Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system

被引:57
作者
Barbosa, Jose Murillo P. [1 ]
Souza, Ranyere L. [1 ]
Fricks, Alini T. [1 ,2 ]
Zanin, Gisella Maria [3 ]
Soares, Cleide Mara F. [1 ,2 ]
Lima, Alvaro S. [1 ,2 ]
机构
[1] Univ Tiradentes, UNIT, BR-49032490 Aracaju, SE, Brazil
[2] ITP, BR-49032490 Aracaju, SE, Brazil
[3] Univ Estadual Maringa, Maringa, PR, Brazil
来源
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2011年 / 879卷 / 32期
关键词
Lipolytic enzyme; Purification; Aqueous two-phase system; EXTRACTION; PARTITION; PROTEINS; ENZYMES;
D O I
10.1016/j.jchromb.2011.10.035
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000 g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4 degrees C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000 g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4 degrees C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution. (C) 2011 Elsevier B.V. All rights reserved.
引用
收藏
页码:3853 / 3858
页数:6
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