title: [Correlation between superior enzymatic properties of β-mannanase AuMan5A/Af and its residue Asp(320)].
abstract: OBJECTIVE
  AuMan5A is a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii. To improve its enzymatic properties, we have previously constructed a mutant with loop substitution, AuMan5A/Af, by substituting a loop of seven residues (316KSPDGGN322) in its substrate binding groove with the corresponding region (PSPNDHF) of A. fumigatus GH family 5 β-mannanase. To reveal the correlation between the superior enzymatic properties of AuMan5A/Af and its residue Asp320, site-directed mutagenesis was used to obtain a new mutant enzyme AuMan5A/Af(D320G).
  
  
  METHODS
  Using megaprimer PCR method, we constructed a new mutant-encoding gene, Auman5A/Af(D320G) by mutating an Asp320 -encoding codon GAC of Auman5A/Af into a Gly320 -encoding GGT. Then, Auman5A/Af(D320G) was extracellularly expressed in Pichia pastoris GS115, and the enzymatic properties of the expressed product were analyzed.
  
  
  RESULTS
  Analytical results indicated that the optimal and melting temperature of AuMan5A/Af(D320G) was 70.0 degrees C and 71.5 degrees C, repectively, higher than those of AuMan5A (T(opt) = 65.0 degrees C, T(m) = 64.5 degrees C) and lower than those of AuMan5A/Af (T(opt) = 75.0 degrees C, T(m) =76.6 degrees C); its half-life at 70.0 degrees C was 40 min, 10 min longer than that of AuMan5A but greatly shorter than 480 min of AuMan5A/Af. Besides, its specific activity was 2.7 fold and 0.3 fold that of AuMan5A and AuMan5A/Af, respectively, and its catalytic efficiency (k(cat)/K(m)) was 3.9 fold and 0.3 fold that of AuMan5A and AuMan5A/Af.
  
  
  CONCLUSIONS
  The mutation of ASP320 into Gly320 greatly affected the temperature characteristics and catalytic activity of AuMan5A/Af, demonstrating that Asp320 plays an improtant role in temperature characteristics, specific activity and catalytic efficiency improving of AuMan5A after loop substitution.
authors.forename: Jianfang
authors.initials: J
authors.lastname: Li
authors.forename: Yunhai
authors.initials: Y
authors.lastname: Dong
authors.forename: Die
authors.initials: D
authors.lastname: Hu
authors.forename: Chunjuan
authors.initials: C
authors.lastname: Wang
authors.forename: Shihan
authors.initials: S
authors.lastname: Tang
authors.forename: Minchen
authors.initials: M
authors.lastname: Wu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001230:Aspergillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D016415:Sequence Alignment; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D001224:Aspartic Acid; D044902:beta-Mannosidase