Study On Breeding Of Naringinase-producing Strain And Characterization Of The Enzyme

Naringin was one of the bitterest components in citrus juice, which obviously decreased the quality of citrus juice. Enzymatic hydrolysis of naringin had many advantages than traditional debittering method, such as simple techniques, wild conditions, little contamination, original nutritional value and so on. In this study, a wild naringinase producing strain was screened from mouldy peel of grapefruit, then UV, UV, UV+NTG, and ion implantation were used to mutagenize the wild strain in order to improve naringinase production potential, and characterization of naringinase was also studied. The main conclusions were as follows:(1)Original Strain ScreeningThrough clarity circle method 52 wild strains with naringinase producing potential were obtained from mouldy peel of grapefruit, and after secondary screening one strain named M 1 was chose for further study due to its higher naringinase activity of 140.8 U/mL. The strain M 1 was identified as Penicillium sp. by morphology and taxonomy methods.(2)Mutation Breeding of Penicillium sp. M 1 by UV, NTG, UV+NTGIn order to improve naringinase producing capacity UV, NTG and UV+NTG were applied to mutagenize Penicillium sp. M 1. The optimum UV treating conditions were as follows:the dilution of the spore suspension (107/mL) was 10"5, the 15 W UV lamp was used as UV light with 30 cm radiation distance and 5 minutes treating time. Through UV mutation a mutant strain named U15 was obtained, the naringinase activity of which reached 334.3 U/mL, about 137.4% higher than original strain.Later Penicillium sp.U 15 was treated with NTG.800μg/mL for 2 h and through the clarity circle screening and secondary screening experiments in rotation-flask, the high-yielding mutant strain Penicillium sp. N 09 was obtained, the naringinase activity of which was 380.5 U/mL, almost 14.4% higher than that of the Penicillium sp.U 15. Then Penicillium sp. N 09 was treated with 800μg/mL NTG and 4 minutes of UV, and a high yield mutant strain Penicillium sp. UN 02 was gotten. After the clarity circle screening and secondary screening experiments in rotation-flask, the naringinase activity of which reached 334.3 U/mL, almost 18.0% higher.(3)N+ ion Mutagenesis of Penicillium sp. UN 02Penicillium sp. UN 02 was treated with 15 KeV and 192 X 1013 ions/cm2 of N+ After the clarity circle screening and secondary screening experiments in rotation-flask, a high-yield strain Penicillium sp.1523 was obtained, the naringinase activity of which reached 573.6 U/mL, almost 27.8% higher than that of Penicillium sp.UN 02. The genetic stability of Penicillium sp.1523 was comparative stable.(4)Characterization of Naringinase from Penicillium sp.1523An extracellular naringinase that hydrolysed naringin into rhamnose and glucose was purified from the culture filtrate of Penicillium sp.1523 The enzyme had an optimum temperature of 50℃and pH of 4.0, Ca2+ Mg2+ stimulated the enzyme activity at a concentration of 2.5～100 mM, Mn2+,Zn2+,EDTA stimulated the enzyme activity at a concentration of 2.5～30 mM, Cu2+ stimulated the enzyme activity at a concentration of 2.5-50 mM, Fe2+,SDS completely inhibited the enzyme activity.