A Cold-Adapted Lipase of an Alaskan Psychrotroph,Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization
A Cold-Adapted Lipase of an Alaskan Psychrotroph,Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization
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DOI：
10.1128/aem.64.2.486-491.1998
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发表时间：
1998-02
影响因子：
4.4
通讯作者：
Dong-Won Choo;T. Kurihara;Takeshi Suzuki;K. Soda;N. Esaki
中科院分区：
文献类型：
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作者：
Dong-Won Choo;T. Kurihara;Takeshi Suzuki;K. Soda;N. Esaki
从阿拉斯加土壤中分离出一种低温生长产生冷适应脂肪酶的嗜冷细菌，并鉴定为假单胞菌菌株。从该菌株中克隆脂肪酶基因（lipP）并测序。从该基因的核苷酸序列（924 bp）推断出的氨基酸序列对应于308个氨基酸残基的蛋白，分子量为33,714。LipP在其他冷适应脂肪酶中也有保守的共识基序，即来自南极moraxella TA144的脂肪酶2 （G. Feller， M. Thiry， J. L. Arpigny, and C. Gerday， DNA细胞生物学，10:31 81 - 388,1991）和哺乳动物激素敏感脂肪酶（D. Langin， H. Laurell， L. S. Holst， P. Belfrage, and C. Holm, Proc. Natl）。学会科学。（USA 90:4897-4901, 1993）：一种五肽，GDSAG，含有假定的活性位点丝氨酸和HG二肽。LipP是从含有编码LipP基因的质粒的重组大肠杆菌C600细胞的提取物中纯化出来的。该酶对三油酸具有1,3位特异性。具有短至中链脂肪酸（C4和C6）的对硝基苯基酯是较好的底物。该酶在pH 6 ~ 9之间较为稳定，水解三丁酸甘油酯的最佳pH为8左右。在5 ~ 35℃的温度范围内，对丁酸对硝基苯基和月桂酸对硝基苯基的水解活化能分别为11.2和7.7 kcal/mol。该酶在高于45℃的温度下不稳定。对硝基苯丁酸酯酶的Km随实验温度的升高而升高。Zn2+、Cu2+、Fe3+和Hg2+对该酶有较强的抑制作用，而苯基甲基磺酰氟和二硝基苯基磷酸对该酶没有影响。各种与水混溶的有机溶剂，如甲醇和二甲基亚砜，浓度为0 - 30%（体积/体积），可激活酶。
ABSTRACT A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from AntarcticMoraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinantEscherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis ofp-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The Km of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn2+, Cu2+, Fe3+, and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.