| Alkane hydroxylase, generally existed in hydrocarbon degrading bacteria,could metabolize a wide range of substrate and plays an important role during the cleaning up of environmental contaminants.Up to now, there are more research about alkane hydroxylase effecting on short chain alkanes, but alkane hydroxylase effecting on mid-long and long chain alkanes has been received little attention. In our previous studies, Pseudomonas aeruginosa NY3, which could rapidly degrade polycyclic aromatic hydrocarbons and middle and long chain alkanes, was isolated from petroleum- contaminated soil samples. The function of two alkane hydroxylase genes of P.aeruginosa NY3 was studied in this paper and the results were important to reveal the mechanism of alkane degradation by P.aeruginosa NY3.By using molecular biological techniques,the three alk B disruption mutants(named NB1 D,NB2D and NB12DD)of NY3 strain were constructed. Compariso n of the differences between alk B disruption mutants and wild-type NY3 strain in the degradation of hydrocarbon by using gas chromatography(GC), Gas chro matography-mass(GC-MS) and other analysis methods.To suggests alk B1 and alk B2 mainly function in alkane utilization by NY3 strain. Main results of this pa per are described as follows:(1)In order to understand more about alkane hydroxylase of P.aeruginosa NY3 strain, two genes homologous to the alk B gene were isolated from the NY3 strain, and three alk B disruption mutants(named NB1 D,NB2D and NB12DD)of NY3 strain were constructed.(2)When wild-type strain NY3 and its mutants grown on n-alkane as the sole carbon and energy source, alk B1 was more effective on the degradation of short-chain alkane(C13 and C14) than alk B2, alk B2 was more effective on the degradation of middle- and long-chain alkane(C16,C19,C21, C22,C24,C25 and C28) than alk B1.NB12 DD can grow on n-alkane as the sole carbon and energy source showed that except alk B1 and alk B2, there are also other genes existed in strain NY3 which could degrade n-alkanes.(3)When P.aeruginosa strain NY3 and its mutants grown on crude oil as the sole carbon and energy source, the difference of alk B1 and alk B2 effected on short-chain n-alkanes was obvious, and the degradation crude oil of alk B1 and alk B2 gene deleted mutant decreased on middle- and long-chain alkane(C16-C32) on the early stage. On the late stage, short-, mid-long and long-chain alkane(C14-C32)was mainly degraded by alk B1 and alk B2, and alk B2 was more effective than alk B1.(4)P.aeruginosa NY3 and its mutants could degrade both n-alkanes and polycyclic aromatic compounds contained in crude oil. The degradation rate of phenanthrene and pyrene contained in crude oil by wild type of P.aeruginosa NY3 was higher than its mutant strains. The results demonstrated that alk B1 and alk B2 played an important role in polycyclic aromatic compounds degradation by wild type of strain NY3.(5)The accumulation pattern of intermediates by wild-type NY3 and its mut ants(NB1D,NB2 D and NB12DD) for 6 days showed that the degradation mech anism of phenanthrene was not identical under the same condition. Trans-2-Bute nedioic acid as an intermediates of phenanthrene degradation could be produced of by all strains through oxidation, decarboxylation and ring cleavages. Althoug h the intermediates accumulated by wild-type NY3 and double mutant strain NB12 DD were more complicated and difference,but the incomplete-ring and red uctive intermediates, including trans-2-Butenedioic, 3(2-hydroxylphenyl) acrylic, succinic Acid, 1,2-dihydrogen-1-phenanthrene,3-(1-formaldehydenaphthyl)acrylic an d 5(2-formaldehy-phenyl)-3-Oxo-4-pentenoic acid, were more easy to be accumul ated. The intermediates accumulated by single mutant strain NB1 D and NB2 D were more simple.The above results laid a solid foundation for further studying on hydroxylase enzymes of Pseudomonas aeruginosa NY3 effecting on middle and long chain alkanes and and developing the degradation capacity of refractory organic compound by bacteria. |
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