Graduation date: 1995

Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme.

The research described in this thesis was aimed at understanding how particle size, porosity, and enzyme accessible surface area influence the rate of saccharification of microcrystalline cellulose. Microcrystalline cellulose (MCC) is a commonly used substrate for the study of cellulolytic enzymes. MCC preparations of different particle size are commercially available. In this study, MCC preparations having average particle sizes of 20, 50, and 90 μm were analyzed with respect to their enzyme accessible surface area, chemical and physical properties and rates of enzymatic saccharification. Saccharification studies were done using a commercially available cellulase preparation from Trichoderma reesei. Pore volume distributions were determined from solute exclusion experiments. Internal surface areas were calculated based on the application of the lamellae model to the pore volume distribution data. External surface areas were calculated based on the average particle size of each MCC preparation assuming that the particles could be represented as solid spheres. The different MCC preparations were found to have nearly equivalent enzyme accessible surface areas per unit weight. Greater than 99 % of the total enzyme accessible surface area for each MCC preparations was found to be within the porous structure of the particles. Enzymatic saccharification experiments demonstrated that the smaller particle size MCCs were more readily digested than those of larger particle size. The similarity of the three MCC preparations with respect to chemical and physical properties (other than particle size), pore volume distribution, and total enzyme accessible surface area suggests that a rate limiting factor in the enzymatic digestion of MCC is a resistance attributable to diffusion within the capillary network of these insoluble substrates. / Graduation date: 1996

Chlorinated phenols are toxic compounds and often released into natural environments from improper disposal or non-point sources. One important factor in assessing the environmental fate and transport is the rates of microbial degradation in soils and natural waters. In this study, eight chlorophenols (4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 3,4-dichlorophenol (3,4-DCP), 3,5-dichlorophenol (3,5- DCP), 2,3,5-trichlorophenol (2,3,5-TCP), 2,4,5-trichlorophenol (2,4,5-TCP), 3,4,5- trichlorophenol (3,4,5-TCP), and 2,4,6-trichlorophenol (2,4,6-TCP)) were chosen as the model compounds because they represent the major degradation products for the anaerobic metabolic products of pentachlorophenol (PCP). Aerobic degradation rates of all eight compounds were determined under controlled laboratory conditions. A mixed bacterial culture was seeded from a municipal wastewater treatment plant and were grown with acetate as a primary substrate and the eight chlorophenols as secondary substrates. Rates of degradation were measured in a batch reactor with an initial concentration of acetate of 1000 mg/l and of the individual chlorophenol of 0.1 mg/l. Rate of acetate and chlorophenols were modelled using a Monod model. The maximum rate of acetate utilization was zero-order in relation to acetate concentration and first-order with respect to organism concentration. The rate of chlorophenol degradation were zero-order in relation to the chlorophenol concentration. The relative rates of degradation were in general as follows: monochlorophenol > dichlorophenols > trichlorophenols. / Graduation date: 1992

Taiwan is an island country. Therefore, it must depend on sea and air transport to communicate with other countried. Especially, the import and export of cargo rely on the sea transport. Nowadays, some ships still use diesel oil as fuel. During the transport, the ship is inevitable for leak, or accidents happening in marine environment. Therefore, in this research, through the man-made sifting way, we will sieve and purify the pure species bacteria, which can degrade the PAHs in diesel fuel floating on the sea surface to decrease its pollution to the marine environment. In this research, eight species of pure bacteria will be sifted to purify. After primarily degrading experiment, we found that the second species of the bacteria performed the best degrading effect. After further identification, it was known that the second species of bacteria was yeast with name of Candida viswanathii. The bacteria were then used to in this study on biodegradation of PAHs in the diesel fuel. According to the experimental results, we found that bacteria could degrade diesel fuel. The OD value and total amount of the bacteria were found to be increased, but showed less degrading effect on PAHs in diesel fuel. However, in the experiment regarding the bacteria against single kind of PAHs, it showed excellent degrading effect on Naphthalene. When the initial concentration of Napthalene was controlled at 10 mg/L, the degrading rates were 0.3 and 0.24 mg/L-day respectively, in two separate experiments. Concerned with the degrading experiment against Anthracene, learned from the chromatography diagram it was observed that the concentration controlled at 10 and 5 mg/L, the production rate of Naphthalene were measured equal to 0.049 and 0.028 mg/L-day respectively. Thus, it was concluded that Anthracene can be degraded by the bacteria, but the degrading rate was slow, and the metabolite might be Naphthalene. As for the Flouranthene, the bacteria presented no degrading effect on it. It was concluded that the bacteria might not degrading ability toward four-ring or above PAHs. In conclusion, it is quite difficult to use single species of bacteria to deal with highly-complicated organic. It is suggested that we should study more about the degrading character of every species of bacteria. After that, we can get better effect by using similar compound bacteria to deal with the PAHs in the diesel oil.

published_or_final_version / Civil and Structural Engineering / Master / Master of Philosophy

Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme.

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