“…57 On the other hand, design expert (version 8.0) is frequently utilized to improve the alpha-amylase production from thermostable and alklophilic alpha-amylase from Bacillus amyloliquefaciens KCP2. 51 Box-Behnken designs: The Box-Behnken designs have been used for RSM experiment to enhance alpha-amylase production from Aspergillus oryzae S2, Aspergillus oryzae CBS 819.72, Bacillus laterosporus, and Bacillus amyloliquefaciens 36,45,52,58 (Figure 4). CCD: Figure 5 shows CCD.…”
Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is widely applied in different industrial sectors. Microorganisms express unique alpha-amylases with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic features. Likewise, genetic engineering methods are applied to produce enzymes with higher stability in contrast to wild types. As there are widespread application of α-amylase in industry, optimization methods like RSM are used to improve the production of the enzyme ex vivo. This study aimed to review the latest researches on the production improvement and stability of α-amylase.
“…57 On the other hand, design expert (version 8.0) is frequently utilized to improve the alpha-amylase production from thermostable and alklophilic alpha-amylase from Bacillus amyloliquefaciens KCP2. 51 Box-Behnken designs: The Box-Behnken designs have been used for RSM experiment to enhance alpha-amylase production from Aspergillus oryzae S2, Aspergillus oryzae CBS 819.72, Bacillus laterosporus, and Bacillus amyloliquefaciens 36,45,52,58 (Figure 4). CCD: Figure 5 shows CCD.…”
Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is widely applied in different industrial sectors. Microorganisms express unique alpha-amylases with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic features. Likewise, genetic engineering methods are applied to produce enzymes with higher stability in contrast to wild types. As there are widespread application of α-amylase in industry, optimization methods like RSM are used to improve the production of the enzyme ex vivo. This study aimed to review the latest researches on the production improvement and stability of α-amylase.
“…The one-factor-at-atime (OFAT) is the classical method that may be simple to plan and execute, but is cumbersome and does not take into account the mutual interaction between parameters (Qua and Wu, 2005). In contrast to this, statistical optimization methods, such as response surface methodology (RSM), which takes the mutual interaction effects of parameters into account and reduces the number of experiments, have been reported widely in the literature for the optimization of media composition aimed at maximizing the fermentative production of microbial metabolites of interest (Agrebi et al, 2009;Liu et al, 2005;Manojkumar et al, 2013;Vuillemin et al, 2014). This paper reports a novel fibrinolytic enzyme production by Bacillus cereus SRM-001 isolated from a chicken dump waste site.…”
Section: Fibrinolytic Enzyme Production By Newly Isolated Bacillus Cementioning
The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO 4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO 4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.
“…al [ 19 ] reported that each organism or strain has its own special condition for maximum enzyme production. Other researcher findings noticed that each microorganism has its own specific culture conditions for maximum α-amylase production [ 3 , 17 , 19 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Fig 19. Optical density of biofilm values of P. aeruginosa isolate before and after treatment with commercial and purified α-amylase…”
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.
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