Manipulation of the Repertoire of Digestive Enzymes Secreted into the Gastrointestinal Tract of Transgenic Mice

Abstract

In non-ruminant livestock the energy which can be derived from dietary cellulose and xylan is limited by the inefficient microbial fermentation of these polymers in the hind-gut. Furthermore, in poultry, cereal-derived plant structural polysaccharides impair normal digestive function through the formation of gel-like structures, which trap nutrients rendering them unavailable to the animal. The nutrition of non-ruminant livestock could be significantly improved by the depolymerization of plant structural polysaccharides, through the introduction of cellulase activity into the small intestines of these animals. Here we describe the expression of Clostridium thermocellum endoglucanase E in the exocrine pancreas of transgenic mice. A non-glycosylated active enzyme is secreted into the small intestines, and is resistant to proteolytic inactivation, demonstrating the feasibility of generating non-ruminant animals with the endogenous capacity to depolymerize plant structural polysaccharides in the small intestines.

The Use of Forced Protein Evolution to Investigate and Improve Stability of Family 10 Xylanases

“…Because the extracellular environment of microbial ecosystems displays high proteinase activity (3), resistance to proteolytic degradation exerts a strong selection pressure on the evolution of extracellular enzymes such as xylanases. This view is consistent with the observation that extracellular xylanases from both mesophilic and thermophilic hosts are resistant to proteinases (3,5), whereas intracellular forms of these enzymes are susceptible to proteolytic attack (1). It is interesting to note that only three amino acid substitutions generate an enzyme that is more thermostable than the wild type xylanase, and thus, the dependence on calcium for this phenotype can easily be selected out.…”

Calcium Protects a Mesophilic Xylanase from Proteinase Inactivation and Thermal Unfolding

“…cellulosa is a modular enzyme comprising an NH 2 -terminal cellulose binding domain linked to a COOH-terminal catalytic domain. The catalytic domain is unique within Family 10 enzymes as it is the only xylanase described to date which contains a calcium binding site (located in loop 7) (11), and it is the only xylanase from either mesophilic or thermophilic microorganisms which has been shown to be sensitive to proteinases (15). The function(s) of the calcium binding domain in XYLA and the structural basis for the enzyme's sensitivity to proteinases remain to be elucidated.…”

Expression of Clostridium thermocellum Endoglucanase Gene in Lactobacillus bulgaricus and Lactobacillus plantarum and in vitro Survival Characteristics of the Transformed Lactobacilli

“…The selection of small intestinal proteinaseresistant cellulase was carried out using the method of Hall et al(1993).…”

“…In mono-gastric animal, the major plant cell wall components of cereals, primarily -glucans and arabinoxylans, form gel-like structures in the small intestines, trap nutrients, and therefore hinder enzymatic hydrolysis and absorption (Hall et al, 1993). The viscous polysaccharides can also cause severe gastrointestinal disorders (Hall et al, 1993).…”

“…The viscous polysaccharides can also cause severe gastrointestinal disorders (Hall et al, 1993). To improve the performances of non-ruminant animals, barley-based diets are often supplemented with endoglucanases and endoxylanases, particularly in poultry.…”