Expression of the Aspergillus aculeatus endo-β-1,4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme
Expression of the Aspergillus aculeatus endo-β-1,4-mannanase encoding gene (man1) in Saccharomyces cerevisiae and characterization of the recombinant enzyme
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DOI：
10.1006/prep.2000.1371
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发表时间：
2001-02-01
影响因子：
1.6
通讯作者：
Stålbrand, H
中科院分区：
文献类型：
--
作者：
Setati, ME;Ademark, P;Stålbrand, H
根据已发表的棘孢曲霉（Aspergillus aculeatus）的甘露聚糖内切酶基因man 1的序列设计引物，通过聚合酶链反应（PCR）从mRNA中扩增出β-1，4-甘露聚糖内切酶基因man 1。将其克隆到Aculeatus KSM 510中，并分别在乙醇脱氢酶（ADH 2（PT））和磷酸甘油酸激酶（PGK 1（PT））启动子和终止子的基因调控下在酿酒酵母中表达。将该man 1基因产物命名为Man 5A。随后，重组酵母菌株的FUR 1基因被破坏以产生自动选择性菌株：酿酒酵母Man 5ADH 2和酿酒酵母Man 5ADH 2。酿酒酵母Man 5 PGK 1。在复合培养基中生长96小时后，菌株分泌521 nkat/ml和379 nkat/ml的活性Man 5A。这些水平分别相当于产生的118和86 mg/l的Man 5A蛋白。研究了天然和重组Man 5A的性质，发现它们相似。由于糖基化，重组酶的表观分子量为50 kDa，而天然酶的表观分子量为45 kDa。测定的Km和V-max值分别为0.3 mg/ml和82 μ mol/min/mg重组酶和0.15 mg/l和180 μ mol/min/mg天然Man 5A。在pH 4-6和50 ℃及以下的范围内观察到最大pH和热稳定性。重组和天然Man 5A的最适pH和温度以及稳定性相对相似。未支化的β-1，4-连接的甘露聚糖聚合物的水解释放甘露糖、甘露二糖和甘露三糖作为主要产物。(C)北京：科学出版社.
The endo-beta -1,4-mannanase encoding gene man1 of Aspergillus aculeatus MRC11624 was amplified from mRNA by polymerase chain reaction using sequence-specific primers designed from the published sequence of man1 from A. aculeatus KSM510, The amplified fragment was cloned and expressed in Saccharomyces cerevisiae under the gene regulation of the alcohol dehydrogenase (ADH2(PT)) and phosphoglycerate kinase (PGK1(PT)) promoters and terminators, respectively. The man1 gene product was designated Man5A. Subsequently, the FUR1 gene of the recombinant yeast strains was disrupted to create autoselective strains: S, cerevisiae Man5ADH2 and S. cerevisiae Man5PGK1. The strains secreted 521 nkat/ml and 379 nkat/ml of active Man5A after 96 h of growth in a complex medium. These levels were equivalent to 118 and 86 mg/l of Man5A protein produced, respectively, The properties of the native and recombinant Man5A were investigated and found to be similar. The apparent molecular mass of the recombinant enzyme was 50 kDa compared to 45 kDa of the native enzyme due to glycosylation, The determined K-m and V-max values were 0.3 mg/ml and 82 mu mol/min/mg for the recombinant and 0.15 mg/l and 180 mu mol/min/mg for the native Man5A, respectively. The maximum pH and thermal stability were observed within the range of pH 4-6 and 50 degreesC and below. The pH and temperature optima and stability were relatively similar for recombinant and native Man5A. Hydrolysis of an unbranched beta -1,4-linked mannan polymer released mannose, mannobiose, and mannotriose as the main products. (C) 2001 Academic Press.