Isolation and characterization of a new Bacillus sp. 50-3 with highly alkaline keratinase activity from Calotes versicolor faeces

Abstract

A new native feather-degrading bacterium has been isolated from the faeces of the agamid lizard Calotes versicolor, collected from the Beijing Zoo in China. The isolate, which has been identified as Bacillus sp. 50-3 based on morphological and biochemical and 16S rDNA tests, was shown to degrade native feather completely at 37°C and pH 7.0 within 36 h when using chicken feathers as the sole carbon and nitrogen source. Bacillus sp. 50-3 presented optimum growth at 37°C and pH 7.0 in feather meal medium. Under these conditions, the maximum keratinase activity (680 ± 25 U/ml) was also achieved. The keratinase of Bacillus sp. 50-3 was active over a broad range of pH values and temperatures toward azokeratin, and presented an optimum pH and temperature of 10.0 and 60°C, respectively. Furthermore, it was relatively heat-and alkalistable. Inhibitor studies showed that it seemed to belong to the serine-metalloprotease type. Therefore, the enzyme from Bacillus sp. 50-3 is a novel, high alkaline keratinase, suggesting its potential use in biotechnological processes.

Production and Characterization of Keratinolytic Protease from New Wool-DegradingBacillusSpecies Isolated from Egyptian Ecosystem

“…Luria-Bertani (LB) agar medium with 1% (w/v) skim milk was used for their cultivation by spreading 0.1 mL of each 10 −5 and 10 −6 dilutions. The plates then were incubated for 24 hours at different temperatures [4, 16]. The colonies which give clear zones formed by hydrolysis of skim milk were picked.…”

“…After 18 hrs, the colony forming units (CFU)/mL culture were 3∗10 6 , and 2% volume of the liquid medium was transferred to the production medium using 250 mL flask containing 100 mL of the production medium. The production medium containing (w/v) NaCl, 0.5 g/L; K 2 HPO 4 , 0.3 g/L; KH 2 PO 4 , 0.4 g/L; wool, 10 g/L; and the pH was adjusted 7.0–7.2 using 2N of NaOH and HCl [16]. The cultivated media were incubated at 37°C and 200 rpm for 5 days.…”

“…The residual activity was measured with standard enzyme reaction. The control is enzyme reacted at zero time (consider as 100%) [16]. …”

“…The residual activity was measured with standard enzyme reaction. The control is enzyme reacted at zero time and considered as 100% [16]. The following buffers were used:

citrate buffer pH 3-4-5-6 (0.1 M)

sodium phosphate pH 7-8 (0.1 M)

glycine/NaOH pH 9-10 (0.1 M)

sodium phosphate dibasic/NaOH pH 11 (0.05 M)

KCl/NaOH pH 12 (0.2 M).

…”

Separation and purification of a keratinase as pesticide against root-knot nematodes

“…50-3 with high keratinase activity has been isolated from Calotes versicolor feces before (Zhang et al 2009). In this study, we report an efficient method for the separation and purification of the keratinase from the strain culture, and the purified keratinase is found to have the activity to kill Meloidogyne incognita.…”