The cloned β-mannanase gene from alkalophilic Bacillus sp. AM-001 produces two β-mannanases in Escherichia coli

The gene encoding β-mannanase was cloned from alkalophilic Bacillus sp. AM-001 into Escherichia coli JM 101 by inserting HindIII-generated DNA fragments into the HindIII site of pUC19. A 2.0 kb XbaI-PstI fragment of the donor strain DNA was sufficient for β-mannanase synthesis. The amount of β-mannanase expressed in E. coli JM101 harboring pMAH3 (containing a 2.4 kb XbaI-HindIII fragment) was about 24% of the activity produced by the donor strain. E. coli JM101 harboring pMAH3 was found to produce two enzymatically active β-mannanases (A and B). These two β-mannanases were purified to electrophoretically homogenous states. The β-mannanase A had enzymatic properties similar to those of the β-mannanases M-I and M-II produced by alkalophilic Bacillus sp. AM-001, and the β-mannanase B resembled its β-mannanase M-III. In contrast to β-mannanase production in the donor strain, that in E. coli was not inducible. The NH2-terminal amino acid sequences from amino acid 1 (Asn) to 9 (Gln) of the three β-mannanases purified from alkalophilic Bacillus sp. AM-001 coincide with those from amino acid 4 (Asn) to 12 (Gln) of the two β-mannanases purified from E. coli transformant.