title: Improving the acidic stability of Staphylococcus aureus α-acetolactate decarboxylase in Bacillus subtilis by changing basic residues to acidic residues.
abstract: The α-acetolactate decarboxylase (ALDC) can reduce diacetyl fleetly to promote mature beer. A safe strain Bacillus subtilis WB600 for high-yield production of ALDC was constructed with the ALDC gene saald from Staphylococcus aureus L3-15. SDS-PAGE analysis revealed that S. aureus α-acetolactate decarboxylase (SaALDC) was successfully expressed in recombinant B. siutilis strain. The enzyme SaALDC was purified using Ni-affinity chromatography and showed a maximum activity at 45 °C and pH 6.0. The values of K m and V max were 17.7 μM and 2.06 mM min(-1), respectively. Due to the unstable property of SaALDC at low pH conditions that needed in brewing process, site-directed mutagenesis was proposed for improving the acidic stability of SaALDC. Homology comparative modeling analysis showed that the mutation (K52D) gave rise to the negative-electrostatic potential on the surface of protein while the numbers of hydrogen bonds between the mutation site (N43D) and the around residues increased. Taken together the effect of mutation N43D-K52D, recombinant SaALDCN43D-K52D showed dramatically improved acidic stability with prolonged half-life of 3.5 h (compared to the WT of 1.5 h) at pH 4.0. In a 5-L fermenter, the recombinant B. subtilis strain that could over-express SaALDCN43D-K52D exhibited a high yield of 135.8 U mL(-1) of SaALDC activity, about 320 times higher comparing to 0.42 U mL(-1) of S. aureus L3-15. This work proposed a  strategy for improving the acidic stability of SaALDC in the  B. subtilis host.
authors.affiliation: The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors.forename: Xian
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Rao
authors.forename: Jingjing
authors.initials: J
authors.lastname: Li
authors.forename: Junping
authors.initials: J
authors.lastname: Zhou
authors.forename: Taowei
authors.initials: T
authors.lastname: Yang
authors.forename: Meijuan
authors.initials: M
authors.lastname: Xu
authors.forename: Teng
authors.initials: T
authors.lastname: Bao
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Zhao
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase