Enzymatic characterization of Bacillus licheniformis γ-glutamyltranspeptidase fused with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) was fused at its C-terminal end with N-terminally truncated forms of Bacillus sp. TS-23 α-amylase. BlGGT and six fusion enzymes, BlGGT/SBD, BlGGT/AMYΔN476, BlGGT/AMYΔN443, BlGGT/AMYΔN376, BlGGT/AMYΔN195, and BlGGT/AMYΔN34, were over-expressed in Escherichia coli M15 cells and purified to apparent homogeneity by metal-affinity chromatography. The fusion constructions had no significant effect on the autocatalytic processing of BlGGT. Progressive decrease in the GGT activity of fusion proteins was associated with an increasing level of truncation, and only BlGGT/AMYΔN34 reserved the amylolytic activity. The protein fusions did not alter the optimal temperature and pH of BlGGT. However, as compared with the parental BlGGT, a significant change in circular dichorism and fluorescence spectra was observed in the fusion enzymes. Thermal unfolding of BlGGT, BlGGT/AMYΔN476, BlGGT/AMYΔN443, and BlGGT/AMYΔN376 followed the two-state unfolding process with a transition point (T(m)) of 61.3-63.1 °C, whereas BlGGT/AMYΔN195 and BlGGT/AMYΔN34 displayed two temperature transitions at 40.6 and 46.7 °C as well as at 62.8 and 62.9 °C, respectively. All of the fusion enzymes exhibited the raw-starch-binding ability, and the adsorbed proteins could be eluted from the adsorbent by 50mM Tris-HCl (pH 9.0) containing 2% soluble starch.     

Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k _cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.     

Sorbitol counteracts temperature- and chemical-induced denaturation of a recombinant α-amylase from alkaliphilic Bacillus sp. TS-23

Enzymes are highly complex systems with a substantial degree of structural variability in their folded state. In the presence of cosolvents, fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways; alternatively, certain conformations can be stabilized or destabilized. To understand the contribution of osmolytes to the stabilization of structural changes and enzymatic activity of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC), we monitored amylolytic activity, circular dichroism, and fluorescence as a function of osmolytes. In the presence of trimethylamine N-oxide (TMAO) and sorbitol, BACΔNC activity was retained significantly at elevated temperatures. As compared to the control, the secondary structures of this enzyme were essentially conserved upon the addition of these two kinds of osmolytes. Fluorescence results revealed that the temperature-induced conformational change of BACΔNC was prevented by TMAO and sorbitol. However, glycerol did not provide profound protection against thermal denaturation of the enzyme. Sorbitol was further found to counteract guanidine hydrochloride- and SDS-induced denaturation of BACΔNC. Thus, some well-known naturally occurring osmolytes make a dominant contribution to the stabilization of BACΔNC.     

Rapid and simple purification of a novel extracellular β-amylase from Bacillus sp.

Bacillus sp. KYJ 963, a local isolate, produced an extracellular amylase with M _r=59 kDa. The amylase was easily purified by adsorption on soluble starch. The analyses of TLC and N-terminal amino acid sequence from the purified protein revealed that the enzyme was a novel -amylase which could not hydrolyze maltose or -cyclodextrin and its N-terminal amino acid sequence was A-V-N-G-Q-S-F-N-S-N-Y-K-T-Y-K-.     

Extensive hydrolysis of raw rice starch by a chimeric α-amylase engineered with α-amylase (AmyP) and a starch-binding domain from Cryptococcus sp. S-2

Recombinant chimeric α-amylase (AmyP-Cr) was constructed by a catalytic core of α-amylase (AmyP) from a marine metagenomic library and a starch-binding domain (SBD_Cr) of α-amylase from Cryptococcus sp. S-2. The molecular fusion did not alter optimum pH, optimum temperature, hydrolysis products, and an ability of preferential and rapid degradation towards raw rice starch, but catalytic efficiency and thermostability were remarkably improved compared with those of the wild-type AmyP. AmyP-Cr achieved the final hydrolysis degree of 61.7 ± 1.2% for 10% raw rice starch and 47.3 ± 0.8% for 15% raw rice starch after 4 h at 40 °C with 1.0 U per mg of raw starch. The catalytic efficiency was very high, with 3.6–4.0 times higher than that of AmyP. The enhanced catalytic efficiency was attributed to the better thermostability and the higher adsorption and disruption to raw rice starch caused by SBD_Cr. The properties of AmyP-Cr open a new way in terms of a new design of raw rice starch processing.

