SCREENING OF FUNGAL MUTANT STRAIN WITH HIGH LACCASE YIELD BY N + 2IMPLANTATION AND ENZYME PRODUCTION CONDITION OPTIMIZATION

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1 ,23 (2) : Journal of Nuclear Agricultural Sciences : (2009) N (1., ; 2., ) : Pleurotus ostreatus WY01, N + RBBR2PDA ATBS, 1 ADW208 (SM), 2180, 7178 UΠg, ADW208, ; ADW208 ;ph ;ABTS, - 80 ;, ph 1510gΠL 012gΠL 512, 8133 UΠg : ;N + ;; SCREENING OF FUNGAL MUTANT STRAIN WITH HIGH LACCASE YIELD BY N + 2IMPLANTATION AND ENZYME PRODUCTION CONDITION OPTIMIZATION WANGLi2sheng 1 ZHU Hong2long 2 LI Chun2feng 1 CHENG Mao2ji 1 CAI Hai2ying 1 (11 College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui ; 21 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu ) Abstract :The basidiospores of Pleurotus ostreatus WY01 were exposed to N + beam and the treated basidiospores were screened in RBBR2containing PDA plates. Then the laccase activities of the selected strains were determined by ABTS, and a mutant named ADW208 with high yield of laccase was obtained. The maximum activity of laccase of ADW208 increased up to 7178 UΠg, 218 times of the original stain, and its laccase production ability was stable in high carbon and low nitrogen rape straw solid medium (SM). Results showed that glucose as carbon source was obviously superior to sucrose, maltose, wheat bran and soluble amylum in soild culture for ADW208. Ammonium tartrate as nitrogen source was more suitable than other nitrogen sources for the laccase secretion. The optimal initial ph was 510 or 6101 The ability of laccase production clearly increased by ABTS and veratryl alcohol induction, but was inhibited by Tween 801 When the optimum parameters for laccase production got from orthogonal design were as follows :glucose 15gΠL, ammonium tartrate 012gΠL,pH 512, the peak value of Lac activity was 8133UΠ g. Key words : Pleurotus ostreatus ; N + implantation mutation ; condition optimization ; laccase (Laccase Lac EC ),,, (780 mv), H 2 O 2, [1 ], [2 ] Pleurotus ostreatus : : : ( ) ;( A) : (19572),,,,, E2mail edu. cn : (19682),,,,, E2mail com ' China Academic Journal Electronic Publishing House. All rights reserved.

2 2 N + 229,,, [3 7 ], Lac 1100 UΠg, Ivana Eichlerova Pleurotus ostreatus Lac 10, 51687UΠg, [8 12 ],,,,, [13 ], [14 18 ],, N + Pleurotus ostreatus, Pleurotus ostreatus, Pleurotus ostreatus WY PDA (/ L) : 20g, KH 2 PO 4 3g,MgSO 4 7H 2 O 115g,VB 1 10mg, 20g, 20g,pH (LM) (ΠL) [19 ] : 1010g, 012g, KH 2 PO 4 210g, CaCl 2 011g, MgSO 4 7H 2 O 015g, 70ml, 011gΠL V B1 10ml,011molΠL (ph412) 100ml (ΠL ) : MgSO 4 7H 2 O 013g, MnSO 4 H 2 O 015g, NaC1 110g, FeSO 4 7H 2 O 011g, CoCl 2 6H 2 O 01185g, CaCl g,ZnSO 4 7H 2 O 0118g,CuSO 4 5H 2 O 011g,A1K(SO 4 ) 12H 2 O 010lg,H 3 BO g,NaMoO 4 2H 2 O 01012g, 115g :12510 g 35, 1 3 (mπv) LM,SM, (121, 30 min), PDA2RBBR : 510 mgπml RBBR 60 PDA, RBBR 625 gπml RBBR2PDA ph : 1molΠL HCl 1molΠL NaOH LM ph , ( Tween280),;ABTS (2,2 2 2 ( ),C 18 H 24 N 6 O 6 S 4 ) (Utra pure), AMRESCO ; (C 9 H 12 O 3 ), RBBR ( Remazo brilliant blue R ), SIGMA2 ALDRICH ; ; TU21901 ;ph225 ph PDA,25 7d, PDA,28 7d [20 ], :,28 Pleurotus ostreatus WY01, 20,, 20,,, Πml, l,, N +, 30min [20 ], ionsπcm , PDA,28, Pleurotus ostreatus WY01 PDA, 60, 20d,, 10 %,, PDA N +, PDA2RBBR,28,

