Psychotrophic bacteria produce heat-stable lipase, protease, and phospholipase. Pervious studies indicate the production of multiple enzymes in several strains of Pseudomonas fluorescens, but conclusive evidence is lacking. The influence of culture conditions on the production and thermostability of phospholipase, protease, and lipase was investigated in 17 raw milk and environmental isolates. Production and thermostability of the enzymes were influenced by strain, stage of growth, and the culture medium. Cross-reactivity of antibodies raised to a purified protease and a commercial lipase indicated the immunological diversity of the enzymes. Protease purification was undertaken to investigate the production of multiple proteases within a single strain. A single monomeric protease with a molecular weight of 52 kDa was purified from P. fluorescens RO98. Biochemical characterization of the enzyme revealed that it was a zinc-metallo acidprotease with pH and temperature optima of 5.0 35°C, respectively. The enzyme was thermostable with a D55 of 41 min and a D62.5 of 18 h. Although sensitive assays exist for proteases, they are not suitable for detection of protease activity in milk in the presence of milk proteins. Existing immunoassays approach the required sensitivity but take about 6 h and cannot distinguish between active and inactive enzyme. An immunoassay that can be completed within 2 h and that can detect and distinguish both total and active enzyme was explored. The ratio of these two forms gives insight into the history of the milk. The ability of the purified protease to hydrolyze hydrophobic peptides associated with bitterness in Cheddar cheese was also investigated. Results demonstrated that the protease had the potential to debitter Cheddar cheese because it was able to hydrolyze the bitter peptides commonly found during aging. Two lipolytic enzymes with molecular weights of 50 (Pf-lip1) and 12 kDa (Pf-lip2) were purified from P. fluorescens RO98. Differences were observed in their biochemical properties. D62.5-values of 12.7 and 29.9 h were determined for Pf-lip1 and Pf-lip2, respectively. Pf-lip1 preferred longer chain length fatty acids, and Pf-lip2 preferred shorter chain length substrates. Pf-lip1 hydrolyzed milk fat and emulsified triolein, but Pf-lip2 did not, indicating that the latter was an esterase. This information is of significance to the dairy industry because activity tests that assay both the lipolytic enzymes need to be used in order to direct raw milk to short shelf-life products during processing and ensure quality of long shelf-life products

Benthic microalgae (BMA) living within the surface sediment of salt marshes are highly productive organisms that provide a significant proportion of organic carbon inputs into estuarine systems. BMA secrete extracellular carbohydrates in the form of low molecular weight carbohydrates and extracellular polymeric substances (EPS) as they migrate within the sediment. EPS plays an important role in the structure and function of BMA biofilms in shallow-water systems as EPS affects habitat structure, stabilizes the sediment, reduces sediment erosion, and is a carbon source for organisms. This study looked at the effect of nutrients and carbohydrate additions on BMA biomass, bacterial biomass, carbohydrate production, and glycosidase activity in the surface 5 mm of intertidal sediment in a subtropical salt marsh (Galveston Bay, Texas). Nitrogen and phosphorus were added to cores collected from the salt marsh and incubated in the lab over four days. Very little change was seen in the biomass of the benthic microalgae or in the different carbohydrate fractions with the added nutrients. The mean chlorophyll a concentration was 13 +/- 5 ug g-1 sediment, the mean saline extractable carbohydrate concentration was 237 +/- 113 ug g-1 sediment, and the mean EPS concentration was 48 +/- 25 ug g-1 sediment. The chlorophyll a and saline extractable carbohydrate concentrations initially decreased over the first 24 hours, but then increased over the rest of the experiment, indicating a possible species compositional shift in the BMA. With no major response with nutrient additions, it is likely that a different environmental factor is limiting for the growth of the benthic microalgae, and therefore the production of sEPS, in this salt marsh. A series of experiments was conducted in situ by adding glucose, alginic acid, and phosphorus to sediment within experimental plots. Samples were taken periodically over three to seven days to determine the biomass of the microbial community, enzyme activities and kinetics, and changes in the concentrations of several sediment carbohydrate pools. u-glucosidase activities (15 +/- 3 nmol g-1 h-1) were significantly higher than u-xylosidase (6 +/- 2 nmol g-1 h-1) and u-galactosidase (8 +/- 2 nmol g-1 h-1) activities within the sediment, and there was no suppression of u-glucosidase activity measured with the glucose addition. These data represent the first measurement of u- xylosidase and u-galactosidase activity in intertidal sediment dominated by BMA. Although preliminary experiments suggested a possible phosphorus limitation within the sediment, there was little change in the bacteria abundance or the benthic microalgae biomass when phosphorus was added in situ. This study begins to illustrate the dynamics of carbohydrate production and loss in this salt marsh, and the ability for the microbial community in the salt marshes of Galveston Bay to adjust to the nutrient and carbohydrate treatments.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 146-162).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographic references (p. [81]-94).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains xv, 205 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 163-190).

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published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

Background. Fungal endophytes inhabit symptomless, living tissues of all major plant lineages to form one of earth's most prevalent groups of symbionts. Many reproduce from senesced and/or decomposing leaves and can produce extracellular leaf degrading enzymes, blurring the line between symbiotrophy and saprotrophy. To better understand the endophyte saprotroph continuum we compared fungal communities and functional traits of focal strains isolated from living leaves to those isolated from leaves after senescence and decomposition, with a focus on foliage of woody plants in five biogeographic provinces ranging from tundra to subtropical scrub forest. Methods. We cultured fungi from the interior of surface-sterilized leaves that were living at the time. of sampling (i.e., dophytes), leaves that were dead and were retained in plant canopies (dead leaf fungi,eDn LF), and fallen. leaves (leaf litter.fungi,LLF) from 3-4 species of woody plants in each of five sites in. North America. Our sampling encompassed 18 plant species. representing. two families of Pinophyta.and five families of Angiospermae. Diversity and composition of fungal communities within and among leaf life stages, hosts, and sites were compared using ITS-partial L SU rDNA data. We evaluated substrate use and enzyme activity by a subset of fungi isolated'onlyfrom living tissues vs. fungi isolated only from non-living leaves. Results Across the diverse biomes and plant taxa surveyed here, culturable fungi living leays were isolated less frequently and were less diverse than those isolated from non-living leaves. Fungal communities in living leaves also differed detectably in composition from communities in dead leaves and leaf litter within focal sites and host taxa, regardless of differential weighting of rare and abundant fungi. All focal isolates grew on cellulose, lignin, and pectin as sole carbon sources, but none displayed igninolytic or pectinolytic activity in vitro. Cellulolytic activity differed among fungal classes. Within Dothideomycetes, activity differed significantly between fungi from living vs. non-living leaves, but such differences were not observed in Sordariomycetes. Discussion. Although some fungi with endophytic life stages clearly persist for periods of time in leaves after senescence and incorporation into leaf litter, our sampling across diverse biomes and host lineages detected consistent differences between fungal assemblages in living vs. non-living leaves, reflecting incursion by fungi from the leaf exterior after leaf death and as leaves begin to decompose. However, fungi found only in living leaves do not differ consistently in cellulolytic activity from those fungi detected thus far only in dead leaves. Future analyses should consider Basidiornycota in addition to the Ascomycota fungi evaluated here, and should explore more dimensions of functional traits and persistence to further define the endophytism-to-saprotrophy continuum.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.