallocarboxypeptidases is mainly determined by the structure of the so-called primary specificity pocket. However, the G215S/A251G/T257A/D260G/T262D mutant of carboxypeptidase T from Thermoactinomyces vulgaris (CPT) with the primary specificity pocket fully reproducing the one in pancreatic carboxypeptidase B (CPB) retained the broad, mainly hydrophobic substrate specificity of the wild-type enzyme. In order to elucidate factors affecting substrate specificity of metallocarboxypeptidases and the reasons for the discrepancy with the established views, we have solved the structure of the complex of the CPT G215S/A251G/T257A/D260G/T262D mutant with the transition state analogue N-sulfamoyl-L-phenylalanine at a resolution of 1.35 Å and compared it with the structure of similar complex formed by CPB. The comparative study revealed a previously underestimated structural determinant of the substrate specificity of metallocarboxypeptidases and showed that even if substitution of five amino acid residues in the primary specificity pocket results in its almost complete structural correspondence to the analogous pocket in CPB, this does not lead to fundamental changes in the substrate specificity of the mutant enzyme due to the differences in the structure of the mobile loop located at the active site entrance that affects the substrate-induced conformational rearrangements of the active site.

Thermoactinoamide A, an Antibiotic Lipophilic Cyclopeptide from the Icelandic Thermophilic Bacterium Thermoactinomyces vulgaris.

The thermophilic bacterium Thermoactinomyces vulgaris strain ISCAR 2354, isolated from a coastal hydrothermal vent in Iceland, was shown to contain thermoactinoamide A (1), a new cyclic hexapeptide composed of mixed d and l amino acids, along with five minor analogues (2-6). The structure of 1 was determined by one- and two-dimensional NMR spectroscopy, high-resolution tandem mass spectrometry, and advanced Marfey's analysis of 1 and of the products of its partial hydrolysis. Thermoactinoamide A inhibited the growth of Staphylococcus aureus ATCC 6538 with an MIC value of 35 µM. On the basis of literature data and this work, cyclic hexapeptides with mixed d/l configurations, one aromatic amino acid residue, and a prevalence of lipophilic residues can be seen as a starting point to define a new, easily accessible scaffold in the search for new antibiotic agents.

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures.

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu-1-Ser1 bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu-12-Lys-11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys32-Cys48 and Cys180-Cys183 cumulatively contributed to the thermostability of TS-GSE.

Kroppenstedtia pulmonis sp. nov. and Kroppenstedtia sanguinis sp. nov., isolated from human patients.

Three human clinical strains (W9323(T), X0209(T) and X0394) isolated from a lung biopsy, blood and cerebral spinal fluid, respectively, were characterised using a polyphasic taxonomic approach. Comparative analysis of the 16S rRNA gene sequences showed the three strains belong to two novel branches within the genus Kroppenstedtia: 16S rRNA gene sequence analysis of W9323(T) showed close sequence similarity to Kroppenstedtia eburnea JFMB-ATE(T) (95.3 %), Kroppenstedtia guangzhouensis GD02(T) (94.7 %) and strain X0209(T) (94.6 %); sequence analysis of strain X0209(T) showed close sequence similarity to K. eburnea JFMB-ATE(T) (96.4 %) and K. guangzhouensis GD02(T) (96.0 %). Strains X0209(T) and X0394 were 99.9 % similar to each other by 16S rRNA gene sequence analysis. The DNA-DNA relatedness was 94.6 %, confirming that X0209(T) and X0394 belong to the same species. Chemotaxonomic data for strains W9323(T) and X0209(T) were consistent with those described for the members of the genus Kroppenstedtia: the peptidoglycan was found to contain LL-diaminopimelic acid; the major cellular fatty acids were identified as iso-C15 and anteiso-C15; and the major menaquinone was identified as MK-7. Differences in endospore morphology, carbon source utilisation profiles, and cell wall sugar patterns of strains W9323(T) and X0209(T), supported by phylogenetic analysis, enabled us to conclude that the strains each represent a new species within the genus Kroppenstedtia, for which the names Kroppenstedtia pulmonis sp. nov. (type strain W9323(T) = DSM 45752(T) = CCUG 68107(T)) and Kroppenstedtia sanguinis sp. nov. (type strain X0209(T) = DSM 45749(T) = CCUG 38657(T)) are proposed.

Thermoactinomyces khenchelensis sp. nov., a filamentous bacterium isolated from soil sediment of a terrestrial hot spring.

