Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

Abstract

The starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70 degrees C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K (m) value. Starch-binding assays showed that the K (d) and B (max) values for the fusion enzyme were 2.3 microM and 0.35 micromol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris-HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption-elution cycle with a purification of 11.4-fold.

Novel Design of an α-Amylase with an N-Terminal CBM20 in Aspergillus niger Improves Binding and Processing of a Broad Range of Starches

“…As expected, the GH13 without CBM has a very low binding capacity, of only about 10%. The fact that not all of the enzyme was retained by starch binding may be explained by the fact that during binding the electrostatic interaction of the enzyme and the starch adsorbent influences the adsorption process as reported for the amylase purification from Bacillus [ 63 , 64 ].…”

Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs)

“…alpha-amylase. The chimeric enzyme and adsorbed raw starch, showing a increase in thermostability and catalytic efficiency [35]; whereas another leucin aminopeptidase from Bacillus kaustophilus fused to an alpha-amylase SBD showed a decrease in catalytic efficiency due to the increase of the K m value [36], indicating that in some cases, the chimeric proteins do not produce the functions expected from the original enzymes, possibly due to a different polypeptide-folding pattern [36]. Thus, although there are several examples to date of hybrid functional proteins between CBMs and a catalytic core, most of them involves CBMs from hydrolytic enzymes linked to bacterial xylanases or amylases catalytic domains [37e39].…”

Role of the invariant Asn345 and Asn435 residues in a leucine aminopeptidase from Bacillus kaustophilus as evaluated by site-directed mutagenesis

“…Expression and purification of all recombinant proteins were conducted according to the procedures described by Huang et al [26]. Briefly, E. coli M15 cells harboring pQE-BkLAP or each of the mutated plasmids were cultivated in 100 ml of LB media containing 100 g/ml ampicillin and 25 g/ml kanamycin to A 600 of 0.6 and then induced with 1 mM isopropyl ␤-d-thiogalactopyranoside (IPTG).…”