Phytase Yielding Strains Mutation Breeding And Integration Of Screening

High active phytase producing fungs-Aspergillus niger were selected by mutiple UV mutation, the definitions of phytase activity were analysised and the measure wavelength of the enzyme was modified, the factors that influence the preparation of protoplast were investigated. Base this, use protoplast-UV mutation and protoplast fusion to filter phytase produce Asp. niger.One Asp. niger with phytase activity about 2250 u/ml which selected by protoplast-UV mutation was used as original, prepared it into fungu-suspend-liquid, through UV mutation,daub on the filter-substract. pre-primary-selection was according to the lucency-circle, primary-selection was one fungus inoculate one flask to ferment, secondary-selection was using several high phytase activity fungus through one fungus inoculate 2-3 flasks to ferment. Then one or two high phytase activity fungus of the secondary selection were used in the next mutation cycle. Such mutation done for 5 circles, Asp-5-12, its phytase activity among 12200-12800 u/ml,improve 5.7 multpile from the original one, were selected out.The 4 definitions and 4 measure ways of phytase activity were analyzed, and their measure wavelength were also modified. In this study the definition is under the conditions of 55,pH4.6,from 0.1 % phytic acid sodium salt,release 1 nmol free phosphorus in one mintiue is one enzyme activity unit and the acetone method is the suitable measure way of the research was also decided. The measure wavelength was modified to 420 nm as preciseA protoplasting technique has been developed for the Aspergillus niger. The fungus age, enzyme system, osmotic stabilizer, Ca2+ concentration influenced on the formation of the protoplast have been confirmed.The result show that 16-18hr fermention, 32 1.0 % cellulase plus 1.0 % snailase confected by PBA(phosphate blend ammonium chloride) contant 0.2 % Ca2+, acted 3 hr is optimal for the protoplast production of Aspergillus. niger.To improve the mutation efficiency, protoplast irradiation by UV was done. Phy-19, which produce phytase that fit for the mono-gastric animal was sought out among these mutated fungus . Differ from others report, first enzyme activity apex of the enzyme located at pH2.6.Since the gastric-acid pH is among 1.5-3.5, the phytase that produced by Phy-19 maybe more fit for using as feed additive.The fusion fungus that produce phytase were also selected by protaplast-fusion methode. The fusion-experiment may be predigested before protaplast-fusion, it can simplify the fusion orperation,and enable the genetic substance single-direction tranfer.The fusion ratio is 0.403 %, This means the fusion was successd but more optimal condtions need further study.