The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis

Abstract

We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic a-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 Bacilus 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 oa-amylase sequences. We found 83% homology with the 48-kDa a-amylase part of the Bacilus polymyxa Pj-a-amylase polyprotein and 50%o homology with Taka amylase A of AspergiUlus 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 Bacilus amyloliquefaciens a-amylase gene. A number of thermostable enzymes have been isolated from Thermoactinomyces vulganis strains. Among these were thermitase (4, 13, 14, 28, 29), a protease from the subtilisin family (2, 5, 36), and a variety of oa-amylases (pullulanases). The a-amylases of strains R47 and 42, like fungal glucoamylases (1, 15, 49, 54), hydrolyze starch and pullulan (1, 54). The a-amylase of T. vulgaris 94-2A, however, utilizes only starch and glycogen as substrates, not pullulan. a-Amylase 1 of T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa and was previously shown to exhibit striking homology to Taka amylase A of Aspergillus oryzae for a short N-terminal sequence (22, 60, 67). Smaller peptides of 33 and 18 kDa have been shown to be products of limited AmyTV1 proteolysis (21). The AmyTV1 amylase is unusual because of its temperature optimum at 62.5°C in a low pH range (4.8 to 6), its relatively short half-life of about 5 min at 70°C, and the production of maltose and maltotriose in the hydrolysate, which lacks glucose (47). In order to overcome problems of poor growth and product yield in T. vulgaris 94-2A, we cloned the gene encoding the AmyTV1 amylase and performed expression studies with Bacillus subtilis. The transcription start site(s) of the gene in B. subtilis was studied after deletion of the 5'-flanking region and after its replacement with the promoter of the B. amyloliquefaciens cx-amylase gene. MATERIALS AND METHODS Bacterial strains, plasmids, and phages. The T. vulgaris strain used was originally isolated and described by Klingenberg et al. (29, 47). The strain 94-2A was selected for higher enzyme production (29). The EMBL3 lambda phage (12) was used for preparation of a T. vulgaris DNA bank in Escherichia

Gene sequence, modeling, and enzymatic characterization of α-amylase AmyLa from the thermophileLaceyellasp. DS3

“…The resulting 69.2 kDa a-amylase may have been formed as a result of limited proteolysis or glycolysis of the 72.5 kDa a-amylase. Generally, the molecular mass of the AmyLa a-amylase was in the range of microbial a-amylases, from 50 to 70 kDa [10,14,15,44], but it was different from other recorded Thermoactinomyces amylases (74.3, 67.5, 54.5, and 135 kDa) [27,28,30].…”

“…This indicates an intermediate temperature stable a-amylase that is useful for the baking industry [46]. In comparing to other Thermoactinomyces amylases, optimum pH, and temperature of AmyLa (pH 7, 50°C) are distinct from AmyTV1 (pH 4.8-6, 62.5°C) [27], TVA I (pH 5, 70°C) [28], and those from Laceyella sacchari (pH 6.5, 45°C) [22]. The activation energy of AmyLa a-amylase was found to be 196.1 kJ.…”

“…4). It is well established that a-amylase, with a few exceptions, contains a conserved Ca 2þ binding site [10,14,27,28,48]. In industry, stable a-amylases in the absence of Ca 2þ is favored [50], so some studies had tried to replace Ca 2þ with other ions [14].…”

“…Based on molecular phylogenetics, Thermoactinomycetaceae has been reclassified into Bacillales (phylum Firmicutes) although it has long been classified in actinomycetales . Hofemeister et al and Shimizu et al characterized different α‐amylases from Thermoactinomyces vulgaris strains 94‐2A and R‐47. Later, other different α‐amylases were characterized from Thermoactinomyces spp.…”

Amylolytic Yeasts: Producers of α-amylase and Pullulanase

“…In contrast, proteolysis decreases the molecular weight. The α-amylase T. vulganis 94-2A (AmyTV1) is a protein of 53 kDa [87].…”