Identification of Thermophilic Aerobic Sporeformers in Bedding Material of Compost-Bedded Dairy Cows Using Microbial and Molecular Methods

Compost-bedded pack barns (CBP) are of increasing interest in dairy farming due to their positive effect on animal welfare. The temperature and the moisture content of the bedding material characterising the composting process can promote the growth of thermophilic aerobic sporeformers (TAS). Therefore, the aim of this study was to determine CBP bedding material characteristics, such as moisture content and temperature, and to determine TAS species. The dilution, the heat inactivation of all non-TAS species and the incubation of 13 bedding samples from four CBP groups resulted in a mean TAS amount over all samples of 4.11 log10 cfu/g bedding material. Based on the subsequent sequencing of parts of the 16S rRNA-gene of 99 TAS colonies, the TAS species Aneurinibacillus thermoaerophilus, Bacillus licheniformis, Geobacillus thermodenitrificans, Laceyella sacchari, Thermoactinomyces vulgaris and Ureibacillus thermosphaericus were identified. The moisture content of the bedding material, the relative humidity above the bedding material and the sampling season significantly affected the amount of TAS. The moisture content or relative humidity above the bedding material significantly influenced the concentration of Ureibacillus thermophaericus or Laceyella sacchari. Consequently, an optimal CBP management including a dry lying surface and an optimal composting process will contribute to a moderate microbial, especially TAS amount, and TAS species distribution.

Study of the Interaction of Sorption and Catalytic Centers in Carboxypeptidase T by X-ray Analysis

Carboxypeptidase T (CPT; EC 3.4.17.18) from Thermoactinomyces vulgaris is a distant homolog of the highly specific pancreatic carboxypeptidase B; but has a broad substrate specificity; the source of which remains unclear. A previous study of the structural bases of the substrate specificity of CPT using stable sulfamoyl analogs of the transition state of the elimination of leucine; phenylalanine; arginine; and glutamic acid; showed that the binding of the C-terminal residue of the substrate to the primary selectivity pocket of CPT leads to a change in the distance between Zn2+ and the sulfur atom. This value is related to the efficiency of catalysis of the corresponding substrate or the inhibition constant of the corresponding stable analog of the transition state. In this work; we obtained crystallographic and kinetic data of the complex of CPT with N-sulfamoyl-L-valine; confirming the effect of the binding of the ligand’s side group by the primary specificity pocket of CPT on the structure of the catalytic center; which can explain the unusual substrate specificity of CPT.

Extracellular Production, Characterization, and Engineering of a Polyextremotolerant Subtilisin-Like Protease From Feather-Degrading Thermoactinomyces vulgaris Strain CDF

Here, the gene encoding a subtilisin-like protease (protease Als) was cloned from Thermoactinomyces vulgaris strain CDF and expressed in Escherichia coli. The recombinant enzyme was released into the culture medium of E. coli as a mature form (mAls). Purified mAls displayed optimal activity at 60–70°C and pH 10.0 using azo-casein as the substrate, and showed a half-life of 13.8 h at 70°C. Moreover, the activity of thermostable mAls was comparable to or higher than those of mesophilic subtilisin Carlsberg and proteinase K at low temperatures (10–30°C). Protease Als was also stable in several organic solvents and showed high compatibility with commercial laundry detergents. Notably, mAls exhibited approximately 100% of its activity at 3 M NaCl, and showed enhanced thermostability with the increase of NaCl concentration up to 3 M. Protease Als possesses an excess of solvent-accessible acidic amino acid residues, which may account for the high halotolerance of the enzyme. Compared with homologous protease C2 from the same strain, protease Als exhibits substantially lower activity toward insoluble keratin substrates but efficiently hydrolyzes soluble keratin released from chicken feathers. Additionally, direct substitution of the substrate-binding site of protease Als with that of protease C2 improves its activity against insoluble keratin substrates. By virtue of its polyextremotolerant attribute and kerationolytic capacity, protease Als may find broad applications in various industries such as laundry detergents, food processing, non-aqueous biocatalysis, and feather processing.

Impact of exogenous α-amylases on sugar formation in straight dough wheat bread

The use of bacterial or fungal α-amylases is common in wheat bread production to improve several quality-related parameters such as loaf volume, crust color or staling behavior. To study the impact of exogenous α-amylases on straight dough wheat bread, we quantitated mono-, di- and oligosaccharides and residual α-amylase activity in bread crumb during storage for up to 96 h. Discovery-driven proteomics of the five α-amylase preparations studied showed that only a few different amylases per preparation were responsible for the hydrolytic effect. Compared to the control, the supplementation with α-amylase from Bacillus amyloliquefaciens in wheat dough preparation led to major changes in the sugar composition of bread crumb during storage with the formation of oligosaccharides like maltopentaose, maltohexaose, maltoheptaose, and maltooctaose. A residual activity corresponding to 4.0% of the applied activity was determined in the breads prepared with α-amylase from B. amyloliquefaciens, but no residual activity was detected for any of the other fungal or bacterial α-amylases from Aspergillus oryzae or Thermoactinomyces vulgaris. Whether the detected residual activity is related to the characteristics of bread staling or bread crumb properties must be clarified in further studies.

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.

Complete Genome Sequence and Methylome Analysis of Thermoactinomyces vulgaris 2H

Here, we report the complete genome sequence and full methylome analysis of a newly isolated, aerobic, thermophilic, Gram-positive actinomycete, a strain of Thermoactinomyces vulgaris designated strain 2H.

Complete Genome Sequence of Thermoactinomyces vulgaris Strain CDF, a Thermophilic Bacterium Capable of Degrading Chicken Feathers

Thermoactinomyces vulgaris strain CDF was isolated from soil and shown to have the ability to degrade chicken feathers at high temperatures. Here, we report the complete genome sequence of this bacterium, which is 2,595,509 bp long with 2,642 predicted genes and an average G+C content of 48.14%.