Molecular and enzymic properties of recombinant 1,2-alpha-mannosidase fromAspergillus saitoi overexpressed in Aspergillus oryzae cells

For the construction of an overexpression system of the intracellular 1,2-a lpha-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now de signated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS ) signal, one of the same strains as described previously. Then the fused 1 ,2-alpha-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergi llus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fus ed f-msdS gene has been overexpressed in a transformant A. oryzae niaD AMI cell. The recombinant enzyme expressed in A. oryzae cells was purified to h omogeneity in two steps. The system is capable of making as much as about 3 20 mg of the enzyme/litre of culture. The recombinant enzyme has activity w ith methyl-2-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside at pH 5.0, wh ile no activity was determined with methyl-3-O-alpha-D-mannopyranosyl a-D-m annopyranoside or methyl-6-O-alpha-D-mannopyranosyl alpha-D-mannopyranoside . The substrate specificity of the enzyme was analysed by using pyridylamin ated (PA)-oligomannose-type sugar chains, Man(9-6)(GlcNAc)(2)-PA (Man is ma nnose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man(8)GlcNAc(2 )-PA (type' M8A ') fastest, and' M6C' {Man alpha 1-3[Man alpha 1-2Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNac-PA} slow est, among the PA-sugar chains. Molecular-mass values of the enzyme were de termined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pi value of the enzyme was 4.6. The N-terminal amino a cid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analy sis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, epsilon, at 280 nm was determined as 91539 M-1 . c m(-1). Contents of the secondary structure (alpha-helix, beta-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 deg rees C: by differential scanning calorimetry. The calorimetric enthalpy, De lta H-cal, of the enzyme was calculated as 13.3 kJ . kg of protein(-1). Det ermination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorp tion spectrophotometry.