Huei-Fen Lo

Enzymic properties of a SDS-resistant Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli

Abstract A novel alkaline α-amylase of Bacillus sp. TS-23 was purified to homogenous state from the culture medium of recombinant Escherichia coli by ammonium sulphate precipitation and successive Sephacryl TM S-100 and PBE TM 94 chromatography. The molecular mass of the purified enzyme was estimated to be 65 kDa by electrophoresis. The pH and temperature optima for amylase activity were pH 9.0 and 60°C, respectively. The enzyme was stimulated by Mn 2+ , Co 2+ and Fe 2+ ions but was strongly inhibited by Hg 2+ and Cu 2+ and by the well-characterized inhibitors, diethylpyrocarbonate and N -bromosuccinimide. The enzyme was active in the presence of 8% sodium dodecyl sulphate (SDS). Bacillus sp. TS-23 α-amylase was stable when it was preincubated with 6% SDS for upto 1 h at 30°C, while inactivation was observed at 60°C. Under optimal condition, this enzyme was able to attack the α-1,4 linkages in soluble starch, amylose, amylopectin and glycogen to generate maltopentaose as the major end product.

Engineering of a truncated α-amylase of Bacillus sp. strain TS-23 for the simultaneous improvement of thermal and oxidative stabilities

BACDeltaNC/Delta RS is a thermostable variant derived from the truncated alpha-amylase (BAC Delta NC) of alkaliphilic Bacillus sp. strain TS-23. With the aim of enhancing its resistance towards chemical oxidation, Met231 of BAC Delta NC/Delta RS was replaced by leucine to create BAC Delta NC/Delta RS/M231L. The functional significance of the 31 C-terminal residues of BAC Delta NC/Delta RS/M231L was also explored by site-directed mutagenesis of the 483 th codon in the gene to stop codon (TAA), thereon the engineered enzyme was named BAC Delta NC/Delta RS/M231L/Delta C31. BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 were very similar to BAC Delta NC in terms of specific activity, kinetic parameters, pH-activity profile, and the hydrolysis of raw starch; however, the engineered enzymes showed an increased half-life at 70 degrees C. The intrinsic fluorescence and circular dichroism spectra were nearly identical for wild-type and engineered enzymes, but they exhibited a different sensitivity towards GdnHCl-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of mutations. Performance of the engineered enzymes was evaluated in the presence of commonly used detergent compounds and some detergents from the local markets. A high compatibility and performance of both BAC Delta NC/Delta RS/M231L and BAC Delta NC/Delta RS/M231L/Delta C31 may be desirable for their practical uses in the detergent industry.

The N-terminal signal sequence and the last 98 amino acids are not essential for the secretion of Bacillus sp. ts-23 α-amylase in Escherichia coli

A truncated Bacillus sp. TS-23 α-amylase gene lacking 96 and 294 bp at its 5′ and 3′ end respectively was prepared by polymerase chain reaction and cloned into Escherichia coli expression vector, pQE-30, under the control of T5 promoter. SDS-PAGE and activity staining analyses showed that the His6-tagged amylase had a molecular mass of approximately 54 kDa. Isopropyl-β-d-thiogalactopyranoside (IPTG) induction of E. coli M15 cells bearing the recombinant plasmid resulted in the extracellular production of active amylase. Western blot analysis also revealed that the truncated amylase was present in the periplasmic space and culture medium.

Replacement of methionine 208 in a truncated Bacillus sp. TS-23 alpha-amylase with oxidation-resistant leucine enhances its resistance to hydrogen peroxide.

