Enhancing the Thermal Tolerance and Gastric Performance of a Microbial Phytase for Use as a Phosphate-Mobilizing Monogastric-Feed Supplement (original) (raw)
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Site Directed Mutagenesis to Improve E. Coli Phytase Activity for Animal Feed
Phytate is largely unavailable to monogastric animal such as swine, poultry and fish, as they lack of sufficient endogenous enzymatic activity to hydrolyze phytate. The result is the elimination of precious nutrients that would be beneficial to their growth; furthermore, they will excrete most of the indigestive phytate which can contribute to phosphorus being over applied to the land. Phosphorus has a beneficial impact on vegetative growth on land as well as marine vegetation, causing an increased growth of weeds. This enhanced vegetation consumes large amounts of oxygen, resulting in the loss of aquatic life and ultimately contributes to water pollution and eutrophication of ground water and aquatic environment. Phytase, a type of histidine acid phosphatase hydrolyzes phytin phosphorus and when present in an animal's digestive tract, benefits the animal while reducing total phosphorus levels in manure. Computer modeling has been used to identify and examine the active site of phytase. The factors influencing the ligand binding strength in the active site were analyzed and computational site directed mutagenesis experiments were carried out to evaluate the effects of mutations on the binding strength before and after mutation. From the directive results of computational studies, point mutation was introduced by site directed mutagenesis using polymerase chain reaction (PCR). The activity was measured by kinetic characterization with phytate as a substrate. Decrease in K M notable in all functional mutants indicates that all mutant shows increment in substrate binding. Two functional mutants showed improvement in phytase activity and thermostability.
Site Directed Mutagenesis to Improve E. Coli Phytase Activity for Animal Feed 1
Phytate is largely unavailable to monogastric animal such as swine, poultry and fish, as they lack of sufficient endogenous enzymatic activity to hydrolyze phytate. The result is the elimination of precious nutrients that would be beneficial to their growth; furthermore, they will excrete most of the indigestive phytate which can contribute to phosphorus being over applied to the land. Phosphorus has a beneficial impact on vegetative growth on land as well as marine vegetation, causing an increased growth of weeds. This enhanced vegetation consumes large amounts of oxygen, resulting in the loss of aquatic life and ultimately contributes to water pollution and eutrophication of ground water and aquatic environment. Phytase, a type of histidine acid phosphatase hydrolyzes phytin phosphorus and when present in an animal's digestive tract, benefits the animal while reducing total phosphorus levels in manure. Computer modeling has been used to identify and examine the active site of phytase. The factors influencing the ligand binding strength in the active site were analyzed and computational site directed mutagenesis experiments were carried out to evaluate the effects of mutations on the binding strength before and after mutation. From the directive results of computational studies, point mutation was introduced by site directed mutagenesis using polymerase chain reaction (PCR). The activity was measured by kinetic characterization with phytate as a substrate. Decrease in K M notable in all functional mutants indicates that all mutant shows increment in substrate binding. Two functional mutants showed improvement in phytase activity and thermostability.
Canadian Journal of Microbiology, 1999
The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44 708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60°C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K m and V max values of 0.79 mM and 3165 U·mg -1 of protein for phytase activity and 5.5 mM and 712 U·mg -1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K m and V max values. The enzyme exhibited similar properties to the phytase purified by , except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the Nterminal amino acid sequence was different. The Bradford assay, which was used by for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U·mL -1 . The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.
Cloning and High-Level Expression of the Enzymatic Region of Phytase in E. coli
International Journal of Peptide Research and Therapeutics, 2019
Phytase is an important enzyme poses great nutritional significance in humans and monogastric animals diets. The phytase production yield using wild sources, including microorganisms , plants, and animals is sorely low. Thus, recombinant expression of phytase has received increasing interest for achieving production rate. Escherichia coli is the most preferred host for expression of heterologous proteins but overexpression of recombinant phytase in E. coli, met with limited success due to the sequestration of the enzyme into inclusion bodies. In the present study, artificial phytases gene with excellent thermostability and activity were designed by detecting the enzymatic region of the E. coli phytase gene by employing bioinformatics tools. Then, the PCR amplified recombinant gene was expressed in E. coli and the active enzyme was recovered from inclusion bodies. Employing cysteine amino acid in the dialysis buffer succeed to the superior activity of the enzyme with a specific activity of 73.8 U/mg. The optimum temperature and pH for enzyme activity were determined at 60 °C and 4, respectively. The novel recombinant enzyme illustrated perfect thermostability up to 70 °C with maintenance 75% of its activity. The enzyme was stable at pH range of 2-10. Moreover, the effects of ions and chemical compounds on enzyme stability and activity were assessed.
