Environmental Engineering Reference
In-Depth Information
Although the Dsz operon is transcriptionally repressed in the presence of bioavailable
sulfur, the enzymes themselves (Dsz A, B, C) are not post-translationally inhibited [7],
opening the possibility of overexpressing the genes under control of strong constitutive
promoters. The first report of this appeared in 1997, in which researchers cloned the dszABC
genes of R. erythropolis IGTS8 behind the constitutive tac promoter into P. putida and P.
aeruginosa species [11]. The resulting strains grew more rapidly than the rhodococci with
DBT as the sole sulfur source and converted DBT to HBP quantitatively, showing that this
approach could provide strains that are quite useful commercially. The first patent on the
incorporation of the Dsz genes into Pseudomonas was issued in 1999 [12], followed by
another incorporating a flavin reductase (to fulfill the function of DszD) as well as the other
Dsz genes into an artificial operon [13].
2.2.5. Improvements in rate and extent
. Despite advances in enzyme expression, further
improvements were necessary to increase the rate and extent of desulfurization to levels
sufficient for commercialization; sustained rates of >20 micromoles of substrate per minute
per gram catalyst were needed, far in excess of the capability of the natural Rhodococcus Dsz
system [7, 1]. Between 1990 and 1998, however, optimization of biocatalyst production
increased the activities of recombinant BDS catalysts 200-fold by increasing concentrations
of DszA, B, and C, and optimizing conditions for DszD, bringing the catalysis rate to within
an order of magnitude of that required for commercial operation [7]. Work in chemostat
selection for gainof-function mutants yielded R. erythropus strains that effectively utilized
octyl sulfide and 5- methyl benzothiophene [8], showing the utility of conventional
approaches. At the same time, novel enzyme engineering was also required, and the
combinatorial method known as RACHITT (see Chapter 2) was developed in the context of
this problem. In the directed evolution of dszC genes from Rhodococcus and Nocardia, new
chimeric enzymes were generated that possessed higher activity, more extensive substrate
oxidation, and broader substrate specificities than either of the parents. These activities were
more than sufficient to meet industrial needs and no longer limited by nutritional needs of the
microbes [14, 15].
2.2.6. Tolerance to industrial conditions
. Microbial desulfurization is currently most
attractive as a step following the conventional HDS, which in turn requires elevated
temperatures. Ideally, therefore, the biocatalytic process would require as little cooling as
possible, saving energy and time; in addition, higher temperatures could afford the advantages
of increased enzymatic rates and diminished contamination by other bacteria. Advances in the
development of thermotolerant Dsz pathways include the discovery of desulfurization in the
thermotolerant Paenibacillus, able to desulfurize DBT at 55°C [7]; the discovery of Bacillus
subtilis WU-S2B, able to desulfurize DBT at 50°C (16); and Mycobacterium phlei GTIS10,
also able to function at temperatures >50°C [8]. An important note in these efforts is that the
thermotolerance appears not to result from the DNA sequences, which are highly similar or
even nearly identical to those of the mesophilic R. erythropus IGTS8, but from other factors
in the whole-cell pathway. Thermophilic catalysis may therefore be constrained to occur
within a thermophilic organism.
2.3. Process Design
An important consideration in BDS process design and in choice of the microbial host is
the transfer of substrate from oil into cells. Close contact between cells and oil requires
emulsification of the oil-water mix that must then be broken to recover the desulfurized oil, at
