Study On The Selection Of Spj0104 By Low-energy N+ion Implantation

Study on synthetic and semi synthetic antibiotic is one of the important ways to explore and develop new antibiotic. D-hydroxyphenylglycine (D-pHPG) is the most important precursor used for the synthesis of semi synthetic lactam types of antibiotic and the product attracting keen competition in the field of pharmacy. Enzyme method is rather better than the chemical one which brings severe pollution and poison. In enzyme method, P-hydroxyphenyldantoin (p-HPH) was transformed into N-Carbamoyl-D-P-hydroxy-phenyldantoin (NC-D-pHPG) by Dhydantoinase (DHase), and then NC-D -pHPG was transformed into D-pHPG by Ncarbamyl Danimo-acid amidohydrolase (DCase). Since the enzyme activity of the existing strain was too low, it was very significant to improve the enzyme activity of the strain by breeding high-yield strain and increasing the enzyme using rate with new techniques.The enzyme activity of the starting strain Spj0104 was validated at first. Then low energy ion implantation and single factor experiments were used to find the optimal energy and dosage. With the reference parameters of energy and dosage orthogonal optimization plan was designed and carried out. Under the optimal condition Spi03 selected from the strains under first ion implantation and Sp902 (one of the MW mutants) were arranged new ion implantation. High-yield strain was screened out among all the mutants. Moreover by orthogonal optimization the culture medium and culture condition were investigated, and then the result of optimization was used to fermentation. In the end genetic stability was conformed. The results of all experiments show as follow:(1) Spj0104 appears like short bacilliform under microscope. Its growth period was rather too long with delaying time from 0 to 21 h. In the logarithm time,21h to 57h, the value of OD6oo increased quickly. While in the stable time,57h to 69h, it kept relatively steady. The enzyme activity was 0.118 955 u/mL determined by HPLC method.(2) In the preparation work of low energy ion implantation, the material should be dried with asepsis air. At 30 min, velum was dry and at 50 min, it was completely dry. The more time increased, the lower the living rate of the strain was. Therefore,30min was the optimal time of drying.(3) Single factor experiments were made basing on fixed pulse, fixed interval time and different dosages of repetition.30KeV was confirmed as the optimal energy. At 30KeV, the curve of the living rates of the strains under variable dosages in the experiment looked like a saddle. According to the papers reported, several dosages (60×1014ions/cm2, 80×1014ions/cm2,100×1014ions/cm2,120×1014ions/cm2,14×1014ions/cm2) that at the two sides of "the saddle" were chosen to investigate the changes of the mutants. Taking enzyme activity, living rate,etc. into consideration,60×1014ions/cm2 was the best dosage. On the basis of optimal energy and dosage, orthogonal optimization was made on energy, dosage, pulse and interval. The results showed the optimal condition was 20KeV,40x1014ions/cm2,15S pulse and 15S interval。(4) Good strain Spi03 whose enzyme activity was 0.2422u/mL, was a mutant strain suffered from ion implantation and Sp902 was a mutant strain had been treated by MW.Both of them were treated with low energy ion implantation. Two good strains Sp208 and Sp201 were screened out. Their enzyme activities are 0.327lu/mL and 0.2382u/ml respectively, and increasing by 35.53 and 28.41 percent.The mutant strains treated by MW and ion implantation changed a lot in forms and many of them were low enzyme producing strain. Strain Sp206, enzyme activity was 0.1536 u/mL. Though total enzyme activity descended, DHase activity increased a lot.(5) The optimal fermentation medium listed below:Corn steep liquor 3.0%, Glucose 1.5%; Inducement 3.0%;Nacl 0.3%;(NH4) SO40.1%; MgSO40.05%;CoCl20.01%.The optimal fermentation conditions were temperature of 30℃, initial pH of 7.0, capacity of 40mL and fermentation time of 22h. The fermentation time was 2h shorter than the original. Being cultured under optimal medium and fermentation conditions, the strain Sp208's enzyme activity reached 0.402u/mL, increasing by 22.89 percent. Five generations'of culture showed good genetic stability.