In vitro properties of phytases from various microbial origins
International Journal of Food Science and Technology, 2002
For the evaluation of the effectiveness of phytase preparations as feed additive, in vitro properties like temperature optimum, temperature stability, pH optimum and pH profile or proteolytic stability are of utmost importance. Although at present all commercial phytase preparations authorized as feed additives in the EU are produced by recombinant filamentous fungi and have similar in vitro properties (acidic pH optimum, narrow pH range, low thermostability) the diversity of microbial phytases is great. Microbial sources for phytases span from fungi and yeasts to bacteria. Some of the naturally occurring phytases were identified to have high thermostability and a broad pH range (e.g. Aspergillus fumigatus phytase). The bacterial Bacillus phytases generally differ from other phytases, having a pH optimum from 7.0 to 8.0, being Ca 2+ dependent and highly specific for phytate. Thermostability can considerably be increased by protein engineering. A so-called Consensus phytase encoded by a synthetic gene was found to be stable in aqueous solutions at 70°C and in feed at pelleting temperatures of 80-90°C. The rate and site of inactivation of feed enzymes in the digestive tract are determined by their susceptibility to proteolytic enzymes. Highest residual activities after incubation in the presence of pepsin or in supernatants of stomach digesta was observed for Escherichia coli and Consensus phytases, while the Bacillus phytase was found to be most resistant to pancreatin. Comparative studies on in vitro properties of enzymes intended for use as feed additives provide valuable information for prediction of in vivo effectiveness.
2010
Phytase is an important enzyme in the food/feed industry. It catalyzes the hydrolysis of phytate, an anti-nutrient compound present in cereals and grains, to release orthophosphate and myo-inositol-6-phosphate with lower degrees of phosphorylation. Phytic acid is a strong chelator capable of complexing with a variety of metal ions under neutral and alkaline conditions, as well as with proteins and starch under acidic conditions. Treatment with phytase increases not only the bioavailability of inorganic phosphorus but also the digestibility of proteins. Moreover, it improves absorption of minerals from food/feed. The action of phytase also contributes towards reducing the pollution in surface-and ground water caused by the phytate and phosphorus run-off from manure in intensive livestock regions.
Molecular Biotechnology, 2008
An extracellular phytase from Bacillus subtilis US417 (PHY US417) was purified and characterized. The purified enzyme of 41 kDa was calcium-dependent and optimally active at pH 7.5 and 55°C. The thermal stability of PHY US417 was drastically improved by calcium. Indeed, it recovered 77% of its original activity after denaturation for 10 min at 75°C in the presence of 5 mM CaCl 2 , while it retained only 22% of activity when incubated for 10 min at 60°C without calcium. In addition, PHY US417 was found to be highly specific for phytate and exhibited pH stability similar to Phyzyme, a commercial phytase with optimal activity at pH 5.5 and 60°C. The phytase gene was cloned by PCR from Bacillus subtilis US417. Sequence analysis of the encoded polypeptide revealed one residue difference from PhyC of Bacillus subtilis VTTE-68013 (substitution of arginine in position 257 by proline in PHY US417) which was reported to exhibit lower thermostability especially in the absence of calcium. With its neutral pH optimum as well as its great pH and thermal stability, the PHY US417 enzyme presumed to be predominantly active in the intestine has a high potential for use as feed additive.
Phytase: A Boom in Food Industry
A B S T R A C T Phytate [myo-inositol(1,2,3,4,5,6)hexakisphosphate] have been considered negative in food industry as it is the main storage form of phosphorus (P) in many plants but this phytate-bound P is not available to non – ruminants as they don't have these endogenous enzyme and hence the availability and digestibility of phytate phosphorous is very low in these animals. Phytic acid has antinutrients behavior and has a potential for binding positively charged multivalent cations, proteins and amino acids in foods. Phytase [myo-inositol(1,2,3,4,5,6)hexakisphosphate phosphohydrolases], a phytate-specific phosphates is an enzyme that can break down the undigestible phytic acid and thus release digestible phosphorus, calcium & other nutrients. Research in the field of animal nutrition has put forth the idea of supplementing phytase enzyme, exogenously, so as to make available bound nutrients from phytic acid and, thereby helps in food processing and digestion in the human ali...