     

Fusion of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis> leucine aminopeptidase II with the raw-starch-binding domain of <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain TS-23 α-amylase generates a chimeric enzyme with enhanced thermostability and catalytic activity

Bacillus stearothermophilus leucine aminopeptidase II (LAPII) was fused at its C-terminal end with the raw-starch-binding domain of Bacillus sp. strain TS-23 -amylase. The chimeric enzyme (LAPsbd), with an apparent molecular mass of approximately 61 kDa, was overexpressed in IPTG-induced Escherichia coli cells and purified to homogeneity by nickel-chelate chromatography. The purified enzyme retained LAP activity and adsorbed raw starch. LAPsbd was stable at 70°C for 10 min, while the activity of wild-type enzyme was completely abolished under the same environmental condition. Compared with the wild-type enzyme, the twofold increase in the catalytic efficiency for LAPsbd was due to a 218% increase in the k _cat value.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Identification of Glutamate Residues Important for Catalytic Activity or Thermostability of a Truncated Bacillus sp. Strain TS-23 α-amylase by Site-directed Mutagenesis

The importance of 17 glutamate residues of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was investigated by site-directed mutagenesis. The Ala- and Asp-substituted variants were overexpressed in the recombinant E. coli cells and the 54-kDa proteins were purified to nearly homologous by nickel-chelate chromatography. Glu-295, which locates in the conserved region III of amylolytic enzymes, mutations resulted in a complete loss of enzyme activity. The specific activity for E151A was decreased by more than 30%, while other variants showed activity comparable to that of BACΔNC. A decreased half-life at 70°C was observed for Glu-219 variants with respective to the wild-type enzyme, suggesting that replacement of Glu-219 by either Ala or Asp might have a significant destabilizing effect on the protein structure.     

Effects of salt and ligand concentrations on the thermal unfolding and refolding of halophilic starch-binding domain from Kocuria varians α-amylase

The starch binding domain of α-amlylase from moderate halophile was expressed in E. coli with His tag (His- SBD12) and characterized for its halophilic properties. His-SBD12 was stable up to 35°C and showed binding activity, although at reduced level, to amylose even in the absence of NaCl. Both NaCl and specific ligands exhibited insignificant influence on the secondary structure of His-SBD12, but showed significant stabilization effects against thermal unfolding concentration-dependently, showing its halophilic properties. NaCl increased thermal stability of His-SBD12 by 4°C at 0.2 M and 15°C at 2 M, and enhanced refolding rate by ~7-fold at 0.2 M and ~170-fold at 2 M. Its specific ligands, β- cyclodextrin (at 3 mM) and maltose (at 470 mM), also stabilized the protein by 11° C, most likely reflecting affinity difference between these two ligands. However, they showed marginal effects on refolding rate. These observations suggest that although binding of NaCl and specific ligands to the native structure can explain their stabilization effects on His- SBD12, it is not a sole factor for modulating their effects on folding of His-SBD12.     

Purification and characterization of a liquefying α-amylase from alkalophilic thermophilic Bacillus sp. AAH-31

α-Amylase (EC 3.2.1.1) hydrolyzes an internal α-1,4-glucosidic linkage of starch and related glucans. Alkalophilic liquefying enzymes from Bacillus species are utilized as additives in dishwashing and laundry detergents. In this study, we found that Bacillus sp. AAH-31, isolated from soil, produced an alkalophilic liquefying α-amylase with high thermostability. Extracellular α-amylase from Bacillus sp. AAH-31 (AmyL) was purified in seven steps. The purified enzyme showed a single band of 91 kDa on SDS-PAGE. Its specific activity of hydrolysis of 0.5% soluble starch was 16.7 U/mg. Its optimum pH and temperature were 8.5 and 70 °C respectively. It was stable in a pH range of 6.4-10.3 and below 60 °C. The calcium ion did not affect its thermostability, unlike typical α-amylases. It showed 84.9% of residual activity after incubation in the presence of 0.1% w/v of EDTA at 60 °C for 1 h. Other chelating reagents (nitrilotriacetic acid and tripolyphosphate) did not affect the activity at all. AmyL was fully stable in 1% w/v of Tween 20, Tween 80, and Triton X-100, and 0.1% w/v of SDS and commercial detergents. It showed higher activity towards amylose than towards amylopectin or glycogen. Its hydrolytic activity towards γ-cyclodextin was as high as towards short-chain amylose. Maltotriose was its minimum substrate, and maltose and maltotriose accumulated in the hydrolysis of maltooligosaccharides longer than maltotriose and soluble starch.     

Thermostable mutant variants of Bacillus sp. 406 α-amylase generated by site-directed mutagenesis

Several mutations are known to increase the thermostability of α-amylase of B. licheniformis and other α-amylases. Site-directed mutagenesis was used to introduce similar mutations into the sequence of the α-amylase gene from mesophilic Bacillus sp. 406. The influence of the mutations on thermostability of the enzyme was studied. It was shown that the Gly211Val and Asn192Phe substitutions increased the half-inactivation temperature (T_m) of the enzyme from 51.94±0.45 to 55.51±0.59 and 58.84±0.68°C respectively, in comparison to the wild-type enzyme. The deletion of Arg178-Gly179 (dRG) resulted in an increase of T_m of the α-amylase to 71.7±1.73°C. The stabilising effect of mutations was additive. When combined they increase the T_m of the wild-type amylase by more than 26°C. Thermostability rates of the triple mutant are close to the values which are typical for industrial heat-stable α-amylases, and its ability to degrade starch at 75°C was considerably increased. The present research confirmed that the Gly211Val, Asn192Phe and dRG mutations could play a significant role in thermostabilization of both mesophilic and thermophilic α-amylases.     

