Gregory L. Côté

A spectrophotometric assay for feruloyl esterases.

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.

Oligosaccharide synthesis by enzymatic transglycosylation

Agricultural Research Service, United States Department of Agriculture, Northern Regional Research Center, 1815 N. University St., Peoria, IL 61604, U.S.A. 2 Biochemical and Food Process Engineering, Agricultural Engineering Department, Purdue University, West Lafayette, IN 47907, U.S.A.

Characterization of the exocellular polysaccharides from Azotobacter chroococcum

Abstract Azotobacter chroococcum produces appreciable amounts of exocellular polysaccharide. Produced by A. chroococcum strain NRRL B-14341 are at least two distinct polysaccharides which can be separated by ion-exchange chromatography on DEAE-Sephadex A-25. Exopolysaccharide I (EPS-I) consists of rhamnose, mannose, and galactose in the molar ratios of 1:2:2, along with a trace of glucose. EPS-I also contains pyruvic acetal and acetic ester groups. Exopolysaccharide II (EPS-II) consists mainly of mannuronic acid, with much smaller proportions of guluronic acid. Acetic ester groups are also present in EPS-II. Methylation analysis of reduced EPS-II shows it to be (1→4)-linked, and 13 C-nuclear magnetic resonance spectroscopy indicates that it is mainly (1→4)-β- d -mannuronan. EPS-II is evidently a low-guluronate form of alginate. Batch-to-batch variation was observed in the relative amounts of the two polysaccharides, the acetyl and pyruvic contents of EPS-I, and the degree of acetylation of EPS-II.

A method for surveying and classifying Leuconostoc spp. glucansucrases according to strain-dependent acceptor product patterns

A number of Leuconostoc spp. strains were screened for their ability to produce glucansucrases and carry out acceptor reactions with maltose. Acceptor products were analyzed by thin-layer chromatography (TLC) and it was discovered that they could be grouped into four distinct categories based on oligosaccharide product patterns. These patterns corresponded with structural features of the dextrans each strain is reported to produce. Strains that produced a typical dextran—characterized by a predominantly linear α(1→6)-linked d-glucan chain with a low to moderate degree of branching—produced a homologous series of isomaltooligosaccharides via acceptor reactions. Strains that produced dextrans with moderate to high levels of α(1→2) branch points, exemplified by NRRL B-1299, synthesized the same isomaltodextrins as well as another series of oligosaccharides migrating slightly faster in our TLC system. Strains that produced dextrans with higher levels of α(1→3)-branches, such as NRRL B-742, synthesized isomaltodextrins plus a series of oligosaccharides that migrated slightly more slowly on TLC. And finally, strains known to produce alternansucrase produced isomaltodextrins plus oligoalternans. Within a given type, variability exists in the relative proportions of each product. The data presented here may be useful in selecting strains for the production of specific types of oligosaccharides, for example as prebiotics.

Purification and properties of an extracellular levansucrase from Erwinia herbicola NRRL B-1678

Levansucrase (EC 2.4.1.10) was purified approximately 40-fold from the extracellular culture fluid of Erwinia herbicola NRRL B-1678. The purified enzyme in its native form occurred as an aggregate. Poly(acrylamide) gel electrophoresis in the presence of sodium dodecyl sulfate showed an active monomer having a molecular weight of 48,000. The enzyme had a Km for sucrose of 28mm and a pH optimum of 6.0, and was stable from −18 to +52°. It was free from α-d-glucosidase, amylase, and glucansucrase activities, but exhibited considerable β-d-fructofuranosidase activity.

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-d-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1 → 6)-linked d-glucose units, and the other was a water-insoluble glucan containing both α-(1 → 6)-linked and α-(1 → 3)-linked d-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1 → 3)-linked d-glucose units than the water-insoluble glucan produced by cell suspensions.

Production of a constitutive, extracellular levansucrase fromErwiniaherbicola NRRL B-1678

SummaryLevansucrase (E.C. 2.4.1.10), which has long been known to be produced intracellularly byErwiniaherbicola, was found in relatively high concentrations in the extracellular culture fluid of bacteria grown in various media. Sucrose was not required for enzyme secretion. A medium consisting of corn steep liquor and sorbitol or mannitol gave the highest yields of enzyme.

Flavorings and other value-added products from sucrose

Publisher Summary This chapter describes the use of biotechnology to produce value-added products from sucrose. It focuses on the use of enzymes to convert sucrose into oligosaccharides or polysaccharides suitable for food use. The last half of the 20th century saw many rapid developments in food technology, including many based on carbohydrate biotechnology. One of the most significant was the introduction of corn syrup-based sweeteners, especially high-fructose corn syrup. The product depends heavily on enzyme technology, including amylases and xylose isomerase. Another area that developed during that period was the introduction of microbial gums to replace the more traditional plant-based gums. Xanthan and gellan have taken over a huge share of the market, with a resultant decrease in market shares for gums tragacanth, ghatti, karaya, and so on. The latter half of the 20th century saw the introduction of artificial, non-caloric sweeteners and the next decade will see an increasing interest in sweeteners that exhibit a slow release of carbohydrate into the bloodstream. The first of the low-glycemic index (low-GI) sweeteners have been introduced in the form of carbohydrates. More can be expected in the future.