The methionine residues at positions 17, 104, 208, 214, 292, 315, 324, and 446 in the primary amino acid sequence of a truncated Bacillus sp. TS-23 α-amylase (His6-tagged BLAΔNC) was changed to oxidative-resistant leucine by site-directed mutagenesis. The mutant enzymes with an apparent molecular mass of approximately 54 kDa were overexpressed in recombinant Escherichia coli. The specific activity for Met315Leu and Met446Leu was decreased by more than 76%, while Met17Leu, Met104Leu, Met208Leu, Met214Leu, Met292Leu, and Met324Leu showed 247, 128, 37, 260, 232, and 241%, respectively, higher activity than the wild-type enzyme. In comparison with wild-type enzyme, a lower Km value was observed for all mutant enzymes. The 3.2- and 4.5-fold increases in the catalytic efficiency (kcat/Km) for Met208Leu and Met324Leu, respectively, were partly contributed by a 68% and 38% decrease in Km values. Wild-type enzyme was sensitive to chemical oxidation, but Met208Leu was stable even in the presence of 500 mM H2O2. Except for Met214Leu, which was quite sensitive to H2O2, the other mutants showed a profile of oxidative inactivation similar to that of the wild-type enzyme. These observations indicate that the oxidative stability of His6-tagged BLAΔNC can be improved by replacement of the critical methionine residue with leucine.

Erratum to: Role of the Conserved Thr399 and Thr417 Residues of Bacillus licheniformis γ-Glutamyltranspeptidase as Evaluated by Mutational Analysis

Role of the conserved Thr399 and Thr417 residues of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was investigated by site-directed mutagenesis. Substitutions of Thr399 and Thr417 of BlGGT with Ser resulted in a dramatic reduction in enzymatic activity. A complete loss of the GGT activity was observed in T399A, T399C, T417A, and T417K mutant enzymes. Furthermore, mutations on these two residues impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments showed that BlGGT mutant precursors, pro-T399S, pro-T417S, and pro-T417A, could precede a time-dependent autocatalytic process to generate the 44.9- and 21.7-kDa subunits; however, the processed T417A had no enzymatic activity. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while Far-UV circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results suggest that residues Thr399 and Thr417 are important for BlGGT in the enzymatic maturation and reaction.

Biophysical characterization of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases: A comparative analysis

The oligomeric states of Bacillus licheniformis and Escherichia coli γ-glutamyltranspeptidases (BlGGT and EcGGT) in solution have been investigated by analytical ultracentrifugation. The results showed that BlGGT has a sedimentation coefficient of 5.12S, which can be transformed into an experimental molecular mass of approximately 62,680Da. The monomeric conformation is conserved in EcGGT. SDS-PAGE analysis and cross-linking studies further proved that the autocatalytically processed BlGGT and EcGGT form a heterodimeric association. Unfolding analyses using circular dichroism and tryptophan emission fluorescence revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and EcGGT had a T(m) value of 59.5 and 49.2°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Chemical unfolding of BlGGT was independent to the pH value ranging from 5 to 10, whereas the pH environment was found to significantly influence the GdnHCl-induced denaturation of EcGGT. Both enzymes did not reactivate from the completely unfolded states, accessible at 6M GdnHCl. BlGGT was active in the presence of 4M NaCl, whereas the activity of EcGGT was significantly decreased at the high-salt condition. Taken together, these findings suggest that the biophysical properties of the homologous GGTs from two mesophilic sources are quite different.

Enzymatic synthesis of γ-l-glutamyl-S-allyl-l-cysteine, a naturally occurring organosulfur compound from garlic, by Bacillus licheniformis γ-glutamyltranspeptidase

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we describe a straightforward enzymatic synthesis of γ-L-glutamyl-S-allyl-L-cysteine (GSAC), a naturally occurring organosulfur compound found in garlic, based on a transpeptidation reaction involving glutamine as the γ-glutamyl donor and S-allyl-L-cysteine as the acceptor. With the help of thin layer chromatography technique and computer-assisted image analysis, we performed the quantitative determination of GSAC. The optimum conditions for a biocatalyzed synthesis of GSAC were 200 mM glutamine, 200 mM S-allyl-L-cysteine, 50 mM Tris-HCl buffer (pH 9.0), and BlGGT at a final concentration of 1.0 U/mL. After a 15-h incubation of the reaction mixture at 60 °C, the GSAC yield for the free and immobilized enzymes was 19.3% and 18.3%, respectively. The enzymatic synthesis of GSAC was repeated under optimal conditions at 1-mmol preparative level. The reaction products together with the commercially available GSAC were further subjected to an ESI-MS/MS analysis. A significant signal with m/z of 291.1 and the protonated fragments at m/z of 73.0, 130.1, 145.0, and 162.1 were observed in the positive ESI-MS/MS spectrum, which is consistent with those of the standard compound. These results confirm the successful synthesis of GSAC from glutamine and S-allyl-L-cysteine by BlGGT.

