A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen

Abstract

Background and aim The purpose of the current study is to isolate a heavily amylase-producing bacteria of the genus Bacillus from soil samples, optimize the production of the enzyme, purify it, and evaluate its activity against biofilm-producing bacteria. A total of 12 soil samples were collected and screened for promising Bacillus species with good amylolytic activity. Isolation was done by serial dilution and plating technique and amylolytic activity was determined by starch agar plate method. Among the 12 Bacillus isolates recovered from soil samples, 7 showed positive α-amylase production. The best isolate that recorded the greatest amylolytic activity was selected for further studies. This isolate was identified by 16S rRNA sequencing as Bacillus cereus and registered under gene bank accession number OP811897. Furthermore, the α-amylase enzyme was produced by a submerged fermentation technique using best production media and partially purified by ammonium sulfate and chilled ethanol and molecular weight had been determined by SDS-PAGE gel electrophoresis. The production of α-amylase was optimized experimentally by one-factor at a time protocol and statistically by Plackett–Burman design as well as RSM CCD design. Data obtained from OFAT and CCD revealed that α-amylase activities were 1.5- and twofold respectively higher as compared to un-optimized conditions. The most significant factors had been identified and optimized by CCD design. Results Among the eleven independent variables tested by PBD, glucose, peptone, (NH4)2SO4, and Mg SO4 were the most significant parameters for α-amylase production with an actual yield of 250U/ml. The best physical parameters affecting the enzyme production were incubation time at 35 °C, and pH 5.5 for 48 h. The partially purified enzyme with 60% ammonium sulphate saturation with 1.38- fold purification showed good stability characteristics at a storage temperature of 4 °C and pH up to 8.5 for 21 days. Antibiofilm activity of purified α-amylase was determined against Pseudomonas aeruginosa (ATCC 35659) by spectrophotometric analysis and CLSM microscopic analysis. Results demonstrated biofilm inhibition by 84% of the formed Pseudomonas biofilm using a microtiter plate assay and thickness inhibition activity by 83% with live/Dead cells percentage of 17%/83% using CLSM protocol. Conclusions A highly stable purified α-amylase from B. cereus showed promising antibiofilm activity against one of the clinically important biofilm-forming MDR organisms that could be used as a cost-effective tool in pharmaceutical industries.

Amylase and Cellulase Production from Newly Isolated Bacillus subtilis Using Acid Treated Potato Peel Waste

“…The plots in this study reported s high amylase production with an increase in pH and less ammonium sulfate. Another report also reported high amylase production using 3D contour plots and depicted the significant effect of multiple variables [ 41 ].…”

“…The plots in this study reported s high amylase production with an increase in pH and less ammonium sulfate. Another report also reported high amylase production using 3D contour plots and depicted the significant effect of multiple variables [41]. For cellulase production, the contour plots illustrate the interaction of different process parameters on enzyme yield.…”

Upstream and Downstream Bioprocessing in Enzyme Technology

“…α-amylase breaks down complex carbohydrates, such as starches and glycogen, into simpler sugars like maltose and glucose. Due to its importance, various investigations have aimed to increase its production, such as the research by Abo-Kamer et al, 2023 [ 47 ]. The researchers isolated α-amylase from the microorganism Bacillus cereus obtained from soil samples.…”

“…Among the independent variables examined, glucose, peptone, (NH 4 ) 2 SO 4 , and MgSO 4 displayed the highest degree of significance, yielding an impressive 250 U/mL of α-amylase. The physical conditions conducive to optimal enzyme production were a temperature of 35 °C and a pH of 5.5, maintained for 48 h [ 47 ].…”