Molecular characterization of α-amylase from Staphylococcus aureus.
ABSTRACT: Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Åand 7.091Å respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria.
Project description:Staphylococcus aureus a natural inhabitant of nasopharyngeal tract mainly survives as biofilms and possess complete Krebs cycle which plays major role in its pathogenesis. This TCA cycle is regulated by Isocitrate dehydrogenase (IDH) we have earlier cloned, sequenced (HM067707), expressed and characterized this enzyme from S. aureus ATCC12600. We have observed only one type of IDH in all the strains of S. aureus which dictates the flow of carbon thereby controlling the virulence and biofilm formation, this phenomenon is variable among bacteria. Therefore in the present study comparative structural and functional analysis of IDH was undertaken. As the crystal structure of S. aureus IDH was not available therefore using the deduced amino sequence of complete gene the 3D structure of IDH was built in Modeller 9v8. The PROCHECK and ProSAweb analysis showed the built structure was close to the crystal structure of Bacillus subtilis. This structure when superimposed with other bacterial IDH structures exhibited extensive structural variations as evidenced from the RMSD values correlating with extensive sequential variations. Only 24% sequence identity was observed with both human NADP dependent IDHs (PDB: 1T09 and 1T0L) and the structural comparative studies indicated extensive structural variations with an RMSD values of 14.284Å and 10.073Å respectively. Docking of isocitrate to both human IDHs and S. aureus IDH structures showed docking scores of -11.6169 and -10.973 respectively clearly indicating higher binding affinity of isocitrate to human IDH.
Project description:An intracellular alpha-amylase from Streptococcus bovis 148 was purified and characterized. The enzyme was induced by maltose and soluble starch and produced about 80% maltotriose from soluble starch. Maltopentaose was hydrolyzed to maltotriose and maltose and maltohexaose was hydrolyzed mainly to maltotriose by the enzyme. Maltotetraose, maltotriose, and maltose were not hydrolyzed. This intracellular enzyme was considered to be a maltotriose-producing enzyme. The enzymatic characteristics and hydrolysis product from soluble starch were different from those of the extracellular raw-starch-hydrolyzing alpha-amylase of strain 148. The deduced amino acid sequence of the intracellular alpha-amylase was similar to the sequences of the mature forms of extracellular liquefying alpha-amylases from Bacillus strains, although the intracellular alpha-amylase did not contain a signal peptide. No homology between the intracellular and extracellular alpha-amylases of S. bovis 148 was observed.
Project description:1. The cell-bound alpha-amylase of Streptococcus bovis has been isolated from other carbohydrases in the cell extract by chromatography on DEAE-cellulose. The enzyme has been compared with the extracellular alpha-amylase produced by this organism. 2. The two amylases had similar action patterns on amylose, the main product being maltotriose with smaller amounts of maltose and a little glucose. 3. The cell-bound amylase hydrolysed maltopentaose and maltohexaose at a similar rate to the hydrolysis of amylose. Maltotetraose was hydrolysed six times more slowly, and maltotriose 280 times more slowly, than amylose. 4. Studies with end-labelled maltodextrins revealed that the cell-bound alpha-amylase preferentially hydrolysed the third linkage from the non-reducing end, liberating maltotriose. The linkage at the reducing end of maltotriose was more easily hydrolysed than the other. 5. Egg-white lysozyme and the extracellular enzymes of Streptomyces albus lysed the cell walls of Streptococcus bovis, releasing amylase into the medium. In the presence of 0.6 m-sucrose 10% of the maximal amylase activity was released by lysozyme. Suspension of the spheroplasts in dilute buffer caused the rupture of the cytoplasmic membrane and the liberation of amylase. 6. A sensitive method for determining the ability of amylases to degrade starch granules is described.
Project description:Glucokinase is classified in bacteria based upon having ATP binding site and 'repressor/open reading frames of unknown function/sugar kinases' motif, the sequence of glucokinase gene (JN645812) of Staphylococcus aureus ATCC12600 showed presence of ATP binding site and 'repressor/open reading frames of unknown function/sugar kinases' motif. We have earlier observed glucokinase of S. aureus has higher affinity towards the substrate compared to other bacterial glucokinase and under anaerobic condition with increased glucose concentration S. aureus exhibited higher rate of biofilm formation. To establish this, 3D structure of glucokinase was built using homology modeling method, the PROCHECK and ProSA-Web analysis indicated this built glucokinase structure was close to the crystal structure. This structure was superimposed with different bacterial glucokinase structures and from the root-mean-square deviation values, it is concluded that S. aureus glucokinase exhibited very close homology with Enterococcus faecalis and Clostridium difficle while with other bacteria it showed high degree of variations both in domain and nondomain regions. Glucose docking results indicated -12.3697 kcal/mol for S. aureus glucokinase compared with other bacterial glucokinase suggesting higher affinity of glucose which correlates with enzyme kinetics and higher rate of biofilm formation.
Project description:Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.
Project description:Bacillus amyloliquefaciens alpha-amylase (1,4-alpha-D-glucan glucanohydrolase. EC 220.127.116.11), which is commercially supplied as 'Bacillus subtilis alpha-amylase' does not cross-react immunologically with B. subtilis alpha-amylase. This enzyme (from B. amyloliquefaciens) was cleaved by treatment with CNBr into seven fragments. Peptide A was selected for sequence determination. It is the longest one, containing 185 amino acids (i.e. approx. 50% of the total molecule) and connects to the hexapeptide of the N-terminus. Its primary structure was aligned by use of various proteolytic enzymes. The sequence of amino acids 181-184 is identical with that of amino acids 14-17 of the alpha-amylase isolated from B. subtilis (except that amino acid 183 is asparagine rather than aspartic acid).
Project description:BACKGROUND:Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low. RESULTS:In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date. CONCLUSIONS:This is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.
Project description:BACKGROUND:PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS:In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS:In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.
Project description:Amylase plays an important role in biotechnology industries, and Gram-positive bacterium Bacillus subtilis is a major host to produce heterogeneous α-amylases. However, the secretion stress limits the high yield of α-amylase in B. subtilis although huge efforts have been made to address this secretion bottleneck. In this question-oriented review, every effort is made to answer the following questions, which look simple but are long-standing, through reviewing of literature: (1) Does α-amylase need a specific and dedicated chaperone? (2) What signal sequence does CsaA recognize? (3) Does CsaA require ATP for its operation? (4) Does an unfolded α-amylase is less soluble than a folded one? (5) Does α-amylase aggregate before transporting through Sec secretion system? (6) Is α-amylase sufficient stable to prevent itself from misfolding? (7) Does α-amylase need more disulfide bonds to be stabilized? (8) Which secretion system does PrsA pass through? (9) Is PrsA ATP-dependent? (10) Is PrsA reused after folding of α-amylase? (11) What is the fate of PrsA? (12) Is trigger factor (TF) ATP-dependent? The literature review suggests that not only the most of those questions are still open to answers but also it is necessary to calculate ATP budget in order to better understand how B. subtilis uses its energy for production and secretion.
Project description:We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene.