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United States Department of Agriculture

Agricultural Research Service

Title: Cloning and Characterization of a Sucrase from Leuconostoc Mesenteroides B-21297

Authors
item Holt, Scott
item Cote, Gregory
item Ahlgren, Jeffrey
item Leathers, Timothy

Submitted to: American Society for Microbiology Branch Meeting
Publication Type: Abstract Only
Publication Acceptance Date: November 6, 1996
Publication Date: N/A

Technical Abstract: Certain Leuconostoc mesenteroides strains can produce industrially significant biopolymers from sucrose metabolism. A sucrase enzyme from L. mesenteroides was cloned to better understand the molecular biology of sucrose utilization by this microorganism. A partial gene library was prepared from L. mesenteroides B-21297 DNA by using the insertion vector lambda-ZAP II. Fourteen plaques expressed sucrase activity on M9-sucrose medium. The cloned sucrases liberated glucose and fructose from sucrose causing heavy E. coli growth and tetrazolium red reduction surrounding the recombinant plaques. Phage lysates from the 14 recombinants did not display polymer-forming activity when assayed on sucrose. One recombinant was subcloned into E. coli and the sonicated bacterial lysate showed sucrase activity when assayed on sucrose as determined by a reducing sugar test. HPLC and GC/MS analyses of the liberated sucrase products indicated the presence of fructose and glucose in equimolar proportions. The E. coli lysate did not possess sucrose phosphorylase activity. Isopropyl beta-D- thiogalactopyranoside induction did not increase sucrase activity in bacterial lysates indicating that the cloned gene may be transcribed from its own promoter. Restriction endonuclease analysis indicated that the cloned DNA fragment was approximately 6.0 kb in size and could be digested by Hincll, Hindlll, EcoRV, Pvull, and Sau3Al. To our knowledge this is the first invertase-type enzyme cloned from L. mesenteroides.

Last Modified: 4/23/2014
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