Activity Measurements for Enzyme Kinetics and Stability

Supplementary Material

Section A

Activity measurements for enzyme kinetics and stability

Macromonolithic silica gels, where β–glucuronidase enzyme (250 U) was immobilised, have been taken from TEOS aging medium after 24 h.Afterwards, the gels were crushed with the help of a metal rod and immersed in the assay solution for activity measurements (Kazan et al. 2017). The hydrolyses of 4-Nitrophenyl β-D–glucuronide (pNP-GluA) to glucuronic acid and p-nitrophenol (pNP) was carried out with free and immobilised GUS catalysing the reaction given below.

The reaction was executed with the final concentration of 50.0mM Na-P buffer (pH 4.5), entrapped 250 U GUS and different final concentrations of pNP-GluA ranging between 0.1 and 30.0mM in a shaking water bath (Wise Bath) at 112 rpm and 37 °C. For the activity assays with free enzyme, the reaction was initiated by adding enzyme solution to the reaction system at the same final conditions as in the case of encapsulated GUS. For free enzyme kinetics, the final concentration of pNP-GluA in assay solution was ranged between 0.1 and 20.0mM for 250 U free GUS. All reactions were implementedin a 1.0 ml-scale volumefor 30 min (pH 4.5 and 37 °C). In order to obtain pNP concentration, reaction mixture was measured at 420 nm (Amersham Biosciences Ultraspec 1100 pro) and a pNP standard curve (y {abs.} = 1,378 x {cons.}, R2=0,999) was employed. The enzyme activity unit was defined as the amount of GUS needed to produce 1.0 µmol of pNP/min at 37 °C, pH 4.5 and activities were calculated as U/ml (according to enzymatic assay of GUS (EC 3.2.1.31) (Sigma-Aldrich, 2016). Michaelis-Menten and Lineweaver-Burk graphs were drawn to obtain KM and VMAX values.

In order to determine the stability of immobilised enzyme, GUS loaded silica macromonolithswere immersed in 37 °C and 4 °C of Na-P buffer (pH 4.5) mimicking storage and reaction environments. The gels were incubated for 3 days and 32 days at 37 °C and 4 °C, respectively. At each 24 h of incubation period, the respective monolith was sampled and crushed, then immersed in the assay solution including 1mMpNP-GluA. In stability tests, the initial activity that immobilised enzyme has shown before the incubation process (Day 0) was considered as 100% and changing enzyme activity against 1mMpNP-GluA during incubation period was expressed as the relative activity taking account of this value.

Section B

Fabrication of microfluidic apparatus

For the fabrication of glass-PDMS microchips, a stainless steel mold that is fabricated at Bilkent University (Micro System Design and Manufacturing Center, Ankara) by computer numerical control (CNC) milling machine (DeckelMaho HSC55) via CNC-based micromachining process as the negative of the desired 0.5 x 0.5 x 400 mm (D x W x L) microfluidic structure was employed (Fig. 1a). PDMS base and curing agent were mixed at a ratio of 10:1 in petri dishes. The mixture was then degassed via a vacuum pump (Rocker 300) inside a desiccator (Bel-Art) before being cast onto the steel-mould and after the casting process, cured inside the mould for 1.5 h at 80 °C within an incubator (NüveEn 025). After the curing process resulting S-shaped PDMS microchannels taken from the mould and were sealed on the glass surface of the microscope slides (Sail Brand) with the plasma treatment surface modification method of which mechanism of action is based on oxidation of PDMS polymer surface via plasma generator (Harrick Plasma, PDC-32G). The resulting microfluidic apparatus were connected to microsyringe pump and used for continuous reactions.

Supplementary Fig. 1:Stability of immobilised GUS within the silica macromonoliths at (a) 4 °C and (b) 37 °C.All values are mean ± SEM, where n≥3; n is the number of samples analyzed. Entrapped GUS exhibited a high stability at 4 °C. Any activity loss was not observed for the first 2 days and the remaining activity was reported as 84% on the 6th day of storage. Even though the activity of the entrapped enzyme in macromonoliths decreased sharply by 62% on the 14thday and continued falling down thereafter, it remained 36% of its initial activity on the 32nd dayof storage. Stability of the enzyme in the silica gelsatthe reaction temperature (37 °C) was lower compared with that of storage conditions. The initial activity of the entrapped enzyme decreased sharply to 62% on the first day of incubation and continued slowly falling down thereafter. It remained 48% of its initial activity on the 3rd day of incubation. It has been reported that after gelation of silica-based hydrogels, remaining terminal –OH or –OR groups in the silica network may bring into contact and continue to condense or become re-esterified depending on thermal fluctuations (Cumana 2013). Therefore, the decreasing enzyme activity behaviour during the storage of silica macromonoliths in water may have been arisen from that re-esterification proceeded and necks at the contact points of silica particles most probably grew depending on temperature fluctuations. In other words, new –Si–O–Si– bonds might have formed within the pore structures where enzyme located. Because of this phenomenon, the resulting new –Si–O–Si– bridges in pore structures may have caused enzyme denaturation by deteriorating protein secondary structure or blocking of the enzyme active sites. Depending on the stability profiles of the encapsulated enzyme at both temperatures, it can be deduced that re-esterification processes proceeded faster at 37 °C and almost completed after 1-day incubation while at slowly proceeded till the 32nd day of incubation at 4 °C.

References

Cumana S (2013) Application of Silica-Based Porous Materials in Microreactors and Chromatographic Separations.Dissertation, Technischen Universität Hamburg-Harburg

Kazan A et al. (2017) Formulation of organic and inorganic hydrogel matrices for immobilization of β-glucosidase in microfluidic platform. Eng Life Sci 17:714-722

Sigma-Aldrich (2016) Enzymatic Assay of β-Glucuronidase (EC 3.2.1.31) from E. coli. Available at: Accessed 24 Jan 2016