• No results found

Although Co/acrylamide induced condition has been tested for the NHase overexpression, yet the acrylamide in different molarities were not tested. In order to exclude the possibility that Co/acrylamide may also overexpress NHase, different molarity concentrations of acrylamide should be tested.

In Co/urea supplemented media with half amount of urea (3.75g/L), the NHase bands still accounted for more than 55% total recovered cytosolic proteins and the protein profile from gradient SDS-PAGE did not shown significant differences from Co/urea (7.5g urea/L). The minimum concentration of urea needed to overexpress NHase should be examined.

In R. rhodochrous DAP 96253, the identity of [amidase]/[nitrilase] was not totally

defined, although according to MALDI TOF/TOF MS it was defined as amidase, and based on the substrates preference and induction method it was considered as [amidase]/[nitrilase]. In order to identify what exactly this enzyme is, a genome sequence is necessary. Amidases from nitrilase/cyanide hydratase family and nitrilases from nitrilase/cyanide hydratase family share similar sequences, and the genome sequences only may not be sufficient to distinguish the posttranslational modification and/or organization differences. Thus, a crystal structure for this [amidase]/[nitrilase] is going to be necessary. Hence, crystallization is needed for the

identification of this enzyme.

Based on the charge, hydrophobicity and size, both NHases from R. rhodochrous DAP

96253 and DAP 96622 are very similar, even though the activities of both purified NHases are not the same. The mechanisms behind this difference or similarities need to be studied. This will need crystallization of both NHases and genome sequence of both enzyme genes. In addition, literature on how NHase α and β subunits were organized in the holoenzyme of NHases from

other strains is not available. Thus, the crystal structure can also be compared between R.

rhodochrous DAP 96253 and DAP 96622.

In order to understand whether [amidase]/[nitrilase] is important in NHase

overexpression in both strains, gene knock-out for [amidase]/[nitrilase] should be studied in R.

rhodochrous DAP 96253. For R. rhodochrous DAP 96622, an inducer for [amidase]/[nitrilase]

overexpression will be necessary for studying the relationship between [amidase]/[nitrilase] and NHase overexpression. A candidate for the overexpression of [amidase]/[nitrilase] (for R.

rhodochrous DAP 96622) will be urea/isovaleronitrile because this has been reported to induced

nitrilase in R. rhodochrous J1 and this method was also proved to be effective with R.

rhodochrous DAP 96253 in current work (Results, Overexpression of [amidase]/[nitrilase] in R.

rhodochrous DAP 96253)(Kobayashi et al., 1992a). Since Co/urea were not able to overexpress

similar level of NHase in R. rhodochrous DAP 96622 compared with R. rhodochrous DAP

96253, if other inducers will overexpress both [amidase]/[nitrilase] and NHase in R. rhodochrous

DAP 96622, a stronger correlation between [amidase]/[nitrilase] and NHase expression will be established.

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