Isolation of a Recombinant Bacillus sp. TS‐23 α‐Amylase by Adsorption‐Elution on Raw Starch

A Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli was adsorbed onto raw starch and the adsorbed enzyme was eluted with maltose or maltodextrin in 50 mM Tris/HCI buffer (pH 8.5). The adsorption-elution procedure resulted in a yield of 53% α-amylase activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) analysis showed that the eluted α-amylase had a molecular mass of approximately 64 kDa. Raw starch could be used repeatedly in the adsorption-elution cycle with good reproducibility. Scanning electron microscopy of the isolated corn starch exhibited a smooth appearance of the granules before adsorption and only a small change in appearance after three adsorption-elution cycles. These results suggest that the raw starch adsorption-elution technique has a great potential in the isolation of Bacillus sp. TS-23 α-amylase from the culture broth of recombinant E. coli.

Experimental evidence for the involvement of amino acid residue Glu398 in the autocatalytic processing of Bacillus licheniformis γ-glutamyltranspeptidase

The role of glutamate 398 in the autocatalytic processing of Bacillus licheniformis γ‐glutamyltranspeptidase (BlGGT) was explored by site‐directed mutagenesis. This glutamate was substituted by either alanine, aspartate, arginine or glutamine and the expressed mutant enzymes were purified to apparent homogeneity with metal‐affinity chromatography. SDS–PAGE analysis showed that E398A, E398D and E398K were unable to process themselves into a large and a small subunit. However, E398Q was not only able to process itself, but also had a catalytic activity comparable to that of BlGGT. As compared with the wild‐type enzyme, no significant change in circular dichroism spectra was observed for the mutant proteins. Thermal unfolding of BlGGT, E398A, E398D, E398K and E398Q followed the two‐state unfolding process with a transition point (T m) of 47.7–69.4 °C. Tryptophan fluorescence spectra of the mutant enzymes were different from the wild‐type protein in terms of fluorescence intensity. Native BlGGT started to unfold beyond ∼1.92 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]0.5, N–U, at 3.07 M equivalent to free energy change ( Δ ⁢ G N − U H 2 ⁢ O ) of 14.53 kcal/mol for the N → U process, whereas the denaturation midpoints for the mutant enzymes were 1.31–2.99 M equivalent to Δ ⁢ G N − U H 2 ⁢ O of 3.29–12.05 kcal/mol. Taken together, these results strongly suggest that the explored glutamate residue is indeed important for the autocatalytic processing of BlGGT.

Cobalt-chelated magnetic particles for one-step purification and immobilization of His6-tagged Escherichia coli γ-glutamyltranspeptidase.

Cobalt-chelated magnetic (Fe3O4-AA-ANTA-Co2 +) particles were prepared and one-step purification and immobilization of His6-tagged Escherichia coli γ-glutamyltranspeptidase (His6-EcGGT) using these particles were evaluated. The optimal conditions for the adsorption of His6-EcGGT to Fe3O4-AA-ANTA-Co2 + particles were found to be 24.7 U g–1 adsorbent, pH 6.5, 300 mM NaCl and 30 min incubation at 4°C, while the elution solution was optimized to be 50 mM phosphate buffer (pH 6.5) containing 150 mM imidazole and 300 mM NaCl. The immobilized His6-EcGGT was recycled five times without significant loss of GGT activity. The average yield rate for the synthesis l-theanine from glutamine and ethylamine reached 56.7%. These results indicate that one-step affinity purification and immobilization of His6-EcGGT by Fe3O4-AA-ANTA-Co2 + particles might serve as an effective process for industrial application.