Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.
Background/Aims: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. Methods: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. Results: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. Conclusion: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.
Abstract:The high cost of fermentation media is one of the technical barriers in amylase production from microbial sources. Amylase is used in several industrial processes or industries, for example, in the food industry, the saccharification of starchy materials, and in the detergent and textile industry. In this study, marine microorganisms were isolated to identify unique amylase-producing microbes in starch agar medium. More than 50 bacterial strains with positive amylase activity, isolated from marine water and soil, were screened for amylase production in starch agar medium. Bacillus sp. BCC 021-50 was found to be the best amylase-producing strain in starch agar medium and under submerged fermentation conditions. Next, fermentation conditions were optimized for bacterial growth and enzyme production. The highest amylase concentration of 5211 U/mL was obtained after 36 h of incubation at 50 • C, pH 8.0, using 20 g/L molasses as an energy source and 10 g/L peptone as a nitrogen source. From an application perspective, crude amylase was characterized in terms of temperature and pH. Maximum amylase activity was noted at 70 • C and pH 7.50. However, our results show clear advantages for enzyme stability in alkaline pH, high-temperature, and stability in the presence of surfactant, oxidizing, and bleaching agents. This research contributes towards the development of an economical amylase production process using agro-industrial residues.
Background: Coconut is a tropical fruit well known for its essential oils that have been recognized for their biological activities since ancient times. There have been no previous investigations on the essential oils from coconut shells. Method: The shell extract of Cocos nucifera (L.) was prepared by the Soxhlet method and total phenolic content (TPC) in the extract was determined by Folin-Ciocalteu (FC) assay. The antioxidant potential of the coconut shell extract was evaluated by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Minimum inhibitory concentration (MIC) of the extract was determined by the strip method against clinically isolated dermal mycosis of 20 infected patients. Result: Total antioxidant activity varied from 92.32% to 94.20% and total phenolic content was found at 5.33 ± 0.02 mg/g in the coconut shell extract. The extract was found to be most effective as an antifungal against human pathogenic fungi, including A. niger, A. flavus, T. rubrum, M. canis, M. gypseum, A. fumigates, T. mentagrophyte and T. vercossum. The crude shell extract was highly effective against all dermal mycosis tested with the MIC ranging from 62 mm to 90 mm, whereas all fungal samples showed good inhibitory effect. Conclusion: The results of the present study provide a potential cure for microbial infections.
Proteolytic enzymes are applied in various industries such as detergent, leather, food, textile, cosmetics, pharmaceutical, and synthetic biotechnology. To meet commercial needs, microbial strains of high value in terms of cost-effective production have been focused. In this study, thermophilic strain, Bacillus sp. BBXS-2 was activated on simple growth medium and then transferred to Luria Bertani (LB) medium. Maximum protease concentration of 6723 U/mL under optimized fermentation conditions (molasses, corn steep liquor, pH 9.0, and 45°C). The results showed, there was no difference observed in bacterial growth and protease titer while replacing yeast extract with corn steep liquor so, in this way, about 90% cost of nitrogen source can be saved. The extracellular enzyme was purified to homogeneity from cell-free supernatant by ammonium sulphate precipitation followed by dialysis and ion exchange chromatography, recovery yield reduced from 100 to 22% and purification fold increased from 1 to 9.82. The enzyme was active in broad pH and temperature range 8-12 pH and 30-60°C, with maximum activity at pH 10.0 and 60°C, respectively. Protease retained more than 90% activity after incubation at 40°C for 2 weeks in the presence of (40% v/v) organic solvents including ethanol, methanol, and isopropanol. Overall, research suggests that this strain is a more promising candidate and possess practical ability to use in industries.
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