Expression of periplasmic α-amylase of Xanthomonas campestris K-11151 in Escherichia coli and its action on maltose

Abstract

A gene encoding the periplasmic a-amylase of Xanthomonas campestris K-11151 was cloned into Escherichia coli using pUC19 as a vector. An ORF of 1578 bp was deduced to be the amylase structural gene. The primary structure of the enzyme had little identity with other a-amylases, except with the enzyme from Bacillus megateriwn. The enzyme was expressed in Em coli from the lac promoter of pUC19 and was found to be transported to the periplasmic space. The expressed enzyme showed the same thermal stability, optimum temperature and substrate specificity as the enzyme from X. campestris. The enzyme formed maltotetraose, but not 6'-nor 6*-maltosyl-rnaItose, from maltose by the reverse reaction, and the tetraose was then hydrolysed to maltotriose and glucose. The addition of maltotriose enhanced the production of glucose from maltose. In addition, maltose was formed by the condensation of glucose by the enzyme. Thus, the periplasmic a-amylase of X. campestris was shown to produce glucose from maltose by hydrolysing maltotetraose and possibly higher maltooligosaccharides, which were the products of a condensation reaction, as a major pathway, and by direct hydrolysis of maltose as a minor pathway.

Oligo-1,6-glucosidase and neopullulanase enzyme subfamilies from the α-amylase family defined by the fifth conserved sequence region

“…1). This is consistent with experimental findings that some of these 'a-amylases' appear to have the mixed substrate specificity of aamylase, cyclomaltodextrinase and neopullulanase [84,105]. The a-amylase from T. maritima was found, however, to be active against soluble starch and pullulan in the ratio 100 :4 [83], indicating that its neopululanase activity is very low.…”

Cloning, sequencing and expression of an α-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme a aThe GenBank accession number for the sequence reported in this paper is AF442961.

“…Pre-incubation samples were set up at 0 % (w\v) NaCl, 0 mM CaCl # ; 10% (w\v) NaCl, 0 mM CaCl # ; 0% (w\v) NaCl, 10 mM CaCl # ; and 10 % (w\v) NaCl, 10 mM CaCl # . Buffered starch\saline solution was then added Bacillus megaterium α-amylase (Metz et al, 1988) ; PPPUL, Paenibacillus polymyxa pullulanase (Yebra et al, 1999) ; DTAMY3, Dictyoglomus thermophilum α-amylase C (Horinouchi et al, 1988) ; XCAMY, Xanthomonas campestris α-amylase (Abe et al, 1996) ; TMGLT, Thermotoga maritima 4-α-glucanotransferase (Liebl et al, 1992). Signature sequence regions (1)(2)(3)(4)(5) to each sample to re-establish standard assay conditions and activity determined by reducing sugar assays after incubation at 65 mC for 30 min.…”

“…The results of this search indicated that AmyA showed high homology to a group of five enzymes : α-amylase from Bacillus megaterium (Metz et al, 1988), neopullulanase from Paenibacillus polymyxa (Yebra et al, 1999), periplasmic α-amylase from Xanthomonas campestris K-11151 (Abe et al, 1996) and α-amylase AmyC from Dictyoglomus thermophilum (Horinouchi et al, 1988). Lower levels of homology were also observed for other α-amylases, glucanotransferases and trehalose synthases from thermophilic bacteria.…”

Characterization of a Novel Amylolytic Enzyme Encoded by a Gene from a Soil-Derived Metagenomic Library

“…However, cyclodextrin glucanotransferase cannot hydrolyze pullulan, which is a substrate for AmyM. Maltogenic amylase, or neopullulanase, is another enzyme with dual activity, but transfer products are formed by the enzyme mainly via ␣-linkages (1,6,15). AmyM, by contrast, transfers the glucanosyl segment of the donor to an acceptor sugar only via ␣-linkages (1,4).…”

“…Maltogenic amylase, or neopullulanase, is another enzyme with dual activity, but transfer products are formed by the enzyme mainly via ␣-linkages (1,6,15). AmyM, by contrast, transfers the glucanosyl segment of the donor to an acceptor sugar only via ␣-linkages (1,4). AmyM can use other acceptor molecules, such as ␣-methyl glucoside, cellobiose, lactose, and maltitol (Fig.…”