Optimization and characterization of alkaliphilic lipase from a novel Bacillus cereus NC7401 strain isolated from diesel fuel polluted soil

Abstract

Five Bacillus cereus strains including B. cereus AVP12, B. cereus NC7401, B. cereus BDBCO1, B. cereus JF70 and B. specie JL47 isolated from the diesel fuel polluted soil adhered to the roots of Tagetes minuta were screened for lipase production with phenol red agar method. B. cereus NC7401 strain successfully expressing and secreting lipase with maximal lipolytic activity was subjected to a submerged fermentation process with five different carbon (starch, glucose, maltose, fructose, and lactose) and five different nitrogen (tryptone, ammonium nitrate, peptone, urea, yeast extract) sources to produce lipase enzyme. Maximum enzyme activity was found with starch (30.6 UmL-1), maltose (40 UmL-1), and tryptone (38.6 UmL-1), and the lipases produced using these sources were named lipase A, B, and C respectively. The total protein content of 8.56, 8.86, and 2.75 μg mL-1 were obtained from B. cereus NC7401 cultured using starch, maltose, and tryptone respectively. Lipase was stable between temperature range 30-80°C and pH 5-10 whereas optimally active at 55°C and pH 8.0. The enzyme was relatively stable for 10 days at 4°C and its optimum reaction time with the substrate was 30 minutes. It was tolerant to 1.5% (v/v) methanol as an organic solvent, 1.5% (v/v) Triton X-100 as a media additive and 1.5% (w/v) Ni2+ as a metal ion. SDS, n-hexane, and Ag+ inhibited lipolytic activity. Oil stains were removed from cotton fabric which showed oil removal efficiency enhancement in the presence of a lipase. Fat hydrolysis of 20, 24, and 30% was achieved following 6 hours of incubation of the fat particles with lipase A, B, and C respectively at a concentration of 20 mg mL-1. To as best of our knowledge, this study on lipases extracted from bacteria of Azad Kashmir, Pakistan origin has never been reported before.

Fig 1

Different washing performances to evaluate the effectiveness of lipase as laundry additive (a) 100 mL water; (b) 100 mL water + 1 mL detergent (7 mg mL^-1); (c, d and e) 100 mL water + 1 mL detergent (7mg mL^-1) + 2 mL solution of Lipase A, B and C respectively.

Fig 2

Screening of B. cereus strains for lipolytic activity with phenol red as pH indicator; a) B. cereus NC7401, b) B.cereus AVP12, c) B. cereus BDBCO1, d) B. specie JL47 and e) B.cereus JF70.

Fig 3. SEM micrograph of B. cereus NC7401, the 24 h old cells that were grown in nutrient broth medium in a shake flasks system.

Fig 4. Statical analysis of different optimum conditions by applying one-way ANOVA.

Fig 5

Cleaning ability of lipase from B. cereus NC7401 on pieces of stained cloth; a) Control; Distilled water with stained cloth), b) Detergent with distilled water, c) Distilled water, detergent, and lipase A, d) distilled water, detergent, and lipase B, e) distilled water, detergent, and lipase C.