NDLI: Improving the acidic stability of Staphylococcus aureus α-acetolactate decarboxylase in Bacillus subtilis by changing basic residues to acidic residues

Content Provider	Springer Nature Link
Author	Zhang, Xian Rao, Zhiming Li, Jingjing Zhou, Junping Yang, Taowei Xu, Meijuan Bao, Teng Zhao, Xiaojing
Copyright Year	2014
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.
Starting Page	707
Ending Page	717
Page Count	11
File Format	PDF
ISSN	09394451
Journal	Amino Acids
Volume Number	47
Issue Number	4
e-ISSN	14382199
Language	English
Publisher	Springer Vienna
Publisher Date	2014-12-28
Publisher Place	Vienna
Access Restriction	Subscribed
Subject Keyword	α-Acetolactate decarboxylase Diacetyl Acidic stability Site-directed mutagenesis Bacillus subtilis Biochemistry Analytical Chemistry Biochemical Engineering Life Sciences Proteomics Neurobiology
Content Type	Text
Resource Type	Article
Subject	Organic Chemistry Biochemistry Clinical Biochemistry

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