A novel thermophilic filamentous bacterium, designated strain T36(T), was isolated from soil sediment sample from a hot spring source collected in Khenchela province, Algeria. Strain T36(T) was identified as a member of the genus Thermoactinomyces by a polyphasic approach. Strain T36(T) was observed to form white aerial mycelium and non-coloured to pale yellow substrate mycelium, both producing endospores, sessile or borne by short sporophores. The optimum growth temperature and pH were found to be 37-55 °C and 7.0-9.0, respectively and the optimum NaCl concentration for growth was found to be 0-7 % (w/v). The diagnostic diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid. The predominant menaquinone of strain T36(T) was identified as MK-7 (H0). The major fatty acids were found to be iso-C15:0 and iso-C17:0. The phospholipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphoglycolipid. The chemotaxonomic properties of strain T36(T) are consistent with those shared by members of the genus Thermoactinomyces. 16S rRNA gene sequence analysis indicated that the sequence similarities between strain T36(T) and Thermoactinomyces species with validly published names were less than 98 %. Based on the combined genotypic and phenotypic evidence, it is proposed that strain T36(T) should be classified as representative of a novel species, for which the name Thermoactinomyces khenchelensis sp. nov. is proposed. The type strain is T36(T) (=DSM 45951(T) = CECT 8579(T)).

Thermoactinomyces guangxiensis sp. nov., a thermophilic actinomycete isolated from mushroom compost.

A novel thermophilic actinomycete, designated strain CD-1(T), was isolated from mushroom compost in Nanning, Guangxi province, China. The strain grew at 37-55 °C (optimum 45-50 °C), pH 6.0-11.0 (optimum pH 7.0-9.0) and with 0-2.0% NaCl (optimum 0-1.0%), formed well-developed white aerial mycelium and pale-yellow vegetative mycelium, and single endospores (0.8-1.0 µm diameter) were borne on long sporophores (2-3 µm length). The endospores were spherical-polyhedron in shape with smooth surface. Based on its phenotypic and phylogenetic characteristics, strain CD-1(T) is affiliated to the genus Thermoactinomyces. It contained meso-diaminopimelic acid as the diagnostic diamino acid; the whole-cell sugars were ribose and glucose. Major fatty acids were iso-C15 :  0, C16 : 0, anteiso-C15  : 0 and iso-C17  : 0. MK-7 was the predominant menaquinone. The polar phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylethanolamine containing hydroxylated fatty acids, ninhydrin-positive glycophospholipid, an unknown phospholipid and glycolipids. The G+C content of the genomic DNA was 48.8%. 16S rRNA gene sequence analysis showed that the organism was closely related to Lihuaxuella thermophila YIM 77831(T) (95.69% sequence similarity), Thermoactinomyces daqus H-18(T) (95.49%), Laceyella putida KCTC 3666(T) (95.05%), Thermoactinomyces vulgaris KCTC 9076(T) (95.01%) and Thermoactinomyces intermedius JCM 3312(T) (94.55%). Levels of DNA-DNA relatedness between strain CD-1T and Lihuaxuella thermophila JCM 18059(T), Thermoactinomyces daqus DSM 45914(T), Laceyella putida JCM 8091(T), Thermoactinomyces vulgaris JCM 3162(T) and Thermoactinomyces intermedius JCM 3312(T) were low (22.8, 33.3, 24.7, 29.4 and 30.0%, respectively). A battery of phenotypic, genotypic and DNA-DNA relatedness data indicated that strain CD-1T represented a novel species of the genus Thermoactinomyces, for which the name Thermoactinomyces guangxiensis sp. nov. is proposed. The type strain is CD-1(T) ( = ATCC BAA-2630(T) = CGMCC 4.7156(T)).

Degradation of intact chicken feathers by Thermoactinomyces sp. CDF and characterization of its keratinolytic protease.

Thermoactinomyces is known for its resistance to extreme environmental conditions and its ability to digest a wide range of hard-to-degrade compounds. Here, Thermoactinomyces sp. strain CDF isolated from soil was found to completely degrade intact chicken feathers at 55 °C, with the resulting degradation products sufficient to support growth as the primary source of both carbon and nitrogen. Although feathers were not essential for the expression of keratinase, the use of this substrate led to a further 50-300 % increase in enzyme production level under different nutrition conditions, with extracellular keratinolytic activity reaching its highest level (∼400 U/mL) during the late-log phase. Full degradation of feathers required the presence of living cells, which are thought to supply reducing agents necessary for the cleavage of keratin disulfide bonds. Direct contact between the hyphae and substrate may enhance the reducing power and protease concentrations present in the local microenvironment, thereby facilitating keratin degradation. The gene encoding the major keratinolytic protease (protease C2) of strain CDF was cloned, revealing an amino acid sequence identical to that of subtilisin-like E79 protease from Thermoactinomyces sp. E79, albeit with significant differences in the upstream flanking region. Exogenous expression of protease C2 in Escherichia coli resulted in the production of inclusion bodies with proteolytic activity, which could be solubilized to an alkaline solution to produce mature protease C2. Purified protease C2 was able to efficiently hydrolyze α- and ß-keratins at 60-80 °C and pH 11.0, representing a promising candidate for enzymatic processing of hard-to-degrade proteins such as keratinous wastes.

Thermoactinomyces daqus sp. nov., a thermophilic bacterium isolated from high-temperature Daqu.

Daqu is a fermentation starter used in the production of Chinese liquors. A thermophilic bacterium, designated strain H-18(T), was isolated from a high-temperature Daqu sample collected from the manufacturing process of a sesame-flavoured liquor in Shandong province, China. It was investigated in a taxonomic study using a polyphasic approach. Strain H-18(T) formed white aerial mycelium and greyish-yellow substrate mycelium, bearing single endospores on aerial and substrate hyphae or on unbranched short sporophores. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids were iso-C15 : 0 and iso-C17 : 0. The predominant menaquinone was MK-7. These chemotaxonomic properties are similar to those of members of the genus Thermoactinomyces. The G+C content of the genomic DNA was 49.1 mol%. 16S rRNA gene sequence comparisons indicated that strain H-18(T) was most closely related to Thermoactinomyces vulgaris KCTC 9076(T) (96.42 % similarity), Thermoactinomyces intermedius KCTC 9646(T) (96.06 %), Laceyella putida KCTC 3666(T) (96.32 %) and Laceyella sacchari KCTC 9790(T) (95.55 %). Strain H-18(T) showed low DNA-DNA relatedness (40.8, 33.4, 20.0 and 14.4 %) with the above strains. Based on morphological and chemotaxonomic characteristics, DNA-DNA hybridization data and physiological properties, strain H-18(T) represents a novel species of the genus Thermoactinomyces, for which the name Thermoactinomyces daqus sp. nov. is proposed. The type strain is H-18(T) ( = DSM 45914(T) = CICC 10681(T)).

Detection of thermoactinomyces species in selected agricultural substrates from Queensland.

Selected overheated substrates commercially available for public use in sub-tropical Queensland, Australia were screened for the presence of Thermoactinomyces species using an air sampler. All substrates with the exception of tea tree mulch were found to contain Thermoactinomyces species. Subsequent 16S rDNA oligonucleotide sequencing of the selected eight isolates indicated that some of these species were closely related to previously reported allergenic Thermoactinomyces vulgaris and Laceyella sacchari. In view of this, the isolates were tested to determine their adhesion ability and cytotoxicity to human lung cells (calu-3 cells). The results indicated that all eight isolates were highly adherent and showed cytotoxicity to this cell line. These findings might indicate that the presence of such species in overheated agricultural materials may constitute a public health risk if storage and handling conditions are not optimal and do not meet criteria defined for sub-tropical climates.

Prevalence of thermoduric bacteria and spores on 10 Midwest dairy farms.

