Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, kcat and kcat/Km higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA

Use of megaprimer and overlapping extension pcr (OE-PCR) to mutagenize and enhance cyclodextrin glucosyltransferase (CGTASE) function

Protein engineering is a very useful tool for probing structure–function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.Bacillus, CGTase, Megaprimer PCR, Overlapping extension PCR, Protein engineering, Rational designProtein engineering is a very useful tool for probing structure–function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1

Protein engineering of selected residues from conserved sequence regions of a novel Anoxybacillus α-amylase

The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t 1/2 43 €...h) and L189I (t 1/2 36 €...h) mutants had a lower thermostability at 65°C than the native ASKA (t 1/2 48 €...h), the mutants F113V and A161D exhibited an improved t 1/2 of 51 €...h and 53 €...h, respectively. Among the mutants, only the A161D had a specific activity, k cat and k cat /K m higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA

COMPUTER SYSTEM FOR SUPPORT OF TERRITORIAL LEVEL STATE ECOLOGICAL MONITORING

The functional model of the State ecological monitoring, methods of making solutions on the planning of the State ecological monitoring activity, structure and functions of the computer system have been developed. The practical results are as follows: effective accumulation and use of the information resources, increase of the State ecological monitoring efficiency in conditions of the limited resources. The work has been put into the Minprirods of Republic Northern Osetia-AlaniaAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca2+ ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca2+ ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite

Sequence arrangements of prophages in <i>Anoxybacillus</i> genome as identified by PHAST.

<p>(<b>A</b>) ProphageWK of <i>Anoxybacillus flavithermus</i> WK1. (<b>B</b>) ProphageG10 of <i>Anoxybacillus kamchatkensis</i> G10. (<b>C</b>) ProphageSK of <i>Anoxybacillus</i> sp. SK3-4. (<b>D</b>) ProphageDT of <i>Anoxybacillus</i> sp. DT3-1. ProphageWK and prophageG10 may not be intact prophages, due to the lack of putative genes encoding morphological proteins. This is in contrast to intact prophageSK and prophageDT, which have more and ordered morphological genes. These morphological genes are arranged in clusters or modules, which is a hallmark of prophage sequences, and in an order typical of temperate tailed-phage genomes. Note that prophageDT is located on the complementary strand of <i>Anoxybacillus</i> sp. DT3-1. The prophage map was reversed for ease of reference. Both prophageSK and prophageDT also share six genes (shown in figure) that appear to be conserved in location and order. The details of ORFs information for prophageSK and prophageDT are provided in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090549#pone.0090549.s001" target="_blank">Table S1</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090549#pone.0090549.s002" target="_blank">Table S2</a></b>.</p

Genome features of the <i>Anoxybacillus</i> species used in this study.

<p>Genome features of the <i>Anoxybacillus</i> species used in this study.</p

Genomes comparison of <i>Anoxybacillus</i> species.

<p>(<b>A</b>) Five-way Venn-diagram showing the number of shared and specific CDS among the <i>Anoxybacillus</i> spp. Orthologous groupings were based on 50% identify cutoff and overlap of at least 70% protein sequence length. (<b>B</b>) BRIG image with <i>Anoxybacillus</i> sp. SK3-4 genome sequence set as the central reference. (<b>C</b>) BRIG image with <i>Anoxybacillus</i> sp. DT3-1 genome sequence set as the central reference.</p

Protein dendrogram of sequences with the encoded genes as a result of horizontal gene transfer.

<p>(<b>A</b>) <i>Anoxybacillus</i> sp. SK3-4 proteinase, (<b>B</b>) <i>Anoxybacillus</i> sp. SK3-4 N-acetyltransferase, (<b>C</b>) <i>Anoxybacillus</i> sp. SK3-4 β-glucosidase, (<b>D</b>) <i>Anoxybacillus</i> sp. SK3-4 α-amylase, (<b>E</b>) <i>Anoxybacillus</i> sp. DT3-1 Na⁺/H⁺ antiporter NhaC, (<b>F</b>) <i>Anoxybacillus</i> sp. DT3-1 Mn²⁺/Fe²⁺/Zn²⁺ transporter, (<b>G</b>) <i>Anoxybacillus</i> sp. DT3-1 Mn²⁺/Fe²⁺ transporter, and (<b>H</b>) <i>Anoxybacillus</i> sp. DT3-1 drug transmembrane transport.</p

List of glycosyl hydrolases (GHs) in <i>Anoxybacillus</i> and their similarity to the other genera.

a<p>The reference used in the protein sequence alignment is denoted as 100%.</p