Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25

Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.     

Deletion analysis of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23

The alpha-amylase from Bacillus sp. strain TS-23 is a secreted starch hydrolase with a domain organization similar to that of other microbial alpha-amylases and an additional functionally unknown domain (amino acids 517-613) in the C-terminal region. By sequence comparison, we found that this latter domain contained a sequence motif typical for raw-starch binding. To investigate the functional role of the C-terminal region of the alpha-amylase of Bacillus sp. strain TS-23, four His(6)-tagged mutants with extensive deletions in this region were constructed and expressed in Escherichia coli. SDS-PAGE and activity staining analyses showed that the N- and C-terminally truncated alpha-amylases had molecular masses of approximately 65, 58, 54, and 49 kDa. Progressive loss of raw-starch-binding activity occurred upon removal of C-terminal amino acid residues, indicating the requirement for the entire region in formation of a functional starch-binding domain. Up to 98 amino acids from the C-terminal end of the alpha-amylase could be deleted without significant effect on the raw-starch hydrolytic activity or thermal stability. Furthermore, the active mutants hydrolyzed raw corn starch to produce maltopentaose as the main product, suggesting that the raw-starch hydrolytic activity of the Bacillus sp. strain TS-23 alpha-amylase is functional and independent from the starch-binding domain.     

Identification of essential histidine residues in a recombinant alpha-amylase of thermophilic and alkaliphilic Bacillus sp. strain TS-23

To understand the structure-function relationships of a truncated Bacillus sp. strain TS-23 -amylase, each of His-137, His-191, His-239, His-269, His-305, His-323, His-361, His-436, and His-475 was replaced with leucine. The molecular masses of the purified wild-type and mutant enzymes were approximately 54 kDa. The specific activity of His323Leu and His436Leu was decreased by more than 52%, while His239Leu, His305Leu, and His475Leu showed activity similar to that of the wild-type enzyme. As compared with the wild-type enzyme, His323Leu and His436Leu exhibited a 62% decrease in the value of k_cat/K_m. Alterations in His-191, His-239, His-305, and His-475 did not cause a significant change in the K_m or k_cat values. At 70°C, a decreased half-life was observed in His436Leu. These results indicate that His-137, His-269, and His-361 of Bacillus sp. strain TS-23 -amylase are important for proper catalytic activity and that His-436 may contribute to the thermostability of the enzyme.Communicated by K. Horikoshi     

A gene encoding for an alpha-amylase from thermophilic Bacillus sp. strain TS-23 and its expression in Escherichia coli

An alpha-amylase gene from Bacillus sp. strain TS-23 was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 in Escherichia coli. A cell fractionation experiment revealed that approximately 60% of the amylase activity was in the periplasmic space. Analysis and activity staining of the concentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed an apparent protein band with a mol. wt of approximately 65,000. The amylase gene (amyA) consisted of an open reading frame of 1,845 bp encoding a protein of 613 amino acids with a calculated mol. wt of 69,543. The predicted amino acid sequence showed high homology with Bacillus species, E. coli and Salmonella typhimurium alpha-amylases. Deletion of 96 amino acids from the C-terminal portion of the amylase did not result in the loss of amylolytic activity. The truncated amylase, deletion of the first 50 amino acids from the N-terminus, was overexpressed in E. coli system and refolded to yield an activable enzyme.     

Phylogenetic Analysis and Biochemical Characterization of a Thermostable Dihydropyrimidinase from Alkaliphilic Bacillus sp. TS-23

Two degenerate primers established from the alignment of highly conserved amino acid sequences of bacterial dihydropyrimidinases (DHPs) were used to amplify a 330-bp gene fragment from the genomic DNA of Bacillus sp. TS-23 and the amplified DNA was successfully used as a probe to clone a dhp gene from the strain. The open reading frame of the gene consisted of 1422 bp and was deduced to contain 472 amino acids with a molecular mass of 52 kDa. The deduced amino acid sequence exhibited greater than 45% identity with that of prokaryotic d-hydantoinases and eukaryotic DHPs. Phylogenetic analysis showed that Bacillus sp. TS-23 DHP is grouped together with Bacillus stearothermophilus d-hydantoinase and related to dihydroorotases and allantoinases from various organisms. His₆-tagged DHP was over-expressed in Escherichia coli and purified by immobilized metal affinity chromatography to a specific activity of 3.46 U mg⁻¹ protein. The optimal pH and temperature for the purified enzyme were 8.0 and 60 °C, respectively. The half-life of His₆-tagged DHP was 25 days at 50 °C. The enzyme activity was stimulated by Co²⁺ and Mn²⁺ ions. His₆-tagged DHP was most active toward dihydrouracil followed by hydantoin derivatives. The catalytic efficiencies (k_cat/K_m) of the enzyme for dihydrouracil and hydantoin were 2.58 and 0.61 s⁻¹ mM⁻¹, respectively.     

