Abstract:

In order to obtain novel β-mannanases with excellent performance, two putative novel GH5 family β-mannanase
genes were excavated from the Aspergillus oryzae RIB40 genome by genome mining, named as Aoman5A and Aoman5B,
respectively. The bioinformatic analysis of the two gene sequences was conducted using corresponding softwares or web
server, and the genes encoding two mature peptides were expressed in Pichia pastoris GS115 with the aid of plasmid
pPIC9KM, and then the expressed products were purified and identified. The results of bioinformatic analysis showed
that AoMan5A contained a signal peptide with 20 amino acid residues, while AoMan5B contained a signal peptide with
21 amino acid residues and a propeptide with 12 amino acid residues. The results of sequence alignment displayed that
the sequences of two enzymes had the highest similarity of 68% and 79% with the reported sequences, and the N-terminal
of AoMan5A also carried a GH1 family CBM. The results of 3-D structure prediction showed that both of them were
in accordance with the (α/β)8 TIM-barrel structure. Under the same expression condition, the enzymes activities in
supernatants from reAoMan5A and reAoMan5B were 2.9 and 12.5 IU/mL, respectively, with specific activity of 8.3
and 104.2 IU/mg, respectively, after purification. The optimum temperature for the former was 35 ℃, while that for
the later was 50 ℃. It turned out that both of them were acidic enzymes. The carbohydrate contents of reAoMan5A
and reAoMan5B were determined to be 25.4% and 12.6% using the phenol sulfuric acid method indicating both to
be glycosylated. This study will lay a solid foundation for further research and application of β-mannanases, and also
provide a referential guidance for the gene mining of other novel enzymes.

Key words: β-mannanase, genome mining, bioinformatic analysis, Aspergillus oryzae, protein expression