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Applicant:
Indian Institute of Technology (IIT) Roorkee 
Title:
A Novel phytase and process for enhancing phytase titer 
Inventor(s):
Sanjoy Ghosh and Richa Rani  
Applicant:
Indian Institute of Technology (IIT) Roorkee 
Application type:
Applied - 2010-04-29 
Application Number:
1024/Del/2010 
Applied at:
India 
Entered by:
Indian Institute of Technology (IIT) Roorkee 
Abstract:
This invention relates to a phytase of low Michaelis-Menten constant (Km) value from a fungal strain, Rhizopus Oryzae, wherein the phytase has the following physiochemical properties: low Michaelis-Menten constant (Km) of 2.42 × 10-4 mM; Molecular weight of 34 kDa; optimum temperature of 45ºC; optimum pH of 1.5 and 5.5; temperature stability up to 100 ºC; pH stability: stable in the range of pH 1.0 to 10.0, excellent enzyme activity at a physiological temperature (39 ºC), Pepsin resistant, low metal ions sensitivity, wide range of substrate specificity; acting on phytate, p-Nitro Phenyl Phosphate (PNPP), Nicotinamide adenine dinucleotide phosphate (NADP), Sodium-1-naphthyl phosphate (SNP), Fructose-1, 6-biphosphate (FBP), Ribose-5-phosphate (RBP), Glucose-1- phosphate (G-1-P), Phosphoenol pyruvic acid (PEP), Adenosine monophosphate (AMP), Adenosine diphosphate (ADP), and Adenosine Triphosphtae (ATP) as a substrate and a process for producing a novel phytase of low Michaelis-Menten constant (Km) value from a fungi by strain improvement process comprising the steps of, a. culturing and maintain the fungal strain on potato dextrose agar pertiplates and slants; b. harvesting viable spores from 6 day old fully sporulated slants by washing with tween-80 containing DW; c. stressing the viable spores of fungal strain by exposing the cells to heat and cold stress, subsequently; d. adjusting the spore suspension to 1×106 cfu (colony forming units on PDA plates) per ml for inoculation; e. inoculation one milliliter spore suspension of step d. to the solid state fermentation medium in a flask; f. subjecting the flask to stationary culture at a temperature of 30 ºC for 72 hrs for phytase production; g. adding 0.1% of tween-80 containing distilled water to the flask with shaking at 200 rpm at temperature of 30 ºC for one hour; h. separating the crude extracts containing the phytase by centrifugation at 12, 000 rpm for 10 min to obtain the supernatant containing crude phytase, i. resulting in enhance enzyme titer up to 60%.  

 
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