General characteristics of thermostable amylopullulanases and amylases from the alkalophilic Bacillus sp. TS-23

An alkalophilic strain of Bacillus sp., designated TS-23, was isolated from a soil sample collected at a hot spring (Tainan, Taiwan). During growth in a medium containing 1% soluble starch as the sole source of carbon, the fermentation broth exhibited both pullulanase and amylase activity. Pullulanase and amylase activities were maximal at 65° C. The pH optima were 8.8 to 9.6 for pullulanase and 7.5 to 9.4 for amylase. Under optimal conditions, a crude preparation hydrolysed pullulan, generating maltotriose as the major product. Strain TS-23 was found to produce five amylases (A_c, A₁, A₂, AP₁, and AP₂), which were visualized by activity staining of proteins that had been separated by native polyacrylamide gel electrophoresis. Both AP₁ and AP₂ had pullulanase activity and A_c, A₁ and A₂ had the ability to adsorb to raw corn-starch. Native corn-starch was partially digested by adsorbed amylases during the course of 12 h at 50° C, with initiation of granular pitting. Further incubation of the reaction mixture resulted in considerable morphological changes in corn-starch granules, and the main soluble products were maltose, maltotriose and higher oligosaccharides.     

Generating oxidation-resistant variants of <Emphasis Type="Italic">Bacillus kaustophilus</Emphasis> leucine aminopeptidase by substitution of the critical methionine residues with leucine

Bacillus kaustophilus leucine aminopeptidase (bkLAP) was sensitive to oxidative damage by hydrogen peroxide. To improve its oxidative stability, the oxidation-sensitive methionine residues in the enzyme were replaced with leucine by site-directed mutagenesis. The variants, each with an apparent molecular mass of approximately 54 kDa, were overexpressed in recombinant Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. The specific activity for M282L, M285L, M289L and M321L decreased by more than 43%, while M400L, M426L, M445L, and M485L showed 191, 79, 313, and 103%, respectively, higher activity than the wild-type enzyme. Although the mutations did not cause significant changes in the K _m value, more than 67.8% increase in the value of k _cat/K _m was observed in the M400L, M426L, M445L and M485L. In the presence of 50 mM H₂O₂, most variants were more stable with respect to the wild-type enzyme, indicating that the oxidative stability of the enzyme can be improved by engineering the methionine residues. This revised version was published online in June 2006 with corrections to the Cover Date.     

Replacement of Methionine 208 in a Truncated <Emphasis Type="Italic">Bacillus</Emphasis> sp. TS-23 α-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 K _m value was observed for all mutant enzymes. The 3.2- and 4.5-fold increases in the catalytic efficiency (k _cat/K _m) for Met208Leu and Met324Leu, respectively, were partly contributed by a 68% and 38% decrease in K _m 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. Received: 12 April 2002 / Accepted: 8 June 2002     

Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H₂O₂. The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H₂O₂ stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H₂O₂, respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H₂O₂ and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.     

Nitric oxide and hydrogen peroxide in tomato resistance. Nitric oxide modulates hydrogen peroxide level in o-hydroxyethylorutin-induced resistance to Botrytis cinerea in tomato.

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for activation of disease resistance reactions of plants to infection with a pathogen or elicitor treatment. However, biochemical mechanisms by which ROS and NO participate in these reactions are still under intensive study and controversial debate. We previously demonstrated that o-hydroxyethylorutin when applied on tomato leaves (Lycopersicon esculentum Mill. cv. "Perkoz") restricted Botrytis cinerea infection development. In this research we investigated ROS and NO generation in tomato plants treated with o-hydroxyethylorutin, non-treated and infected ones. The NO content was enhanced or decreased in the studied plants by supplying them with NO generator-SNP or scavenger-cPTIO. NO detection was carried out using diaminofluorescein diacetate (DAF-DA) in conjunction with confocal laser scanning microscopy. The influence of elevated and decreased levels of NO on B. cinerea infection development and ROS generation was studied. The elevated NO concentration in tomato leaves strongly decreased hydrogen peroxide concentration without affecting other studied ROS (superoxide anion and hydroxyl radical) levels. H2O2 concentrations in NO-supplied leaves were low regardless of further treatment of tomato leaves with o-hydroxyethylorutin or inoculation with B. cinerea. The low H2O2 concentration coincided with quick and severe infection development in NO-supplied leaves. As activities of enzymes generating (SOD EC 1.15.1.1)) and removing (APX EC 1.11.1.11, CAT EC 1.11.1.6) H2O2 were unchanged in the studied plants, the decrease in H2O2 concentration was probably due to a direct NO-H2O2 interaction.     

Binding of small, acid-soluble spore proteins to DNA plays a significant role in the resistance of Bacillus subtilis spores to hydrogen peroxide.

