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Over the past 12 months we have addressed parts of all three Aims defined in the original proposal.  Specifically, we have:

Aim 1:  Encapsulate both PtNHase and CtNHase in sol-gel materials and determine if they are catalytically active.  Initially, we needed to develop new expression systems for the Co-type NHase from Psuedonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Comamonas testosteroni N1(CtNHase).  We expressed the α-and β-subunit genes of PtNHase and CtNHase with a polyhistidine affinity tag (His₆-tag) on the C-terminus of the β-subunit.  We have characterized the recombinant CtNHase and PtNHase enzymes kinetically and spectroscopically as well as obtained ICP-MS data on each.  These functional expression systems now allow large amounts (~25 mg/L) of CtNHase and PtNHase to be purified quickly and easily.  We have also obtained the His₆-tagged expression system for the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) and have purified this enzyme to homogeneity.

With these new expression systems in hand, we sought to fully characterize both the CtNHase and ReNHase enzymes crystallographically.  At this time, we have obtained the X-ray crystal structures of CtNHase at of 2.4 Å and the ReNHase enzyme at 2.8 Å resolution.  Although both structures are new, they are very typical of Fe-type nitrile hydratases.  The structures show the α- and β-subunits interacting with the active site at the interface of the two subunits with the Fe(III) ions residing in five coordinate and is bound by three Cys residues and two backbone amide nitrogen’s, where the two axial Cys residues are both oxidized to cysteine-sulfenic acids.

With active PtNHase, CtNHase and ReNHase in hand, we have incorporated each into sol-gel matrixes.  In general, TMOS was used as the silica precursor and ~200 μl of NHase:sol-gels monoliths were cast on the bottom of a glass vials.  The activity of the PtNHase, CtNHase and ReNHase:sol-gels were investigated by running reactions with 600 mM acrylonitrile in 50 mM Tris/HCl pH 7.5.  All of the encalsulated NHase enzymes were active under these conditions towards acrylnitrile.  These encapsulated enzymes have been proven to retain their catalytic activity weeks after being removed from the reaction vessel, rinsed, and dried.  Furthermore, the catalytic pellets can be treated with trypsin, which proteolytically digests all surface accessible proteins, and shown to retain some activity, indicating that active enzyme is trapped inside the pellets (and is not just attached to the pellet surface).  The encapsulated enzymes are also protected from denaturants such as guanidine-HCl whereas WT NHase enzymes quickly denature under identical reaction conditions.  Reactions have also been run as a function of temperature with the NHase materials remaining active at more than 60 ^oC for up to 10 hours while WT enzyme looses activity within a minute.  Finally, the morphology of the NHase:sol-gels were examined by SEM which showed that the NHase:sol-gels have a porous structure with pore diameters of 116-140 nm, large enough for substrate and product to  diffuse into and out of the material. 

Over the next 12 months, we will fully characterize the ReNase and CtNHase:sol-gel encapsulated enzymes as well as explore alternative encapsulation matrices in order to increase the observed activity levels.  We have begun examining gel formation using tetraethoxysilane (TEOS) as a less expensive sol-gel precursor.  Our preliminary work on this alternative shows that the enzyme is fully functional in gels made from TEOS.  The quantitative comparison is underway. 

Aim 2.  Investigate the breadth and selectivity of substrates that can be degraded.   CtNHase and PtNHase, have been successfully encapsulated in a solid support material and their specificity and activity towards a variety of nitrile substrates has been (and are continuing to be) characterized.  Typical reactions are set-up in which a NHase:sol-gel pellets are placed in a sealed vial and allowed to run for an extended period of time to ensure that the reaction runs to completion.  The subsequent reaction mixture was analyzed using a newly developed HPLC assay to characterize the product(s) formed.  The LC traces show no evidence of any product other than the amide of choice.  (Note: butyric acid was not added to these samples).  Using an acrylic acid standard, we were able to demonstrate that our limit of detection for acrylic acid is roughly 2.8 nmol.  Therefore, we can report that we do not see any sign of acrylic acid from which we can conclude that it is not present as a contaminant of greater than 2.8 nmol.  This is not surprising.  The enzyme should not catalyze the side reactions that might result in acrylic acid formation.  These results also confirm that we do not see any other side reactions occurring.  At this time, we have examined acrylamide, benzylamide, methylacrylamide, and 2-nitro-5-thiocyanatobenzoic acid (NTCB) and the expected amide is the only product detected.  Over the next 12 months, we will examine a wide variety of nitrile and compare the reaction rates and observed product(s) to soluble enzyme and these studies are currently underway. 

Aim 3.  Examine the reactivity of these novel biomaterials in a continuous reactor with both protic and aprotic solvent mixtures.  The production of acrylonitrile to acrylamide in organic solvents was investigated.  The organic solvents used for the assay were dry methanol, hexanes, isopropanol, DMSO, and pentane.  Production of acrylamide was observed in methanol but no acrylamide was produced using the other solvents.  The activity in methanol was 90% of that observed in aqueous conditions.  Since the observed activity levels in methanol decrease with time, it appears that enough water may exist in the sol-gel or in the methanol to allow the encapsulated enzyme to be catalytic.  Over the next 12 months, we will explore these observations further as well as place NHase:sol-gel material at the bottom of a 10 cm chromatography column and pump a continuous flow of fresh nitrile substrate through the column.  We will examine product formation with time to determine if a continuous flow reactor can be prepared with NHase sol-gel materials.