Biochemical properties of a cold-adapted recombinant lipase from Proteus sp. Pw

• Main Authors: Su, Po-Husan, 蘇柏瑄
• Other Authors: Chang, Rey-Chang
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/hm3af3

碩士 === 國立高雄科技大學 === 海洋生物技術系 === 107 === In this study, we have isolated a bacterium with lipase activity from Lotus Pond. The 16S rRNA sequence analysis of the bacterium indicated that have 99 % similarity to Proteus vulgaris AD_AR5. Therefore, we name the strain as Proteus sp. Pw. The full length lipase gene (864 bp) was cloned and encoded a mature lipase with 287 amino acids. The molecular weight is 31.2 kDa. The lipase DNA and amino acid sequences showed 94 % and 96 % identity with a lipase of Proteus sp. K19 (JN594058.1). The biochemical analysis showed not only highest activity toward p-nitrophenyl butyrate esters (C4) with short chain fatty acids but also toward p-nitrophenyl myristate (C14) with long chain fatty acids. Effect of temperature on the activities of Pw lipase activities is 25°C with p-nitrophenyl butyrate and 20 °C with p-nitrophenyl myristate. The temperature stability of Proteus sp. Pw recombinant lipase in the range 10 °C to 55 °C and keep high activity below 10 °C. Proteus sp. Pw belong cold-adapted lipase. The Pw show optimum pH is pH 8.5 with p-nitrophenyl butyrate and pH 8.0 with p-nitrophenyl myristate. The Pw is stable in the range pH 6 – 11. Among the natural oil substrates, Pw show the highest activity with olive oil and high activity with fish oil. EDTA does not affect Pw lipase hydrolyze short chain fatty acids and long chain fatty acids. Non-ionic surfactants are inhibite Pw lipase activity a little. Gum Arabic is enhanced Pw lipase activity with short chain fatty acids and long chain fatty acids. Pw activity is enhanced 1.65-fold and 1.49-fold by 10 mM Li + and Na + with short chain fatty acids. Pw activity is enhanced 1.53-fold and 1.46-fold by 10 mM Mn 2+ and Mg 2+ with long chain fatty acids.Pw is stable in polar organic solvent. Pw is stable in some non-polar organic solvent too. For example, benzene, n-hexane and toluene. Ester synthesis from oleic acid and various alcohol. We find Synthetic ability for tert-butanol. At a substrate ratio of 1 : 1, the most efficient synthetic esters in both n-hexane and toluene. When adding 40 μg of enzyme to n-hexane, a 91% transesterification rate can be obtained. And adding 10 μg of enzyme to toluene, a 88% transesterification rate can be obtained. The highest transesterification rate is obtained at 50% n-hexane and 30% toluene. The results of current study illustrated that this specific lipase is potentially useful for industrial application.