Rapid screening method to select microorganism strains that produce a high proportion of alternan to dextran

• US 5,789,209 A
• Filed: 04/29/1996
• Issued: 08/04/1998
• Est. Priority Date: 08/02/1994
• Status: Expired due to Fees

• Current Assignee: United States Department Of Agriculture, United States Incorporated
• Original Assignee: United States Department Of Agriculture
• Inventors: Hayman, G. Thomas, Cote, Gregory L., Leathers, Timothy D.
• Primary Examiner(s): Marx, Irene
• Application Number:
  US08/641,082
• Time in Patent Office: 827 Days
• Field of Search: 435/101, 435/103, 435/34, 435/172.1
• US Class Current: 435/101
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12R 2001/01 Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• CPC Class Codes: G01N 2400/14 alpha-D-Glucans, i.e. having alpha 1,n (n=3,4,6) linkages between saccharide units, e.g. pullulan
• CPC Class Codes: G01N 2400/22 Dextran

Abstract

A rapid screening procedure to select microorganism strains that produce a high proportion of alternan to dextan and a high proportion of alternansucrase to dextransucrase is described. Strains selected by the method are useful to produce a high proportion of alternansucrase to dextransucrase and to produce a high proportion of alternan to dextran, either fermentatively by cultivation of the strains on sucrose, or enzymatically by using alternansucrase produced by the strains of the invention. The selected strains can also be used to obtain microorganism strains having further improvements.

Claims

1. A rapid screening method to identify a bacterial test strain that produces a greater proportion of alternan to dextran, wherein said test strain is a strain of Leuconostoc mesenteroides or L. dextranicum, said method comprising in sequence:

(a) growing a test strain culture of a bacterial species known to produce alternan and dextran in a sucrose-containing liquid growth medium under conditions effective for production of polysaccharides;

(b) treating the liquid culture produced in step (a) with crude dextranase, wherein said crude dextranase contains enzymes which convert dextran and sucrose unused by the bacterial cells into reducing sugars and wherein bacterial cells have not been removed from said liquid culture produced in step (a);

(c) reacting the liquid culture produced in step (b), which contains bacterial cells, with a reagent that produces color in the presence of reducing sugars;

(d) measuring the optical density of the liquid test culture produced in step (c), from which bacterial cells have not been removed, at 540 nm and comparing the color produced by said test strain with the color produced by a negative control comprising a known alternan and dextran producing strain that has been grown and reacted with the color producing reagent under the same conditions as the test strain but that has not been treated with said crude dextranase; and

(e) based on the comparison in step (d), identifying the test strain as a strain which produces a greater proportion of alternan to dextran if the optical density of the test strain liquid culture at 540 nm is no greater than about 0.3 optical density units higher than that of said negative control.

2. The method of claim 1 wherein said test strain is a mutant of Leuconostoc mesenteroides or L. dextranicum.

3. The method of claim 2 wherein said test strain is a mutant of L. mesenteroides strains NRRL B-1355, NRRL B-1498, or NRRL B-1501.