3 RBBR, PDA, - 4 (10 8 Πml), 3ml, 28 30d, g, 1210ml 4,, 200rpm 1h, ; 410ml 200rpm 15min, 1,, 5000rpm 15min,, Lac Lac ABTS [21 ] :25,pH412,1 molπl ABTS 1 (U) N + N + Pleurotus ostreatus WY01 1, ( ionsπcm 2 ),,, ionsπcm 2,,,,,,,,,,, ;, 1 N + Fig. 1 Effect of N + implantation on survival rate of basidispones ;,,,,,,,,, 2,, ionsπcm 2, ;,, ionsπcm 2 :DNA, ; DNA,,,,DNA,, 2 N + Fig. 2 Effect of N implantation on mutation rate , ADW201 ADW220, Pleurotus ostreatus RBBR ( 1) 2 ADW208 ADW211, SM 0-30d 3,ADW208 ADW211 Pleurotus ostreatus, 7178UΠg 6111UΠg, 2178UΠg ADW208 Pleurotus ostreatus, 22d

4 2 N d,ADW208 26d 2 ADW208 ADW211, PDA 5,, ADW208 2 ( P 1 P 5 ) ( 4), 1 RBBR2PDA Table 1 Discoloration of original and some primary strains original strain screening strains of P. ostreatus cultured on PDA medium containing RBBR culture time (d) ADW ADW ADW ADW ADW ADW ADW208 ADW211 P 1 P 2 P 3, P 4 ( ) ADW ADW208 (LM),, ( 5), ADW208,, 22 (7178 UΠg), ( 0156 UΠg) : - ; ; Note : - no discoloration, discoloration gradually, whole discoloration 5 P. ostreatus ADW208 Fig. 5 Effect of carbon sources on Lac production of mutant ADW208 of P. ostreatus 3 ADW208 ADW211 Fig. 3 The Lac production of original strain, ADW208 and ADW211 of P. ostreatus on solid cultivation 4 ADW208 Fig. 4 The Lac production of subcultures of mutant ADW208 of P. ostreatus 214 ADW208 LM,, 6,,7178UΠg, % 215 ph ADW208 LM ph , ph ADW208 ( 7), ph , 216 ADW208 ABTS (1mmolΠL) - 80 (1 %) (1mmolΠL) 3, 4 LM, ADW208 ( 8),ABTS ADW208,, ADW208,,

5 P. ostreatus ADW208 Fig. 6 Effect of nitrogen sources on Lac production of mutant ADW208 of P. ostreatus 7 ph P. ostreatus ADW208 Fig. 7 Effect of initial ph on Lac production of mutant ADW208 of P. ostreatus ph, L 16 (4 3 ) , : ( P < 0101), ( P < 0105), ph ( P > 0105) A3B2C1, 1510gΠL, 012gΠL,pH 512,12, UΠg, ( 9) No 2 Table 2 The results of the orthogonal test A glucose (gπl) and analysis of its range B ammonium tartrate (gπl) C ph Lac (UΠg) 1 1(5) 1 (011) 1(512) (5) 2 (012) 2(514) (5) 3 (014) 3(516) (5) 4 (018) 4(518) (10) 1 (011) 2(514) (10) 2 (012) 1(512) (10) 3 (014) 4(518) (10) 4 (018) 3(516) (15) 1 (011) 4(518) (15) 2 (012) 1(512) (15) 3 (014) 2(514) (15) 4 (018) 4(518) (20) 1 (011) 3(516) (20) 2 (012) 2(514) (20) 3 (014) 1(512) (20) 4 (018) K K K K R P. ostreatus ADW208 Fig. 8 Effect revulsives on Lac production of mutant ADW208 of P. ostreatus sources A glucose B ammonium tartrate C ph initial ph 3 Table 3 Results of anova analysis of Lacs DF anova SS mean Square F F value Pr > F

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