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Recombinant expression, purification, and characterization of an α-amylase from Massilia timonae.

ABSTRACT: This work reports the amy1 gene cloning from Massilia timonae CTI-57, and its successful expression in Escherichia coli Rosetta™ (DE3) from the pTRCHis2B plasmid. The recombinant AMY1 protein had 47 kDa, and its modeled structure showed a monomer composed of three domains. An N-terminal domain with the characteristic (β/α)₈-barrel structure of α-amylases, which contained the catalytic amino acid residues. The second domain was small, and the C-terminal domain was similar to those found in the barley α-amylase. A phylogenetic analysis demonstrated a high sequence identity of the studied protein with bacterial and plant α-amylases from the GH13_6 subfamily. This is the first characterized bacterial α-amylase from this glucoside hydrolase subfamily. Besides starch, the enzyme was also active against maltodextrin, amylopectin, and blocked p-nitrophenyl α-d-maltoheptaoside, but could not use β-cyclodextrin or 4-nitrophenyl α-d-glucopyranoside. The K _M for highly pure grade soluble starch from potato and V _max values were 0.79 mg/mL and 0.04 mg/min, respectively. The calcium ion showed to be essential for the purified enzyme's activity, while EDTA, molybdenum, cobalt, and mercury were strong inhibitors. The enzyme was almost fully active in SDS presence. The enzyme's optimal pH and temperature were 6.0 and 60 °C, respectively, and its denaturation T _m was 79 °C. A TLC analysis revealed that glucose and maltose are products of the enzyme's action on starch. In conclusion, this work described the M. timonae GH13_6 subfamily α-amylase, which showed to be thermostable and anionic detergent-resistant.

SUBMITTER: Tagomori BY

PROVIDER: S-EPMC7778672 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.

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Recombinant expression, purification, and characterization of an α-amylase from Massilia timonae.

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