Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria.

Present study was aimed at screening and characterizing thermostable amylase-producing bacteria from water and sediment samples of unexplored hot spring of Tatta Pani Kotli Azad Kashmir. Four thermophilic isolates were characterized on morphological, biochemical, physiological basis and were authenticated by molecular analysis. By 16S rDNA sequencing, isolates were identified as Anoxybacillus mongoliensis (MBT001), Anoxybacillus flavithermus (MBT002), Bacillus (MBT004). Among all identified strains, MBT003 showed maximum homology with both Anoxybacillus mongoliensis and Anoxybacillus flavithermus. Amylase activity was analyzed qualitatively in starch agar and quantitatively by DNS method. The optimal enzyme production was observed and authenticated by Response Surface Methodology at 7 pH, 70 °C, 1.25% substrate concentration, 300 µL of inocula volume after 48 h of incubation. Optimum amylase activity (4.4 U/mL) and stability (3.3 U/mL) was observed with 1.5% soluble starch at 70 °C. Maximum activity (3.7 U/mL) and stability (1.5 U/mL) was found at pH 8. Enzyme activity was increased in the presence of MgSO4 and CaCl2. Amylase was stable with surfactants and commercial detergents for 30 min. Supplementation of the enzyme with commercial detergent improved the washing ability of the detergent. This investigation has revealed that these thermostable bacteria are excellent source of amylase which can be used commercially for generating economic activity on sustainable basis.

Statistical factorial designs for optimum production of thermostable α-amylase by the degradative bacterium Parageobacillus thermoglucosidasius Pharon1 isolated from Sinai, Egypt.

BACKGROUND: This study aimed to isolate potent thermophilic and amylolytic bacteria from a hot spring of Pharaoh's bath, Sinai, Egypt, and screen its degradative activity. The amylolytic activity was further optimized using a statistical full factorial design followed by response surface methodology. RESULTS: A thermophilic bacterium was isolated from the hot spring of Pharaoh's Bath, Sinai, Egypt. The isolate produced amylase, cellulase, and caseinase and was identified by 16S rRNA gene sequencing as Parageobacillus thermoglucosidasius Pharon1 (MG965879). A growth medium containing 1% soluble starch was found to optimize the amylase production. Dinitrosalycalic acid method (DNS) was used to estimate the amount of reducing sugar produced. Statistical full factorial and response surface designs were employed to optimize physical variables affecting the α-amylase production and determine the significant interactions of the studied variables during the fermentation process. According to the results obtained by the response optimizer, the maximum amylase activity reached 76.07 U/mL/ min at 54.1°C, pH 5.6 after 98.5 h incubation under aerobic conditions. Moreover, the produced enzyme was thermostable and retained most of its activity when exposed to a high temperature of 100°C for 120 min. Maximum enzyme activity was attained when the enzyme was incubated at 70°C for 30 min. CONCLUSIONS: This is the first report of the production of thermostable α-amylase by the potent thermophilic Parageobacillus thermoglucosidasius. The enzyme endured extreme conditions of temperature and pH which are important criteria for commercial and industrial applications.

Development of thermostable amylase enzyme from Bacillus cereus for potential antibiofilm activity.

The marine bacterial strain Bacillus cereus was used to produce amylase enzyme and has excellent alkali-stable and thermostable enzymatic activity. The combined effects of pH, temperature and incubation time on amylase activity were studied using response surface methodology. The amylase enzyme activity was also determined in the presence of various metal ions, chelating agents, detergents and the results showed that the maximum enzyme activity was observed in the presence of calcium chloride (96.1%), EDTA (63.4%) and surf excel (90.6%). The amylase enzyme exhibited excellent antibiofilm activity against marine derived biofilm forming bacteria Pseudomonas aeruginosa and Staphylococcus aureus in microtiter plate assay and congo red assay. Light and confocal laser scanning microscopic (CLSM) analysis were also used to confirm the potential biofilm activity of amylase enzyme. The CLSM analysis showed the inhibition of complete biofilm formation on amylase enzyme treated glass surface. Further in vivo toxicity analysis of amylase enzyme was determined against marine organisms Dioithona rigida and Artemia salina. The results showed that there is no morphological changes were observed due to the minimal toxicity of amylase enzyme. Overall these findings suggested that marine bacterial derived amylase enzyme could be developed as potential antibiofilm agent.

Effect of ultraviolet radiation on physiological and biochemical properties of yeast Saccharomyces cerevisiae during fermentation of ultradispersed starch raw material

Background: Study of correlation between pretreatment of yeast with ultraviolet radiation and efficiency of further fermentation of wort made of ultrafine grain particles to ethanol. Results: We investigated three races of industrial yeast Saccharomyces cerevisiae (native and irradiated by ultraviolet). Physiological properties during fermentation of starchy wort were tested in all variants. It was shown that activation of the yeast by ultraviolet radiation allows to further increase the ethanol yield by 25% on average compared with the native yeast races when using thin (up to micro- and nano-sized particles) or standard grain grinding. Conclusions: Using mechanical two-stage grinding of starchy raw materials and ultraviolet pretreatment of yeast, the efficiency of saccharification of starch and fermentation of wort to ethanol was increased.