Statistical Optimization of Fibrinolytic Enzyme Production Using Agroresidues byBacillus cereusIND1 and Its Thrombolytic ActivityIn Vitro

Abstract

A potent fibrinolytic enzyme-producing Bacillus cereus IND1 was isolated from the Indian food, rice. Solid-state fermentation was carried out using agroresidues for the production of fibrinolytic enzyme. Among the substrates, wheat bran supported more enzyme production and has been used for the optimized enzyme production by statistical approach. Two-level full-factorial design demonstrated that moisture, supplementation of beef extract, and sodium dihydrogen phosphate have significantly influenced enzyme production (P < 0.05). A central composite design resulted in the production of 3699 U/mL of enzyme in the presence of 0.3% (w/w) beef extract and 0.05% (w/w) sodium dihydrogen phosphate, at 100% (v/w) moisture after 72 h of fermentation. The enzyme production increased fourfold compared to the original medium. This enzyme was purified to homogeneity by ammonium sulfate precipitation, diethylaminoethyl-cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography, and casein-agarose affinity chromatography and had an apparent molecular mass of 29.5 kDa. The optimum pH and temperature for the activity of fibrinolytic enzyme were found to be 8.0 and 60°C, respectively. This enzyme was highly stable at wide pH range (7.0–9.0) and showed 27% ± 6% enzyme activity after initial denaturation at 60°C for 1 h. In vitro assays revealed that the enzyme could activate plasminogen and significantly degraded the fibrin net of blood clot, which suggests its potential as an effective thrombolytic agent.

Novel Sequential Screening and Enhanced Production of Fibrinolytic Enzyme byBacillussp. IND12 Using Response Surface Methodology in Solid-State Fermentation

“…The carbon, nitrogen, and other nutrients' content in cow dung are an indication that it could be a good nutrient source for the microbes [50]. The agroindustrial residues such as rice chaff [51], shrimp shells [4], cane molasses [52], and wheat bran [7] were used as the substrate for the production of fibrinolytic enzyme. The availability of cow dung substrate is higher than most of the reported substrates.…”

Thrombolytic Potential of Novel Thiol-Dependent Fibrinolytic Protease from Bacillus cereus RSA1

“…Biochemical characterization revealed no indole production, methyl red, or oxidase activity, while positive results were obtained for the voges-proskauer and catalase test. It utilized citrate, exhibited gelatinase and catalase activities, along with nitrate reduction, and did not show starch utilization or oxidase activity but utilized sugars with gas production [30,43]. The selected strain was further subjected to molecular characterization and was identified as Bacillus cereus RSA1 (NCBI Accession number MK288105).…”

“…The Plackett-Burman Design was used to evaluate the most significant variables for enhanced production of fibrinolytic protease from a number of carbon sources (glucose and sucrose), nitrogen sources (peptone, casein, and yeast extract), inorganic salts (K 2 HPO 4 , MgSO 4 ), and cultivation parameters (rpm, time of incubation, inoculums size, and initial pH) [30,[44][45][46]. The results including the actual level of variables with symbol codes (as mentioned in Section 2.8) are displayed in Tables 1 and 2.…”

“…The analyses of effect of numerous physico-chemical parameters require a lot of experimentation and consumption of chemicals and time. Hence, Plackett-Burman Design and response surface methodology are applied for the enzyme production process to select the optimal levels of these parameters [30,44].Plackett-Burman design is an extensively used statistical tool for screening of numerous parameters available to select the most profound ones [45]. This is followed by response surface methodology, which aids in determining the optimal level of the key variables involved in enhanced production of fibrinolytic protease [46].…”

“…The analyses of effect of numerous physico-chemical parameters require a lot of experimentation and consumption of chemicals and time. Hence, Plackett-Burman Design and response surface methodology are applied for the enzyme production process to select the optimal levels of these parameters [30,44].…”