Thermoduric bacteria (TDB), including sporeformers and their spores, can be present in milk and dairy products even after pasteurization. They have the potential to adversely affect the quality and shelf life of products. The objectives of this study were to identify the origin and common species of heat-resistant bacteria occurring during summer and winter on Midwest dairy farms. Bulk tank milk samples were taken from 10 dairy farms located along the South Dakota section of Interstate 29, with herd sizes ranging from 650 to 3,500 lactating dairy cows. Milk samples were profiled for the prevalence of TDB and spore counts (SC). Corn silage samples and swabs of the milking clusters were also taken at the dairies to further profile the potential sources of TDB and SC. The samples were taken 3 times during 2 seasons [winter (January-March) and summer (June-August)] to track seasonal changes in the farm bacterial flora. During winter, the average TDB counts in bulk tank milk were 2.61 log compared with 2.76 log TDB counts in the summer. The SC was 1.08 log in the winter, which was half the 2.06 log SC present in the summer season. Corn silage sampled in winter contained a 7.57 log TDB count compared with an increased 10.77 log TDB count during summer sampling. Concentrations of SC in corn silage reached an average of 6.3 log in winter compared with 11.81 log for summer. The seasonal effect was evident with an increase in summer counts across the board for TDB and SC, both in the feed and bulk tank milk samples. Bacillus licheniformis was the predominant species identified in 62.4% of winter (85 total) and 49.4% of summer (83 total) samples. Bacillus subtilis made up 9.4% of the remaining winter isolates, followed by Bacillus sonorensis at 8.2%. Conversely, B. sonorensis made up 12% of the summer isolates followed by Bacillus pumilus at 10.8%. Bacillus licheniformis is a ubiquitous microbe and was isolated from both TDB and sporeformer categories in all 3 sample types. There were larger increases in SC than TDB, indicating that summer temperatures and conditions may favor proliferation of sporeforming bacteria over that of TDB. In conclusion, samples from bulk tank milk, milking cluster swabs, and corn silage samples at each of the 10 sites indicated that B. licheniformis was the major contaminant species, regardless of season. In this experiment, corn silage was the major environmental source of both TDB and SC with higher concentrations in summer when compared with winter.

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine.

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO4(2-) ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 subsites in these three carboxypeptidases was revealed. It was found that SO4(2-) ion occupies the same position in the S1' subsite as the C-terminal carboxy group of the substrate.

Engineering of phenylalanine dehydrogenase from Thermoactinomyces intermedius for the production of a novel homoglutamate.

The dramatic increase in healthcare costs has become a significant burden to this era. Many patients are unable to access medication because of the high price of drugs. Genetic engineering has made advances to increase the yield, titer, and productivity in the bio-based production of chemicals, materials of interest, and identification of innovative targets for drug discovery. Currently, the production of homoglutamate (α-Aminoadipic acid) involves petrochemical routes that are costly with low yield and often not suitable for industrial production. Here, we established the development of NADH-dependent homoglutamate by engineering NADH-dependent phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius, which provides a novel tool for in-vivo metabolic engineering and in-vitro catalysis. Based on computational insight into the structure, we proposed the site-specific directed mutagenesis of the two important residues of PDH through docking simulations by AutoDock Vina which elucidated the binding mode of PDH with α-Ketoadipic acid and ligands. Our results demonstrated that the catalytic efficiency Km/Kcat of the final mutant Ala135Arg showed a 3-fold increase amination activity towards the ketoadipic acid as compared to the other mutant Gly114Arg, a double mutant Gly114Arg/Ala135Arg, and wild type TiPDH. Furthermore, we have introduced formate dehydrogenase as a cofactor regenerative system in this study which further made this study economically viable. Our study unfolds the possibility of biosynthesis of other non-proteinogenic amino acids that might be valuable pharmaceutical intermediaries.

Structures and function of a tailoring oxidase in complex with a nonribosomal peptide synthetase module.

Nonribosomal peptide synthetases (NRPSs) are large modular enzymes that synthesize secondary metabolites and natural product therapeutics. Most NRPS biosynthetic pathways include an NRPS and additional proteins that introduce chemical modifications before, during or after assembly-line synthesis. The bacillamide biosynthetic pathway is a common, three-protein system, with a decarboxylase that prepares an NRPS substrate, an NRPS, and an oxidase. Here, the pathway is reconstituted in vitro. The oxidase is shown to perform dehydrogenation of the thiazoline in the peptide intermediate while it is covalently attached to the NRPS, as the penultimate step in bacillamide D synthesis. Structural analysis of the oxidase reveals a dimeric, two-lobed architecture with a remnant RiPP recognition element and a dramatic wrapping loop. The oxidase forms a stable complex with the NRPS and dimerizes it. We visualized co-complexes of the oxidase bound to the elongation module of the NRPS using X-ray crystallography and cryo-EM. The three active sites (for adenylation, condensation/cyclization, and oxidation) form an elegant arc to facilitate substrate delivery. The structures enabled a proof-of-principle bioengineering experiment in which the BmdC oxidase domain is embedded into the NRPS.

Production and partial characterization of high molecular weight extracellular alpha-amylase from Thermoactinomyces vulgaris isolated from Egyptian soil.