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. “FVHLFEW” was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.     

Effect of polyethylene glycol on Bacillus subtilis α-amylase purification with raw and modified starch

Polyethylene glycol was found to enhance adsorption of Bacillus subtilis -amylase on starch in optimum concentration 10 % (w/w). Degree of adsorption at 12°C was increased from 83 to 98 % and from 30 to 81 % for cross-linked and raw starch, resp. Higher sorption capacity and easy desorption of -amylase without temperature or pH change was reached at 22 °C. Yield of -amylase 95 % and purification factor 8.3 were achieved on the cross-linked starch column. The method is suitable for -amylase isolation from PEG phase after its microbial production in aqueous two-phase systems.     

Construction of an amylolytic industrial strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis α-amylase gene

The gene encoding Schwanniomyces occidentalis -amylase (AMY) was introduced into the chromosomal sequences of an industrial strain of Saccharomyces cerevisiae. To obtain a strain suitable for commercial use, an -integrative cassette devoid of bacterial DNA sequences was constructed that contains the AMY gene and aureobasidin A resistance gene (AUR1-C) as the selection marker. The AMY gene was expressed under the control of the alcohol dehydrogenase gene promoter (ADC1p). The -amylase activity of Sacc. cerevisiae transformed with this integrative cassette was 6 times higher than that of Sch. occidentalis. The transformants (integrants) were mitotically stable after 100 generations in nonselective medium.     

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. Received: 23 December 2000/Accepted: 26 January 2001     

The potential of brewer's spent grain to improve the production of α-amylase by Bacillus sp. KR-8104 in submerged fermentation system

Brewer's spent grain (BSG) was used as a solid substrate for the production of α-amylase by Bacillus sp. KR-8104 in a submerged fermentation system. The production of α-amylase was maximized through statistical optimization of the BSG concentration and incubation time using the Doehlert experimental design. The highest tested amount of BSG (5%, w/v) in the optimization process resulted in a 5.1-fold enhancement of the response. Subsequently, we studied the role of the water-soluble and -insoluble fractions of BSG in the production of α-amylase. The results revealed that whole BSG had a greater effect on the production of α-amylase than each fraction had separately. Finally, when we examined the potential of BSG to replace the constituents of a medium formula, we observed that simultaneously adding BSG, omitting dextrin, and reducing the other ingredients concentration in the culture medium improved the production of α-amylase and made the production process more economical.     

Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp. strain: purification and properties

The purpose of this study was purification and characterization of catechol 1,2-dioxygenase from Geobacillus sp. G27 strain, which degrades α-naphthol by the β-ketoadipate pathway. The catechol 1,2-dioxygenase (C1,2O) was purified using four steps of ammonium sulfate precipitation, DEAE-celullose, Sephadex G-150 and hydroxylapatite chromatographies. The enzyme was purified about 18-fold with a specific activity of 7.42 U mg of protein⁻¹. The relative molecular mass of the native enzyme estimated on gel chromatography of Sephadex G-150 was 96 kDa. The pH and temperature optima for enzyme activity were 7 and 60°C, respectively. A half-life of the catechol 1,2-dioxygenase at the optimum temperature was 40 min. The kinetic parameters of the Geobacillus sp. G27 strain catechol 1,2-dioxygenase were determined. The enzyme had apparent K_m of 29 μM for catechol and the cleavage activities for methylcatechols were much less than for catechol and no activity with gentisate or protocatechuate was detected.     

Purification and properties of the raw starch-degrading α- amylase of Bacillus sp. IMD 434

The raw starch-degrading a-amylase of Bacillus sp. IMD 434 was purified to homogeneity by acetone precipitation, ion- exchange chromatography and hydrophobic interaction chromatography. The enzyme had a relative molecular mass of 69,200, displayed maximum activity at pH 6.0 and 65°C and released large amounts of glucose and maltose on hydrolysis of starch.     

Characterization and application of a detergent-stable alkaline α-amylase from Bacillus subtilis strain AS-S01a

A strain AS-S01a, capable of producing high-titer alkaline α-amylase, was isolated from a soil sample of Assam, India and was taxonomically identified as Bacillus subtilis strain AS-S01a. Optimized α-amylase yield by response surface method (RSM) was obtained as 799.0 U with a specific activity of 201.0 U/mg in a process control bioreactor. A 21.0 kDa alkaline α-amylase purified from this strain showed optimum activity at 55 °C and pH 9.0, and it produced high molecular weight oligosaccharides including small amount of glucose from starch as the end product. The K_m and V_max values for this enzyme towards starch were determined as 1.9 mg/ml and 198.21 μmol/min/mg, respectively. The purified α-amylase retained its activity in presence of oxidant, surfactants, EDTA and various commercial laundry detergents, thus advocating its suitability for various industrial applications.