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Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli.
Appl Environ Microbiol 1996; 62(10):3861-3AE

Abstract

We report an improvement of a published procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose (B. Brand and W. Boos, Appl. Environ. Microbiol. 55:2414-2415, 1989). Instead of inducing the expression of the trehalose-synthesizing enzymes encoded by the chromosomal genes otsAB by high osmolarity, we now induce their expression from a plasmid under normal growth conditions by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside). Instead of using a pgi zwf double mutant to prevent glucose utilization, we use a pgi::Tn10 insertion only. In addition to being defective in treA, which encodes a periplasmic trehalase, the strain is now also defective in treF, which encodes a newly discovered cytoplasmic trehalase. This strain is genetically stable; it has no growth defects; and after induction with IPTG, it will transform [14C]glucose to [14C]trehalose in minimal medium without any carbon source under aerobic conditions at a rate of 3 nmol/min/10(9) cells. With the improved method, the overall yield of trehalose from glucose is about 80% and the process takes place without dilution of the specific radioactivity of the glucose residues. The accumulated trehalose is extracted from the bacteria by 70% hot ethanol and can easily be purified radiochemically by chromatographic techniques.

Authors+Show Affiliations

Department of Biology, University of Konstanz, Germany.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

8837441

Citation

Horlacher, R, et al. "Improved Method for the Preparative Synthesis of Labeled Trehalose of High Specific Activity By Escherichia Coli." Applied and Environmental Microbiology, vol. 62, no. 10, 1996, pp. 3861-3.
Horlacher R, Peist R, Boos W. Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli. Appl Environ Microbiol. 1996;62(10):3861-3.
Horlacher, R., Peist, R., & Boos, W. (1996). Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli. Applied and Environmental Microbiology, 62(10), pp. 3861-3.
Horlacher R, Peist R, Boos W. Improved Method for the Preparative Synthesis of Labeled Trehalose of High Specific Activity By Escherichia Coli. Appl Environ Microbiol. 1996;62(10):3861-3. PubMed PMID: 8837441.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Improved method for the preparative synthesis of labeled trehalose of high specific activity by Escherichia coli. AU - Horlacher,R, AU - Peist,R, AU - Boos,W, PY - 1996/10/1/pubmed PY - 1996/10/1/medline PY - 1996/10/1/entrez SP - 3861 EP - 3 JF - Applied and environmental microbiology JO - Appl. Environ. Microbiol. VL - 62 IS - 10 N2 - We report an improvement of a published procedure using Escherichia coli to synthesize 14C-labeled trehalose from [14C]glucose (B. Brand and W. Boos, Appl. Environ. Microbiol. 55:2414-2415, 1989). Instead of inducing the expression of the trehalose-synthesizing enzymes encoded by the chromosomal genes otsAB by high osmolarity, we now induce their expression from a plasmid under normal growth conditions by the addition of IPTG (isopropyl-beta-D-thiogalactopyranoside). Instead of using a pgi zwf double mutant to prevent glucose utilization, we use a pgi::Tn10 insertion only. In addition to being defective in treA, which encodes a periplasmic trehalase, the strain is now also defective in treF, which encodes a newly discovered cytoplasmic trehalase. This strain is genetically stable; it has no growth defects; and after induction with IPTG, it will transform [14C]glucose to [14C]trehalose in minimal medium without any carbon source under aerobic conditions at a rate of 3 nmol/min/10(9) cells. With the improved method, the overall yield of trehalose from glucose is about 80% and the process takes place without dilution of the specific radioactivity of the glucose residues. The accumulated trehalose is extracted from the bacteria by 70% hot ethanol and can easily be purified radiochemically by chromatographic techniques. SN - 0099-2240 UR - https://www.unboundmedicine.com/medline/citation/8837441/Improved_method_for_the_preparative_synthesis_of_labeled_trehalose_of_high_specific_activity_by_Escherichia_coli_ L2 - http://aem.asm.org/cgi/pmidlookup?view=long&pmid=8837441 DB - PRIME DP - Unbound Medicine ER -