Studies on structure and activity of recombinant 1,2-α-mannosidase from Aspergillus saitoi

为了构建斋藤曲霉胞内1，2-α-甘露糖苷酶（EC3.2.1.113）基因（msdS）的过表达系统，对斋藤曲霉（Aspergillussaitoi，现命名为A. phoenglycin），该基因的N-末端序列被曲霉胃蛋白酶I（EC 3.4.23.18）基因（apnS）信号的N-末端序列替换，该信号是与先前描述的相同菌株之一。将融合的1，2-α-甘露糖苷酶基因（f-msdS）插入到质粒pNAN 8142（9.5kbp）的P-No 8142和T-agdA之间的NotI位点，构建了Aspergillus sp. AM 1（11.2kbp）表达质粒pNAN AM 1。融合的f-msdS基因在大肠杆菌中过表达。AM 1细胞。重组酶在A.经两步纯化获得均一的细胞。该重组酶在pH5.0时对甲基-2O-α-D-吡喃甘露糖基-α-D-吡喃甘露糖苷有活性。用吡啶胺化（PA）-低聚甘露糖型糖链Man_（GlcNAc）_2-PA分析了该酶的底物特异性<9-6>。酶水解 ...更多信息 在PA糖链中，Man_8GlcNAc_2-PA（M8 A型）水解最快，M6 C型最慢。SDS/PAGE测得该酶的分子量为63 kDa，Superrose 12凝胶过滤测得该酶的分子量为65 kDa。该酶的pI值为4.6。N端氨基酸序列为GSTQSRADAIKAAFSHAWDGYLQY，序列分析表明apnS基因的信号肽被去除。远紫外圆二色性分析表明，酶的二级结构（α-螺旋、β-结构和剩余部分）的含量分别为55%、38%和7%。差示扫描量热法测得该酶的熔点Tm为71 ℃。用原子吸收分光光度计测定每摩尔酶中1g原子的Ca^&lt;2+&gt;，用定点突变法测定在曲霉细胞中表达的酶的活性位点。将Asp-269替换为Asn，Glu-273、Glu-411、Glu-414、Glu-474和Glu-504替换为Gln，可使Man_9-GlcNAc_2-PA和Man_1 - 2 Man-Ome的比活性急剧下降。从目前的结果来看，Asp-269、Glu-273、Glu-414和Glu-474可能是Ca^2+结合配体，而Glu-124和Glu-411可能是1，2-α-甘露糖苷酶的催化残基。少

For the construction of an overexpression system of the intracellular 1,2-α-mannosidase (EC3.2.1.113) gene (msdS) from Aspergillus saitoi(now designated A. phoenicis), the N-terminal sequence of the gene was replaced with that of the aspergillopepsin I (EC3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1,2-α-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 andT-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1(11.2 kbp) was constructed. The fused f-msdS gene has heen overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The recombinant enzyme has activity with methyl-2O-α-D-mannopyranosyl α-D-mannopyranoside at pH 5.0. The substate specificity of the enzyme was analyzed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man_<9-6>(GlcNAc)_2-PA. The enzyme hydr … More olysed Man_8GlcNAc_2-PA (type'M8A') fastest, and 'M6C'slowest, among the PA-sugar chains. Molecular mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superrose 12 respectively. The pl value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. Contents of the secondary structure (α-helix, β-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7 % respectively. The melting temperature, T_m of the enzyme was 71 ℃ by differential scanning calorimetry. Determination of 1 g-atom of Ca^<2+>/mol of enzyme was performed by atomic-absorption spectrophotometer.Active site determination of the enzyme expressed in Aspergillus oryzae cells was performed by site-directed mutagenesis. Substitutions of Asp-269 to Asn and of the Glu-residues, Glu-273, Glu-411, Glu-414, Glu-474 and Glu-504, to Gln altered the drastic decrease of specific activities with Manα1-2Man-Ome and Man_9-GlcNAc_2-PA as substrates. From the present results, Asp-269, Glu-273, Glu-414 and Glu-474 are probably in the Ca^<2+>-binding ligands, whereas Glu-124 and Glu411 and are probably the catalytic residues for the 1,2-α-mannosidase. Less