Optimizing production of alpha-amylase production by Thermoactinomyces vulgaris isolated from Egyptian soil was studied. The optimum incubation period, temperature and initial pH of medium for organism growth and enzyme yield were around 24 h, 55 degrees C and 7.0, respectively. Maximum alpha-amylase activity was observed in a medium containing starch as carbon source. The other tested carbohydrates (cellulose, glucose, galactose, xylose, arabinose, lactose and maltose) inhibited the enzyme production. Adding tryptone as a nitrogen source exhibited a maximum activity of alpha-amylase. Bactopeptone and yeast extract gave also high activity comparing to the other nitrogen sources (NH4CI, NH4NO3, NaNO3, KNO3, CH3CO2NH4). Electrophoresis profile of the produced two alpha-amylase isozymes indicated that the same pattern at about 135-145 kDa under different conditions. The optimum pH and temperature of the enzyme activity were 8.0 and 60 degrees C, respectively and enzyme was stable at 50 degrees C over 6 hours. The enzyme was significantly inhibited by the addition of metal ions (Na+, Co2+ and Ca2+) whereas CI- seemed to act as activator. The enzyme was not affected by 0.1 mM EDTA while higher concentration (10 mM EDTA) totally inactivated the enzyme.

Structure of the microbial carboxypeptidase T complexed with the transition state analog N-sulfamoyl-l-lysine.

Carboxypeptidase T (CPT) from Thermoactinomyces vulgaris (EC 3.4.17.18) has a broad substrate specificity, the mechanism of which remains unclear. It cleaves off arginine residues by 10, and lysine residues by 100 times worse than hydrophobic leucine residues despite the presence of negatively charged Asp260 at the bottom of the primary specificity pocket. To study the relationship between the structure and specificity the 3D structure of CPT in complex with the stable transition state analog N-sulfamoyl-l-lysine (SLys) was determined in which the S-atom imitates the sp3-hybridized carbon in the scissile-bond. Crystals grown in microgravity has the symmetry of space group P6322. The present complex structure was compared with the previously reported complex structure of CPT and N-sulfamoyl-L-arginine (SArg). The location/binding of SLys in the active site of CPT very closely resembled that of SArg, and the positively charged N-atom of SLys was at the same position as the corresponding positively charged N-atom of SArg. The SLys complex is stabilized by the hydrogen bond between the nitrogen atom and OH-group of Thr257. The contact areas of the residues Tyr255, Leu211, and Thr262 with SLys were reduced in comparison with the same of SArg. This difference in bonding of SArg and SLys side chains in the primary specificity pocket induces shifts differences within the catalytic center (especially Tyr255-O20 and S18-Arg129 N1 gap) that may influence the enzyme's catalytic reaction. Therefore, this information may be useful for the design of carboxypeptidases with improved selectivity towards Arg/Lys for biotechnological applications.

Bacterial and fungal communities and their predictive functional profiles in kinema, a naturally fermented soybean food of India, Nepal and Bhutan.

Bacterial and fungal communities in kinema, a naturally fermented soybean food of the Eastern Himalayan regions of India, Nepal and Bhutan were profiled by high-throughout sequence analysis. Firmicutes (78.4%) was the most abundant phylum in kinema, followed by Proteobacteria (14.76%) and other phyla. Twenty seven species of Bacillus were detected, among which Bacillus subtilis (28.70%) was the most abundant bacterium, followed by B. licheniformis, B. thermoamylovorans, B. cereus, Ignatzschineria larvae, Corynebacterium casei, B. sonorensis, Proteus vulgaris, Brevibacillus borstelensis, Thermoactinomyces vulgaris, Lactobacillus fermentum and Ignatzschineria indica. Ascomycota was the most abundant fungal phylum in kinema. Wallemia canadensis, Penicillium spp., Aspergillus spp., Exobasidium spp., Arthrocladium spp., Aspergillus penicillioides, Mortierella spp., Rhizopus arrhizus and Mucor circinelloides, were major moulds, and Pichia sporocuriosa, Trichosporon spp., Saccharomycopsis malanga and Rhodotorula cycloclastica were abundant yeasts in kinema. We detected 277 species of bacteria among which, 99.09% were culturable and 0.91% were unculturable; and 80 fungal species among which, 33.72% were culturable and 66.28% were unculturable. Several unique bacterial genera to each country were observed, whereas no unique fungal genus was observed in kinema. Maximum coverage of sequencing depth was observed in all samples. Based on PCA plot, close relation was observed between samples of India and Nepal, whereas samples of Bhutan was clearly distinctive. Predictive functional features of bacterial and fungi related to metabolisms were inferred by the KEGG Orthology and MetaCyc databases, respectively.