24 3 2004 5 ACTA SCIENTIAE CIRCUMSTANTIAE Vol. 24,No. 3 May,2004 :025322468 (2004) 0320482205 :X523 :A PNAN5 ( Rhodococcus sp. strain PNAN5),,, 3 (, 100080) :, 16S rrna PNAN5 ( Rhodococcus sp. strain PNAN5). 20 40 ph 710 910 PNAN5 80 % 100 %, 2 10 mmol L - 1. 1,22, ( R. opacus) 2, 32. K m 35194 mol L - 1,V max 0184 mol L - 1 min - 1 mg - 1. : ; ;, 1,22 Isolation, identification of phenol2degrading Rhodococcus sp. strain PNAN5 and characterization of its ring2cleavage dioxygenases SHEN Xihui, LIU Zhipei, WANGBaojun, LIU ShuangJiang 3 Beijing 100080) ( Institute of Microbiology, Chinese Academy of Sciences, Abstract :A bacterial strain using phenol, naphthalene, benzoate and p2cresol as sole carbon and energy sources was isolated from waste sludge and was identified as Rhodococcus sp. strain PNAN5 according to morphological, physiological and its 16S rrna gene sequence. This strain is able to degrade both polycyclic aromatics such as naphthalene and monocyclic aromatics such as phenol. Under experimental condi2 tions, strain PNAN5 degraded 80 100 % of the added phenols at temperatures of 20 40 and ph of 710 910. The concentrations of phe2 nol at 2 10 mmol L - 1 did not significantly affect its removal efficiency. Its aromatic2degradation pathway is different from catechol 2,32dioxy2 genase of the R. opacus, but adopted to that of catechol 1,22dioxygenase. The kinetic constants of the catechol 1,22dioxygenase in cellular lysates were determined. The K m and V max of catechol 1,22dioxygenase for catechol were 35. 94 mol L - 1 and 0. 84 mol L - 1 min - 1 mg - 1 (protein), respectively. Keywords : Rhodococcus ; phenol ; naphthalene ; catechol 1,22dioxygenase,,. Gennaro [1 ] R7 ( Rhodoccocus opacus R7),,,. :2003207204 ; :2003212228 : 863 (2002AA601170) ; ( KSCXZ2SW2113) : (1972 ),, ; 3
3 : PNAN5 ( Rhodococcus sp. strain PNAN5) 483 PNAN5 ( Rhodoccocus sp. strain PNAN5).,. 1 111, 1 % (ph = 710 10 mmol L - 1, MgSO 4 7H 2 O 0103 g L - 1, [2 ] 5 ml L - 1, NH 4 Cl 10 mmol L - 1, 4 mmol L - 1 ),30 180 r min - 1 3 d, 3,,, ;,.,, 4 mmol L - 1. LB, 15 g L - 1. 600 nm. 112 ph ph, PNAN5 : (1) 2 mmol L - 1 ph 810, 20 h ; (2) 2 mmol L - 1 ph 30 20 h ; (3) ph 810 30 60 h. 113 [3,4 ]. 114 16S rrna DNA [5 ]. PCR 16S rrna, P f Pr, 16S rrna 8227 149521514. PCR, Blast. PNAN5 16S rrna GenBank AY090021. 115 42 [6 ]. 116 6000 g 15 min, ph 810 50 mmol L - 1 2,,20000 g 1 h,,. 117 1,22 2,32 260 nm( 260 = 16000 mol L - 1 cm - 1 ) 22 375 nm( 375 = 12000 mol L - 1 cm - 1 ) [7,8 ]. 1 mol. ph [9 ] :50 mmol L - 1 (ph = 410 517),50 mmol L - 1 (ph = 610 717),50 mmol L - 1 Tris2HCl (ph = 715 815),50 mmol L - 1 NaOH2 (ph = 815 1010). Bradford [10 ].
484 24 2 211 PNAN5. LB,,, (015 018) m (110 214) m,,,. (, ), ( R. pyridinivorans) [11 ] ( 1). 16S rrna 16S rrna 99 %. 16S rrna PNAN5 ( Rhodoccocus sp. strain PNAN5). 1 PNAN5 Table 1 Comparison of morphological and physiological characteristics between Rhodoccocus sp. strain PNAN5 and R. pyridinivorans Strain PNAN5 R. pyridinivorans Strain PNAN5 R. pyridinivorans + + + + - - - - - - + + + + + + VP - - - + - - + + + + - + + + 1 PNAN5 Fig. 1 Growth curves of strain PNAN5 with different substrates as sole carbon sources 212 PNAN5 PNAN5,. (4 mmol L - 1 ) PNAN5, ( 1), (115 d). PNAN5,OD 600 018. 213 ph PNAN5 2 mmol L - 1, PNAN5 24 h 100 %. ph PNAN5., 20 40, ph 710 910, PNAN5 80 % 100 %, ph, PNAN5., 2 10 mmol L - 1, 16 mmol L - 1,,
3 : PNAN5 ( Rhodococcus sp. strain PNAN5) 485. 2 ( A) ph( B) PNAN5 ( ) ( ) Fig. 2 Effects of temperature (A) and ph (B) changes on the growth ( ) of strain PNAN5 and on its degradation of phenol ( ) 214 PNAN5 1,22 (, A A), 2,32 (, ), [12 ]. 2,32. PNAN5, 1,22 2,32,,,PNAN5 1,22 0180 0111 units mg - 1, 2,32, PNAN5 1,22 ; 1,22 2,32,. 215 1,22 PNAN5, 1,22. 2, ( 32 ), 42. ph 2 PNAN5 1,22 Table 2 Substrate spectrum of catechol 1,22dioxygenase from Rhodococcus sp. strain PNAN5 Π(units mg - 1 ) 01110 32 01106 42 01106 42 0 0, ph 810 910 30 35,. 1,22, Lineweaver2Burk K m 35194 mol L - 1,V max 0184 mol L - 1 min - 1 mg - 1. 3,,, DNA shuffling
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