Brünker P, Altenbuchner J, Kulbe K D, Mattes R
Institut für Biotechnologie, ETH Zürich, Switzerland.
Biochim Biophys Acta. 1997 Mar 20;1351(1-2):157-67. doi: 10.1016/s0167-4781(96)00189-3.
A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.
从荧光假单胞菌DSM50106中纯化得到了一种依赖NAD的甘露醇脱氢酶（MtlD），并测定了其N端氨基酸序列。根据该肽段序列推导的寡核苷酸用作探针，从荧光假单胞菌的基因组文库中分离出甘露醇脱氢酶基因（mtlD）。对包含整个mtlD基因的1.8 kb NruI片段进行核苷酸序列分析，发现一个1482 bp的开放阅读框，编码一种计算分子量为54.49 kDa的蛋白质。该酶与球形红杆菌的甘露醇脱氢酶以及酿酒酵母的一种假定甘露醇脱氢酶具有高度相似性，氨基酸序列的总体同一性分别为44%和42%，而与大肠杆菌或粪肠球菌的甘露醇-1-磷酸脱氢酶的相似性仅约为23%的相同氨基酸。通过构建诱导表达质粒，大肠杆菌中合成的甘露醇脱氢酶的比活性从0.02 U（mg蛋白）⁻¹提高到10 U（mg蛋白）⁻¹。在mtlD基因的3'端融合六个组氨酸密码子并在大肠杆菌中表达后，活性甘露醇脱氢酶可以通过使用Ni²⁺基质柱的亲和色谱两步法进行纯化。纯化后的酶表现出46 U（mg蛋白）⁻¹的比活性，并且被证明是一种具有广泛底物谱的多元醇脱氢酶，能够高效氧化甘露醇、山梨醇和阿拉伯糖醇。
