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

为了构建saiitoi曲霉胞内1,2-α-甘露醇苷酶（EC3.2.1.113）基因（msdS）过表达系统，将该基因的n端序列替换为与上述菌株相同的aspergillopepsin I （EC3.4.23.18）基因（apnS）信号序列。将融合的1,2-α-甘露糖苷酶基因（f-msdS）插入到pNAN 8142质粒（9.5 kbp） P-No8142和t - agda之间的NotI位点，构建了米曲霉表达质粒pNAN- am1 （11.2 kbp）。融合的f-msdS基因在转化的m.o ryzae niaD AM1细胞中过度表达。重组酶在m.o ryzae细胞中表达，分两步纯化至均匀性。重组酶在pH 5.0下对甲基- 20 -α- d -甘露糖吡喃糖基α- d -甘露糖吡喃苷具有活性。利用pyridylaminated (PA)-寡甘露糖型糖链Man_<9-6>(GlcNAc)_2-PA分析酶的底物特异性。在pa -糖链中，酶水解Man_8GlcNAc_2-PA （‘ m8a ’型）的速度最快，而水解‘M6C’型的速度最慢。经SDS/PAGE和Superrose 12凝胶过滤测定，酶的分子量分别为63 kDa和65 kDa。该酶的pl值为4.6。该酶的n端氨基酸序列为GSTQSRADAIKAAFSHAWDGYLQY，序列分析表明该酶去除了apnS基因的信号肽。远紫外CD法测定的二级结构（α-螺旋结构、β-结构和酶的剩余部分）含量分别为55.8%、38%和7%。差示扫描量热法测定酶的熔化温度T_m为71℃。用原子吸收分光光度计测定酶中Ca^<2+>/mol的1 g原子。该酶在米曲霉细胞中表达的活性位点测定采用定点诱变法。以Manα1-2Man-Ome和Man_9-GlcNAc_2-PA为底物，将Asp-269替换为Asn，将Glu-273、Glu-411、Glu-414、Glu-474和Glu-504替换为Gln，使特异性活性急剧下降。从目前的研究结果来看，Asp-269、Glu-273、Glu-414和Glu-474可能是Ca^<2+>-结合配体，而Glu-124和glu - 411和可能是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