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Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein.
J Biol Chem. 1986 Feb 25; 261(6):2946-53.JB

Abstract

Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase.

Authors

No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

3005273

Citation

Freundlieb, S, and W Boos. "Alpha-amylase of Escherichia Coli, Mapping and Cloning of the Structural Gene, malS, and Identification of Its Product as a Periplasmic Protein." The Journal of Biological Chemistry, vol. 261, no. 6, 1986, pp. 2946-53.
Freundlieb S, Boos W. Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. J Biol Chem. 1986;261(6):2946-53.
Freundlieb, S., & Boos, W. (1986). Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. The Journal of Biological Chemistry, 261(6), 2946-53.
Freundlieb S, Boos W. Alpha-amylase of Escherichia Coli, Mapping and Cloning of the Structural Gene, malS, and Identification of Its Product as a Periplasmic Protein. J Biol Chem. 1986 Feb 25;261(6):2946-53. PubMed PMID: 3005273.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Alpha-amylase of Escherichia coli, mapping and cloning of the structural gene, malS, and identification of its product as a periplasmic protein. AU - Freundlieb,S, AU - Boos,W, PY - 1986/2/25/pubmed PY - 1986/2/25/medline PY - 1986/2/25/entrez SP - 2946 EP - 53 JF - The Journal of biological chemistry JO - J Biol Chem VL - 261 IS - 6 N2 - Mal+ lacZ operon fusions, inducible by maltose, were isolated in Escherichia coli, strain MC4100. One fusion strain, SF1707, was analyzed in detail. This fusion did not map in any of the known genes of the malA or malB region, but its expression was under control of malT, the positive regulator gene of the maltose regulon. The gene in which the fusion occurred mapped between xyl and mtl at 80 min on the linkage map and was transcribed clockwise. We define this gene as malS. The malS-lacZ fusion was transferred onto a phage lambda vector and the 5' portion of malS was subcloned into pBR322. The resulting plasmid was used as a probe to identify the intact malS gene in a lambda library of E. coli chromosomal HindIII fragments. The phage that hybridized with the probe contained a 12-kilobase insert. The malS containing portion was subcloned into pBR322 as a 4-kilobase ClaI-HindIII fragment. This plasmid directed the malT and maltose-dependent synthesis of a periplasmic protein of 66,000 apparent molecular weight. The purified enzyme hydrolyzed maltodextrins longer than maltose including cyclic dextrins. The primary products of hydrolysis were glucose, maltose, and maltotriose, even when maltotetraose was used as a substrate. These properties differentiate this periplasmic enzyme from the cytoplasmic amylomaltase and define it as an alpha-amylase. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/3005273/Alpha_amylase_of_Escherichia_coli_mapping_and_cloning_of_the_structural_gene_malS_and_identification_of_its_product_as_a_periplasmic_protein_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(17)35878-7 DB - PRIME DP - Unbound Medicine ER -