Cloning, expression, and purification of a recombinant cold-adapted β-galactosidase from antarctic bacterium Pseudoalteromonas sp 22b
Cloning, expression, and purification of a recombinant cold-adapted β-galactosidase from antarctic bacterium Pseudoalteromonas sp 22b
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DOI：
10.1016/j.pep.2004.09.002
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发表时间：
2005-01-01
影响因子：
1.6
通讯作者：
Turkiewicz, M
中科院分区：
文献类型：
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作者：
Cieslinski, H;Kur, J;Turkiewicz, M
革兰氏阴性菌南极假交替单胞菌。22b是从磷虾Thyssanoessa macrura的消化道中分离出来的，它能合成一种胞内冷适应的β-半乳糖苷酶。编码该β-半乳糖苷酶的基因已被聚合酶链式反应扩增、克隆、在大肠杆菌中表达、纯化和鉴定。该酶是一种同源四聚体蛋白，每个单体由1028个氨基酸残基组成。采用亲和层析油PABTG-Sepharose两步法对酶进行了纯化，纯化后的酶纯度为50%。重组蛋白的酶学性质与天然假单胞菌相同。22bβ-半乳糖苷酶。该酶是冷适应的，在10℃时保持了20%的最大活性。纯化后酶的最大活性接近40℃，最适pH为6.0~8.0。PNPG是其首选底物(活性比ONPG高58%)。该酶特别不耐热，在50℃下10分钟内失去所有活性。乳糖的测定结果表明，乳糖在30℃下6h和15℃下28h内分别有90%的乳糖被水解。由于其特性，重组假交替单胞菌属具有较高的生物学活性。22bβ-半乳糖苷酶可以在冷藏温度下应用于低乳糖乳制品的生产。(C)2004 Elsevier Inc.保留所有权利。
The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase. The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography oil PABTG-Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Psendoalteromonas sp. 22b beta-galactosidase. The enzyme is cold-adapted and at 10degreesC retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40degreesC and at pH of 6.0-8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10min at 50degreesC. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30degreesC and during 28 h at 15degreesC. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products. (C) 2004 Elsevier Inc. All rights reserved.