Saha Badal C
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois 61604, USA.
Biotechnol Prog. 2004 Mar-Apr;20(2):537-42. doi: 10.1021/bp034277p.
Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.
甘露醇2-脱氢酶（MDH）催化依赖吡啶核苷酸将果糖还原为甘露醇。中间乳杆菌（NRRL B-3693）是一种异型发酵乳酸菌（LAB），被发现是甘露醇的优良生产者。该细菌的MDH通过DEAE生物凝胶柱色谱、Bio-Gel A-0.5m凝胶过滤、辛基琼脂糖疏水相互作用色谱和Bio-Gel羟基磷灰石HTP柱色谱从细胞提取物中纯化至同质。纯化后的酶（比活性为331 U/mg蛋白质）是一种异源四聚体蛋白，天然分子量（MW）约为170000，亚基分子量分别为43000和34500。该酶的等电点为pH 4.7。两个亚基具有相同的N端氨基酸序列。纯化后的MDH还原作用的最佳温度为35℃，50℃时活性为44%，60℃时仅为15%。该酶在pH 5.5时活性最佳，在pH 6.5时活性为50%，在pH 5.0时还原果糖的活性仅为35%。将甘露醇氧化为果糖的最佳pH为7.0。纯化后的酶在pH 4.5 - 8.0和温度高达35℃时相当稳定。该酶将果糖还原为甘露醇的K(m)和V(max)值分别为20 mM和396 μmol/min/mg蛋白质。它对葡萄糖、木糖和阿拉伯糖没有任何还原活性。以NADPH为辅因子时，该酶对果糖的活性比以NADH为辅因子时高4.27倍。这是首个来自LAB的高度依赖NADPH的MDH（EC 1.1.1.138）。还介绍了该酶与其他微生物MDH的比较特性。
