Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.

Biodiesel is one of the important biofuels as an alternative to petroleum-based diesel fuels. In the current study, enzymatic transesterification reaction was carried out for the production of biodiesel from waste cooking oil (WCO) and experimental conditions were optimized, in order to reach maximum biodiesel yield. Bacillus stearothermophilus and Staphylococcus aureus lipase enzymes were individually immobilized on CaCO3 to be used as environmentally friendly catalysts for biodiesel production. The immobilized lipases exhibited better stability than free ones and were almost fully active after 60 days of storage at 4 °C. A significant biodiesel yield of 97.66 ± 0.57% was achieved without any pre-treatment and at 1:6 oil/methanol molar ratio, 1% of the enzyme mixture (a 1:1 ratio mixture of both lipase), 1% water content, after 24 h at 55 °C reaction temperature. The biocatalysts retained 93% of their initial activities after six cycles. The fuel and chemical properties such as the cloud point, viscosity at 40 °C and density at 15 °C of the produced biodiesel complied with international specifications (EN 14214) and, therefore, were comparable to those of other diesels/biodiesels. Interestingly, the resulting biodiesel revealed a linolenic methyl ester content of 0.55 ± 0.02% and an ester content of 97.7 ± 0.21% which is in good agreement with EN14214 requirements. Overall, using mixed CaCO3-immobilized lipases to obtain an environmentally friendly biodiesel from WCO is a promising and effective alternative for biodiesel production catalysis.

Bacteriophages of thermophiles are of increasing interest owing to their important roles in many biogeochemical, ecological processes and in biotechnology applications, including emerging bionanotechnology. However, due to lack of in-depth investigation, they are underrepresented in the known prokaryotic virosphere. Therefore, there is a considerable potential for the discovery of novel bacteriophage-host systems in various environments: marine and terrestrial hot springs, compost piles, soil, industrial hot waters, among others. This review aims at providing a reference compendium of thermophages characterized thus far, which infect the species of thermophilic \'Bacillus group\' bacteria, mostly from Geobacillus sp. We have listed 56 thermophages, out of which the majority belong to the Siphoviridae family, others belong to the Myoviridae and Podoviridae families and, apparently, a few belong to the Sphaerolipoviridae, Tectiviridae or Corticoviridae families. All of their genomes are composed of dsDNA, either linear, circular or circularly permuted. Fourteen genomes have been sequenced; their sizes vary greatly from 35,055 bp to an exceptionally large genome of 160,590 bp. We have also included our unpublished data on TP-84, which infects Geobacillus stearothermophilus (G. stearothermophilus). Since the TP-84 genome sequence shows essentially no similarity to any previously characterized bacteriophage, we have defined TP-84 as a new species in the newly proposed genus Tp84virus within the Siphoviridae family. The information summary presented here may be helpful in comparative deciphering of the molecular basis of the thermophages\' biology, biotechnology and in analyzing the environmental aspects of the thermophages\' effect on the thermophile community.

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here we used Bacillus stearothermophilus as an indicator strain in the format of the carbapenem inactivation method (CIM) procedure to develop a rapid carbapenemase phenotype detection method: CIMB.S. Methods: The CIMB.S test was derived from the mCIM, where B. stearothermophilus replaced Escherichia coli as the indicator strain. The test bacteria were incubated in the presence of imipenem for 30 min, and then, aliquots were placed on colorimetric plates, and incubation was continued for 3.5 h at 60°C. We examined 134 clinical strains to evaluate the CIMB.S performance. Results: The CIMB.S can be completed in 4 h, and we successfully identified 38/39 (97.4%) carbapenemase-producing Enterobacteriaceae, including 17/18 (94.4%) carbapenemase-producing Pseudomonas aeruginosa and 18/19 (94.7%) carbapenemase-producing Acinetobacter baumannii. All non-carbapenemase producers we tested were negative and included Enterobacteriaceae (n = 36), P. aeruginosa (n = 17), and A. baumannii (n = 5). Conclusions: The CIMB.S test is a rapid carbapenemase phenotype detection method requiring only 4 h of total work time and displays high sensitivity and specificity.

Odor emissions represent one of the important issues of aerobic composting. The addition of microbial agents to compost is an important method for solving this problem, but this process is often unstable when a single microbial agent is added to the compost. Therefore, in this study, five treatments comprising different proportions of Bacillus stearothermophilus, Candida utilis, and Bacillus subtilis were tested to determine the best combination of the three microbial agents for ammonia reduction, as follows: control group (CK), 2:1:1 (A), 1:1:2 (B), 1:2:1 (C), and 1:1:1 (D). Compared with the CK group, the A, B, C, and D groups reduced ammonia emissions by 17.02, 9.68, 53.11, and 46.23%, respectively. The total ammonia emissions were significantly lower in C and D than in CK (p < 0.05). These two treatment groups had significantly increased nitrate nitrogen concentrations and decreased pH values and ammonium nitrogen concentrations (p < 0.05). Throughout the composting process, the total bacterial number was significantly higher in C and D than in CK (p < 0.05). Therefore, it is likely that B. stearothermophilus, C. utilis, and B. subtilis compounded from 1:2:1 (C) to 1:1:1 (D) reduced the ammonia emissions due to (1) a reduction in the pH and (2) the promotion of the growth of ammonia-oxidizing bacteria and the conversion of ammonium nitrogen to nitrate nitrogen. This study provides a theoretical basis and technical support for the odor problem of layer manure compost and promotes the development of composting technology.

