ACTA SCIENTIAE CIRCUMSTANTIAE. 16S rrna PNAN5 ( Rhodococcus sp. strain PNAN5). 20. ( Institute of Microbiology, Chinese Academy of Sciences,

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1 ACTA SCIENTIAE CIRCUMSTANTIAE Vol. 24,No. 3 May,2004 : (2004) :X523 :A PNAN5 ( Rhodococcus sp. strain PNAN5),,, 3 (, ) :, 16S rrna PNAN5 ( Rhodococcus sp. strain PNAN5) ph PNAN5 80 % 100 %, 2 10 mmol L ,22, ( R. opacus) 2, 32. K m 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 ) ( 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 % of the added phenols at temperatures of and ph of 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 mol L - 1 and mol L - 1 min - 1 mg - 1 (protein), respectively. Keywords : Rhodococcus ; phenol ; naphthalene ; catechol 1,22dioxygenase,,. Gennaro [1 ] R7 ( Rhodoccocus opacus R7),,,. : ; : : 863 (2002AA601170) ; ( KSCXZ2SW2113) : (1972 ),, ; 3

2 3 : PNAN5 ( Rhodococcus sp. strain PNAN5) 483 PNAN5 ( Rhodoccocus sp. strain PNAN5)., , 1 % (ph = 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 ), r min d, 3,,, ;,.,, 4 mmol L - 1. LB, 15 g L nm. 112 ph ph, PNAN5 : (1) 2 mmol L - 1 ph 810, 20 h ; (2) 2 mmol L - 1 ph h ; (3) ph h. 113 [3,4 ] S rrna DNA [5 ]. PCR 16S rrna, P f Pr, 16S rrna PCR, Blast. PNAN5 16S rrna GenBank AY [6 ] g 15 min, ph mmol L - 1 2,,20000 g 1 h,, ,22 2, nm( 260 = mol L - 1 cm - 1 ) nm( 375 = mol L - 1 cm - 1 ) [7,8 ]. 1 mol. ph [9 ] :50 mmol L - 1 (ph = ),50 mmol L - 1 (ph = ),50 mmol L - 1 Tris2HCl (ph = ),50 mmol L - 1 NaOH2 (ph = ). Bradford [10 ].

3 PNAN5. LB,,, ( ) m ( ) 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 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 ph PNAN5 2 mmol L - 1, PNAN5 24 h 100 %. ph PNAN5., 20 40, ph , PNAN5 80 % 100 %, ph, PNAN5., 2 10 mmol L - 1, 16 mmol L - 1,,

4 3 : PNAN5 ( Rhodococcus sp. strain PNAN5) ( 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, units mg - 1, 2,32, PNAN5 1,22 ; 1,22 2,32, ,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 ) , ph ,. 1,22, Lineweaver2Burk K m mol L - 1,V max 0184 mol L - 1 min - 1 mg ,,, DNA shuffling

5 486 24, [13 ].,. PNAN5,,. PNAN5 R7, R7 2,32, PNAN5 1,22,,. 4 1) PNAN5,. 2) PNAN ph % 100 %, 2 10 mmol L ) 2,32,PNAN5 1,22,, K m mol L - 1, V max 0184 mol L - 1 min - 1 mg - 1, : [ 1 ] Gennaro P D, Rescalli E, Galli E. Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o2xylene isolated from a polycyclic aromatic hydrocarbon contaminated soil [J ]. Res Microbiol, 2001, 152 (9) : [ 2 ] Konopka A. Isolation and characterization of a subsurface bacterium that degrades aniline and methylanilines [J ]. FEMS Microbiol Lett, 1993, 111 (1) : [ 3 ] Krieg R, Holt J G. Bergey s manual of determinative bacteriology ( 9th edition) [ M]. Baltimore, Phladelphia : Williams g Wilkins,1994 [ 4 ],. [M]. :,2001 [ 5 ] Treadway S L, Yanagimachi K S, Lankenau E. Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24 [J ]. Appl Microbiol Biotechnol, 1999, 51 (6) : [ 6 ] Folsom B R, Chapman P J, Pritchard P H. Phenol and trichloroethylene degradation by Pseudomonas cepacia G4 : Kinetics and in2 teractions between substrates [J ]. Appl Environ Microbiol, 1990, 56 (5) : [ 7 ] Liu Z, Yang H, Huang Z. Degradation of aniline by newly isolated, extremely aniline2tolerant Delftia sp. AN3 [J ]. Appl Microbiol Biotechnol, 2002, 58 (6) : [ 8 ] Strachan P D, Freer A A, Fewson C A. Purification and characterization of catechol 1,22dioxygenase from Rhodococcus rhodochrous NCIMB and cloning and sequencing of its cata gene [J ]. Biochem J, 1998, 333 (3) : [ 9 ] Briganti F, Pessione E, Giunta C. Purification, biochemical properties and substrate specificity of a catechol 1,22dioxygenase from a phenol degrading Acinetobacter radioresistens [J ]. FEBS Lett, 1997, 416 (1) :61 64 [ 10 ] Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein2 dye binding [J ]. Anal Biochem, 1976, 72 (1) : [ 11 ] Yoon J H, Kang S S, Cho Y G. Rhodococcus pyridinivorans sp. nov., a pyridine2degrading bacterium[j ]. Int J Syst Evol Microbi2 ol, 2000,50 (6) : [ 12 ] Pessione E, Divari S, Griva E. Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme[j ]. Eur J Biochem, 1999,265 (2) : [13 ] Furukawa K. Engineering dioxygenases for efficient degradation of environmental pollutants [J ]. Curr Opin Biotechnol, 2000, 11 (3) : [ 14 ] Hinteregger C, Leitner R, Loidl M. Degradation of phenol and phenolic compounds by Pseudomonas putida EKII [J ]. Appl Microbi2 ol Biotechnol, 1992, 37 (2) :