Purification and biochemical characterization of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in detergent formulations

Abstract

An extracellular alkaline protease producing strain was isolated from alkaline soil and identified as Bacillus alcalophilus TCCC11004 on the basis of 16S rDNA gene sequencing and biochemical properties. The most appropriate medium for the protease production was composed of (g/l): maltodextrin 110, yeast extract 17.5, cotton seed meal 29.3, K₂HPO₄ 18, trisodium citrate 3.3 and CaCl₂ 2.6. The alkaline protease TC4 was purified from the culture supernatant by ammonium sulfate precipitation, Sephadex G-75 gel filtration and SP-Sepharose HP ion exchange chromatography, with a 6.8 fold increase in specific activity and 15.2% recovery. The molecular weight was estimated to be 26 kDa on SDS-PAGE. The protease was highly active from pH 9.0-12.0 with an optimal at pH 11.0. It was active at 30 - 60°C and exhibited maximal activity at 50°C. The thermostability of the protease was increased by the addition of CaCl₂. It retained 70 and 81% of its initial activity after heating for 2 h at 50°C, in the absence or presence of 2 mM CaCl₂, respectively. The enzyme was inactivated by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suggesting that it is a serine protease. The protease was stable in 0.5% SDS and retained 70.3% of its initial activity after 1 h of incubation. It was active in the presence of 3% Triton X-100 with 100% activity and stable towards oxidizing agent with 69.2% activity in the presence of 1% H₂O₂. The enzyme showed excellent compatibility with commercial detergents such as TaiZi, BiLang, DiaoPai and TianQing, retaining more than 90% of its initial activity in the tested detergents after 1 h of preincubation at 40°C.

Keywords: Serine alkaline protease, Bacillus alcalophilus, stability, detergent compatibility.