Many feed ingredients in use in monogastric diets contain significant quantities of antinutritional factors (ANF) which limit both their feed value and their use. Almost all enzymes currently being used address such factors to varying degrees, allowing for more economic utilization of raw materials. However, animal response to xylanase, beta-glucanase and even phytase utilization reported in the literature tends to vary. Factors such as enzyme source, ingredient variety and environment under which the ingredient was grown, stored and processed into animal feed, age of animal, interaction with other dietary ingredients, and health status are shown to affect significantly the response obtained. As a result, the mode of action of xylanases and beta-glucanases is still debated due to too much emphasis being placed on interpretation of individual trial results without regard to the interactive factors or the literature dataset as a whole. Better understanding of such factors will improve data interpretation. While results with phytase are not subject to such extreme variation, they are nevertheless inconsistent in the degree to which inorganic phosphorus can be replaced by this enzyme. Greater understanding of the ANF and factors which interact to govern the response to added exogenous enzymes will undoubtedly improve the economic return and confidence in their use. Improved knowledge of ANF structure will result in development of enzymes directed towards far more specific targets, which enhances the likelihood of success and should reduce the overall enzyme usage.

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Exogenous enzymes for pigs and poultry

The review deals with phytase-producing microorganisms along with optimum conditions for its production. Various methods used for purifying phytases and their characteristics are discussed. Heterologous gene expression, cost-effective large-scale phytase production, and various biotechnological applications of the enzyme in animal feed and food industries are also discussed.

http://www.protocol-online.org/forums/uploads/monthly_07_2010/msg-6644-055766200%201280492228.ipb

Phytases: microbial sources, production, purification, and potential biotechnological applications.

We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces. An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at −29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.

Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification

This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 473-484, 1997.

Production of recombinant proteins in transgenic plants: practical considerations

The Bacillus subtilis strain VTT E-68013 was chosen for purification and characterization of its excreted phytase. Purified enzyme had maximal phytase activity at pH 7 and 55°C. Isolated enzyme required calcium for its activity and/or stability and was readily inhibited by EDTA. The enzyme proved to be highly specific since, of the substrates tested, only phytate, ADP, and ATP were hydrolyzed (100, 75, and 50% of the relative activity, respectively). The phytase gene (phyC) was cloned from the B. subtilis VTT E-68013 genomic library. The deduced amino acid sequence (383 residues) showed no homology to the sequences of other phytases nor to those of any known phosphatases. PhyC did not have the conserved RHGXRXP sequence found in the active site of known phytases, and therefore PhyC appears not to be a member of the phytase subfamily of histidine acid phosphatases but a novel enzyme having phytase activity. Due to its pH profile and optimum, it could be an interesting candidate for feed applications.

Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis.

Phytate is the main storage form of phosphorus in many plant seeds, but bound in this form it is a poor nutrient for monogastric animals Its availability can be significantly increased by addition of the enzyme phytase, which releases phosphate from the substrate, phytate We have engineered a phytase from Aspergillus niger in tobacco seeds, providing a stable and convenient packaging of the enzyme that is directly applicable in animal feed The enzyme was expressed as 1 percent of the soluble protein in mature seeds In in vitro tests that simulated chicken crop and stomach conditions, release of phosphate from feed by addition of transgenic seeds was demonstrated Supplementation of broiler diets with transgenic seeds resulted in an unproved growth rate, comparable to diets supplemented with fungal phytase or phosphorus Addition of phytase-transgenic seeds to animal feed thus obviates the need for inorganic phosphorus supplementation, and is environmentally desirable because of the reduced excretion of phosphorus

Hans A. J. Versteegh

Papers

Phytase-containing Transgenic Seeds as a Novel Feed Additive for Improved Phosphorus Utilization