57th Annual Report on Research 2012 Under Sponsorship of the ACS Petroleum Research Fund

Over the past 24 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. We have now developed 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 with a polyhisitidine affinity tag (His₆-tag) on the C-terminus of the α-subunit and CtNHase with a His₆-tag on the C-terminus of the β-subunit. We have characterized the recombinant CtNHase and PtNHase enzymes kinetically, spectroscopically, and crystallographically. These functional expression systems now allow large amounts (~10 mg/L) of CtNHase and PtNHase to be purified quickly and easily.

We have focused on PtNHase as it is thermally stable and more resistant to air oxidation. We have incorporated it into sol-gel matrixes using tetramethyl orthosilicate (TMOS) as the silica precursor for the encapsulation process. Briefly, hydrolyzed TMOS sol, 0.100 mL was mixed with an equal volume of pure PtNHase (1. 8 mg) in 50 mM Tris-HCl, pH 7.5. The mixture was placed on ice until gelation occurred to create0.200 mL PtNHase:sol-gel monoliths on the bottom of a glass vial. Following gelation, the monoliths were washed with 1.2 mL of 50 mM Tris-HCl, pH 7.5 (sol-gel buffer) and stored at 4 °C overnight in 0.400 mL of the same buffer. These monoliths were washed with 1.2 mL of buffer and then crushed into small beads. SEM images indicate 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.

Catalytic activity of the PtNHase:sol-gels were investigated by running reactions with 600 mM acrylonitrile in 50 mM Tris/HCl pH 7.5. These encapsulated enzymes are active and retain their catalytic activity weeks after being removed from the reaction vessel, dried, and rehydrated. In fact, the encapsulated enzyme retains >80% of its catalytic activity after being removed from the reaction vessel, rinsed, and re-used in more than six reaction cycles. Encapsulated enzymes are also protected from denaturants such as guanidine-HCl whereas the soluble PtNHase enzymes quickly denature under identical reaction conditions. Moreover, the PtNHase:sol-gels retain ~90% of their observed activity after treatment with trypsin and chymotrypsin, two proteases that will digests all surface accessible proteins, whereas the soluble PtNHase showed <15% activity. These data indicate that PtNHase is trapped inside sol-gel pellets (and is not just attached to the pellet surface). Reactions have also been run as a function of temperature with PtNHase:sol-gels remaining active at more than 60 ^oC for up to 10 hours while the soluble enzyme loses activity within a minute at this temperature. Moreover, these biomaterials convert >90% of the acrylonitrile to acrylamide over a 50 min reaction period at 35, 45, and 55 °C.

Over the next 12 months, we will examine encapsulated and explore alternative encapsulation matrices. We have begun examining gel formation using tetraethoxysilane (TEOS) as a less expensive sol-gel precursor as well as alginates, which gel under more mild conditions. Our preliminary work shows that the CtNHase enzyme is fully functional in gels made from TEOS and alginates.

Aim 2. Investigate the breadth and selectivity of substrates that can be degraded. Now that CtNHase and PtNHase have been successfully encapsulated in a solid support material their specificity and activity towards a variety of nitrile substrates will be examined. Typical reactions will be 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 will analyzed using an HPLC assay developed during the granting period 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. As NHase enzyme should not catalyze the undesirable side reaction that might result in acrylic acid formation, it is not surprising that we do not detect any acid formation. At this time, we have examined acrylamide, and the expected amide is the only product detected. Over the next 12 months, we will examine a wide variety of nitriles and compare the reaction rates and observed product(s) to soluble enzyme.

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 is also being 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. Moreover, the only product observed is acrylamide which is the result of hydration of acrylonitrile. We have determined the retention time for the product of acrylonitrile if methanolysis were occurring, which is significantly different from acrylamide. Therefore, methanolysis is not occurring. Over the next 12 months, we will explore these observations further as well as place PtNHase: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.