Ammonia emissions is an important issue during composting because it can cause secondary pollution and a significant of nitrogen loss. Based on research adding Bacillus stearothermophilus can reduce ammonia emissions during composting because it can use sugar in organic matter fermentation to produce organic acids over 50 °C. This study conducted the batch experiments by adding different concentrations of Bacillus stearothermophilus to reduce the ammonia emissions and find out its characteristic during layer manure composting by using an aerobic composting reactor with sawdust as a bulking agent. The results show that the application of Bacillus stearothermophilus can accelerate the rate of temperature and significantly decrease pH, the warming period was 2 days in the treatment with Bacillus stearothermophilus, while it was 4 days in the treatment without Bacillus stearothermophilus. Ammonia emissions were mainly occurred in warming and high temperature period during composting. The ammonia emissions in the treatment with 8.00 g/kg initial Bacillus stearothermophilus were significantly lower than the other lower Bacillus stearothermophilus treatment and control during composting (p < 0.05), and it can significantly increase ammonium-nitrogen and nitrate-nitrogen concentration, reduce pH (p < 0.05), but the average number of Bacillus stearothermophilus copies in treatment with different initial Bacillus stearothermophilus concentration had no significant difference (p > 0.05). MiSeq System Sequencing results find that the addition of Bacillus stearothermophilus changed the bacterial community structure under warming and high-temperature periods during composting, increased the relative abundance of lactic acid bacillus and nitrification bacteria. Therefore, the reason for the low ammonia emission in 8.00 g/kg initial Bacillus stearothermophilus treatments might be not only due to the Bacillus stearothermophilus itself, but also Bacillus stearothermophilus can change the indigenous microorganism community, including increase the relative content of lactic acid Bacillus and nitrification bacteria, thus reducing the pH and promoting nitrification, and reducing ammonia emissions.

BACKGROUND: To evaluate the feasibility of high-temperature solid-state fermentation (SSF) using soybean meal (SBM) during the non-sterile process, Bacillus stearothermophilus was employed to assess the nutritional quality and bioactivity of SBM after fermentation.
RESULTS: The fermented SBM (FSBM) without autoclaving showed significant improvements in nutritional quality and bioactivity. The contents of peptides and crude and soluble proteins increased by 131.21%, 5.3% and 15.52%, respectively. Meanwhile, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging ability, reducing ability and hydroxyl free radical-scavenging activity rose by 57.07%, 238.92% and 368.26%, respectively. The inhibitory activity of angiotensin I-converting enzyme increased from 1.43 ± 0.83% to 26.89 ± 1.03%, while the trypsin inhibitor activity decreased by 74.05%. The contents of neutral and alkaline proteases and the growth of microorganisms in FSBM without autoclaving were higher and better than in steam-treated FSBM. After steam treatment, the water-holding capacity of SBM decreased, and a high crosslink density was observed on the surface of SBM particles.
CONCLUSIONS: It is feasible to ferment SBM by high-temperature SSF using B. stearothermophilus under non-sterile conditions. Adverse effects of SSF using sterile SBM might be owing to the low water-holding capacity caused by autoclaving. © 2018 Society of Chemical Industry.

Treatment with KI and its subsequent removal induced disassembly of Bacillus stearothermophilus pyruvate dehydrogenase complex (PDC) and association of disassembly products, respectively. The disassembly yielded neither completely dissociated components nor aggregate, but did yield a few molecular forms smaller than PDC. Depending on the KI concentration, these changes were of three phases: K-1, below 0.6 M; K-2, 1.0-1.5 M; K-3, above 1.8 M. PDC was disassembled in K-1 to C1 comprising pyruvate decarboxylase and lipoate acetyltransferase mainly and C2 comprising the decarboxylase and dihydrolipoamide dehydrogenase. In K-1, the removal of KI resulted in an apparent reconstitution of PDC. The mixing of C1 with an excess of C2 yielded an assembly larger than PDC and restored enzyme activities, but specific activities were different from those of PDC. In K-2 and K-3 phases, complexes smaller than PDC were yielded from disassembly products, and activities except for that of the acetyltransferase were not restored.

Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions.