<p><b>Yeast Assimilable Nitrogen (YAN) is an important component in winemaking. It primarily consists of ammonium and free amino acids, in particular glutamine and arginine. Low levels of YAN in grape juice are associated with stuck or sluggish fermentation. The current tests for YAN are too complicated for most winemakers to do on-site, instead requiring samples to be sent to commercial laboratories, which is costly and time-consuming. </b></p>
<p>My goal was to develop an enzyme-based biosensor that provides a simple, robust, and inexpensive on-site test for YAN. In previous work at the Victoria University of Wellington, the blue pigment synthetase A (BpsA) enzyme was developed to quantify glutamine in human biological samples by converting it into a blue dye, indigoidine. Preliminary tests had shown that BpsA can also quantify glutamine in grape juice, giving a way to quantify one of the main components of YAN.</p>
<p>In this thesis, I aimed to find two other enzymes to sense and report on ammonium and arginine – the other major components of YAN. The targets were a glutamine synthetase (GS) for ammonium detection and either an arginine deiminase (ADI) or an arginine dihydrolase (ArgZ) for arginine detection. The key consideration was the ability of the enzymes to function in a one-pot cascade reaction with grape juice samples that did not require pre-treatment. </p>
<p>The role of GS in the biosensor was to catalyse the conversion of grape-derived ammonium into glutamine (in the presence of exogenously-added glutamate). This glutamine could then be converted to indigoidine by BpsA. Of the six enzymes screened, the Helicobacter pylori GS (HpGS) was selected as the most suitable candidate. It was easy to express recombinantly in Escherichia coli and purify with high yields (70 mg per litre of culture), and importantly it was highly active in the same assay conditions as those used for BpsA, having steady-state kinetic parameters of kcat = 0.78 ± 0.02 s-1 and Khalf = 97 ± 6 µM for ammonium. For the arginine sensing enzyme, both ADI and ArgZ were considered. ADI catalyses the hydrolysis of one ammonium from the side chain of arginine, while ArgZ catalyses the hydrolysis of two. The ammonium released could then be incorporated into glutamine, and then indigoidine, via the HpGS- and BpsA-catalysed reactions. In total, 3 ADI enzymes and 4 ArgZ enzymes were characterised. The Rhodospirillum centenum ArgZ (RcArgZ) was the most suitable candidate. It could be produced in high yield (100 mg per litre of E. coli culture) and was active in a one-pot reaction with HpGS and BpsA. </p>
<p>After extensive optimisation, the three-enzyme biosensor was compared with commercial test kits manufactured by Unitech and Megazyme. These commercial test kits quantify ammonium and primary amino acids in separate spectrophotometric assays. I compared the ammonium test kits with a simplified HpGS-BpsA version of my biosensor (with the BpsA-only signal for glutamine subtracted). The results showed all three methods to be comparable. The primary amino acid test kits were compared with the three-enzyme RcArgZ- HpGS- BpsA biosensor (with ammonium levels subtracted). The three-enzyme biosensor reported lower primary amino acid levels than the commercial test kits, as expected since it only detects arginine and glutamine, while the commercial test kits detect all primary amino acids. However, because arginine and glutamine are particularly abundant in grape juice, my biosensor reported 35-61% of the total amount of primary amino nitrogen in the samples tested. Winemakers only required an estimate that YAN is ‘high enough’ in their freshly-harvested grapes and my biosensor is able to provide that information. Thus, the RcArgZ–HpGS–BpsA biosensor I developed is a good alternative for winemakers who do not have the capacity to measure YAN spectrophotometrically, on-site. </p>
<p>Finally, feedback from winemakers suggested that my biosensor would be most useful if it could be formulated as tablets. The essential step in developing such a product was to determine conditions for freeze-drying each enzyme so that it maintained activity. A series of experiments showed that the activity of each enzyme could be preserved if it was dialysed against 40 mg/ml mannitol and 10 mg/ml sucrose before freeze-drying. The freeze-dried enzymes could be stored at -20°C for at least 3 months and resuspended for use with near 100% activity.</p>
<p>Overall, this research has demonstrated the power of searching for new enzymes to use in semi-quantitative, colorimetric biosensors. The prototype described here, involving a novel combination of three enzymes, has the potential to be of use for winemakers in New Zealand, and around the world.</p>