Dormant spores of Bacillus subtilis which lack the majority of the alpha/beta-type small, acid-soluble proteins (SASP) (termed alpha- beta- spores) that coat the DNA in wild-type spores are significantly more sensitive to hydrogen peroxide than are wild-type spores. Hydrogen peroxide treatment of alpha- beta- spores causes DNA strand breaks more readily than does comparable treatment of wild-type spores, and alpha- beta- spores, but not wild-type spores, which survive hydrogen peroxide treatment have acquired a significant number of mutations. The hydrogen peroxide resistance of wild-type spores appears to be acquired in at least two incremental steps during sporulation. The first increment is acquired at about the time of alpha/beta-type SASP synthesis, and the second increment is acquired approximately 2 h later, at about the time of dipicolinic acid accumulation. During sporulation of the alpha- beta- strain, only the second increment of hydrogen peroxide resistance is acquired. In contrast, sporulation mutants which accumulate alpha/beta-type SASP but progress no further in sporulation acquire only the first increment of hydrogen peroxide resistance. These findings strongly suggest that binding of alpha/beta-type SASP to DNA provides one increment of spore hydrogen peroxide resistance. Indeed, binding of alpha/beta-type SASP to DNA in vitro provides strong protection against cleavage of DNA by hydrogen peroxide.     

Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance.

A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherichia coli and in Bacillus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.     

Isolation and characterization of a cis-acting mutation conferring catabolite repression resistance to alpha-amylase synthesis in Bacillus subtilis.

Bacillus subtilis 168GR10 was shown to contain a mutation, gra-10, which allowed normal temporal activation of alpha-amylase synthesis in the presence of a concentration of glucose that is inhibitory to activation of amylase synthesis in the parent strain, 168. The gra-10 mutation was mapped by phage PBS-1-mediated transduction and by transformation to a site between lin-2 and aroI906, very tightly linked to amyE, the alpha-amylase structural gene. The gra-10 mutation did not pleiotropically affect catabolite repression of sporulation or of the synthesis of extracellular proteases or RNase and was unable to confer glucose-resistance to the synthesis of chloramphenicol acetyltransferase encoded by the cat-86 gene driven by the amyE promoter region (amyR1) inserted into the promoter-probe plasmid pPL603B. It therefore appears that gra-10 defines a cis-regulatory site for catabolite repression, but not for temporal activation, of amyE expression. The evidence shows that temporal activation and glucose-mediated repression of alpha-amylase synthesis in B. subtilis 168 are distinct phenomena that can be separated by mutation.     

Production of extracellular alkaline alpha-amylase by solid state fermentation with a newly isolated Bacillus sp

Production of alkaline alpha-amylase employing our laboratory isolate, Bacillus sp., under solid state fermentation, was optimized. The effect of wheat bran and lentil husk was examined. Lentil husk exhibited the highest enzyme production. The appropriate incubation time, inoculum size, moisture level, and buffer solution level were determined. Maximum yields of 216,000 and 172,800 U/g were achieved by employing lentil husk and wheat bran as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10.0 with 30% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20% and 1:0.5 for two solid substrates.     

Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp. No. 195.

Newly isolated Bacillus sp. No. 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases. The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200. The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE. The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively. The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase. It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1. The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No. 195 amylase.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

Contribution of catalase to hydrogen peroxide resistance in Enterococcus faecalis

Enterococcus faecalis exhibits high resistance to oxidative stress. Several enzymes are responsible for this trait. The role of alkyl hydroperoxide reductase (Ahp), thiol peroxidase (Tpx), and NADH peroxidase (Npr) in oxidative stress defense was recently characterized. Enterococcus faecalis, in contrast to many other streptococci, contains a catalase (KatA), but this enzyme can only be formed when the bacterium is supplied with heme. We have used this heme dependency of catalase activity and mutants deficient in KatA and Npr to investigate the role of the catalase in resistance against exogenous and endogenous hydrogen peroxide stress. The results demonstrate that in the presence of environmental heme catalase contributes to the protection against toxic effects of hydrogen peroxide.     

Cloning and expression of a Bacillus sp. 79-23 cellulase gene

Summary A gene for encoding cellulase was cloned from Bacillus sp. 79-23 into Escherichia coli and the nucleotide sequence was determined. The cellulase gene, designated as celS, was composed of 1,497 base pairs and the nucleotide sequence of the celS gene was highly homologous to those of other B. subtilis cellulase genes. The enzyme encoded by celS was highly active on carboxymethylcellulose but also exhibited activity towards avicel and p-nitrophenyl--spd-cellobiopyranoside. When its native promoter was replaced with a strong B. subtilis promoter, the extracellular cellulase was produced up to 8.5 units per ml in B. subtilis DB104.