Isolation And Identification Of An Alkaline Lipase-producing Strain Aspergillus Awamori Hb-03 And Its Lipase Characterization

Lipases can catalyse the hydrolysis of long chain triglycerides, which are one of the most important industrial enzymes. Microbial lipases are currently receiving much attention with the rapid development of enzyme technology. Lipases constitute the most important group of biocatalysts for biotechnological applications. Recently, the microbial lipases are widely applied in the detergent, food, flavour industry, biocatalytic resolution of pharmaceuticals, esters and amino acid derivatives, making of fine chemicals, agrochemicals, use as biosensor, bioremediation and cosmetics and perfumery.In this paper, a lipase-producing strain HB-03 from organic-rich soil samples from Changsha city was isolated and identified. The growth conditions and nutritional factors for lipase production by strain HB-03 were optimized. Lipase produced by the strain was purified and its partial properties were investigated.(1) After samples cultured in the enrichment medium, the transparent circle method was adopted to do the primary screening and 18 strains were obtained when olive oil was used as the sole carbon source of selective solid medium to form clear transparent circle. After secondary screening of the 18 strains, a high-activity strain named HB-03 was isolated.(2) Morphological and molecular biological methods were used in the colony characterization, morphological observation and 18S rDNA sequence analysis of the isolate, and the strain HB-03 was identified as Aspergillus awamori.(3) The growth conditions and nutritional factors for lipase production by strain HB-03 were optimized, and the maximum lipase production of (45.9±2.3) U/mL was obtained at 30℃and pH 7.0 after 36 h Incubation using olive oil (1%) and sucrose (0.5%) as carbon sources and combination of peptone (2%), yeast extract (0.5%) and ammonium sulfate (0.1%) as nitrogen sources.(4) The the extracelluar lipase secreated by the strain Aspergillus awamori HB-03 was precipited by 90% ammonium sulfate purified by followed SephadexG-75 gel filtration chromatography. Purifications of 10.6 fold and 18.84% yield over the initial material were achieved. The specific activity of the lipase was 1,862.2 U/mg. The molecular weight of the purified lipase was 68 kDa.(5) The optimum pH and temperature for the purified lipase was found to be pH 8.5 and 40℃respectively. And the lipase kept almost 90% of activity in pH 7.0-10.0 and the temperatures up to 45℃. The metal ions of Mn2+, Ba2+ significantly enhanced the lipase activity, whereas Cu2+, Fe3+ and Mg2+ strongly reduced its activity. The Km and Vmax value of the purified enzyme for p-nitrophenyl palmitate were 0.13 mmol/L and 60.6 mmol/L/min respectively.An alkaline lipase-producing stain Aspergillus awamori HB-03 was isolated from oil rich samples of Changsha city and identified in this article. Lipase produced by this strain was purified and analyzed. Results showed that this lipase has potential industrial application.