The beta-galactosidase enzyme, also known as lactase, is an industrial biocatalyst used to produce lactose free milk (Horner, 2011). The activity of this enzyme is easy to measure and it is commercially available (Biosynth FL31718) making it an excellent model to test Solidzymes’ carrier screening methodology and equipment.

Enzyme carriers were first assessed for their ability to bind lactase. To do this carrier samples were equilibrated with phosphate buffer until the pH was between 6.5 – 7.5. Then the capacity of carriers was compared by binding 5 mg of lactase to 50 mg carrier samples (10% w/w). Binding was calculated by measuring the mg of lactase remaining in the supernatant after 16 hrs (Table 1). A second set of 50 mg equilibrated carrier samples was used to bind 1 mg (2% w/w) lactase. These were used for activity measurements in order to allow both low and high capacity enzyme carriers to be screened together. Conditions for activity measurements were designed for similarity to cold milk using an available study (Akkerman, 2019). A specialized activity assay was developed in order to measure the activity of all immobilized samples simultaneously (see Materials and Methods).

Initial activity measurements showed that immobilized lactase had very different activities between 0 and 3.19 umol / min / mg depending on which carrier it was immobilized on. The highest activities were obtained from carriers that used covalent attachment followed by ionic interactions and aromatic interactions (SZ #13). No purely hydrophobic adsorption based carriers performed well underscoring the preference of this enzyme for hydrophilic surfaces.

After initial activity measurements the samples were washed thoroughly with 1X reaction buffer and stored at 4^oC. Immobilized lactase samples that performed in the lowest 10% were discarded leaving 18 out of the original 43 samples for re-use experiments. The activity of these 18 remaining biocatalysts was re-measured weekly in order to ascertain their stability (Figure 1). During storage the samples were checked for microbial infection, but none was found (data not shown). The daily yield (in g lactose / g lactase) from each sample was estimated with the equation:

Daily yields were used to estimate the total potential yield over the duration of the experiment according to the following equation:

The potential yield represents the lactose that might have been hydrolyzed by a given biocatalyst in a continuous reactor. The time that immobilized lactase worked in this experiment was of course much less than the total possible time since it’s activity was assessed periodically in batch mode (Figure 1). The activity was also certainly affected by the low temperature and presence of calcium. The main purpose of this experiment was not to maximize activity but to compare the relative yields of lactase on different carriers in order to identify the support that best fits this enzyme (Figure 2). At 4^oC there were six similar carriers (#49-52) that high yield and similar stability, so carrier #52 was used to represent this group as a whole.

Figure 1. Stability of immobilized lactase stored at 4^oC in reaction buffer.

Figure 2. Calculated yields from lactase immobilized on the best 18 carriers then stored at 4^oC. Carriers from Figure 1 are indicated with circles.

Immobilization on a solid surface is often used as a means to improve the thermostability of enzymes. Because these lactase samples lost so little activity over time at 4^oC we were not able to compare their relative stabilities. Therefore, it was decided to continue using them at 37^oC in hopes of comparing the relative stability of lactase molecules on different surfaces. Accordingly, all eighteen samples were assayed at 4^oC and at 37^oC in order to capture temperature effects on the activity assay. Then a second 35 day stability experiment was undertaken with the samples stored at 37^oC between measurements (Figure 3). Most of the carriers still did not lose significant activity at 37^oC, except for carriers #49-52 which lost about 34% of their activity on average over the first two weeks. Interestingly, this activity loss did not continue beyond the other carriers suggesting two populations of enzymes with different temperature stabilities. Thus, carriers #49-52 started with a “bonus” population of enzymes that were active at 4^oC but not at 37^oC. The lost activity significantly effected the relative yields during the 35 day experiment at 37^oC (Figure 4).

Figure 3. Stability of immobilized lactase stored at 37^oC in reaction buffer. These are the same samples used previously in Figure 1. On Day 0 activity was initially measured at both 4^oC and 37^oC in order to differentiate between temperature effects on the activity assay and temperature effects on long term stability of the lactase enzyme structure.

Figure 4. Calculated yields from lactase immobilized on the best 18 carriers then stored at 37^oC. Carriers from Figure 1 are indicated with circles. Yields are from the same >35 day old samples used for Figure 3. Top five carrier candidates from Figure 1 and Figure 3 are indicated with circles.

Conclusion

This case study illustrates Solidzymes’ approach to biocatalyst screening. From an initial forty-three immobilized lactase candidates twenty-five were eliminated due to poor starting activity. Seven proved unstable in longer term stability tests. Amongst the remaining eleven SZ #49-#54 were clear favorites in terms of catalytic yield at 4^oC. These candidates are similar and use relatively expensive materials which may, or may not, be acceptable to the customer. One goal of our screening process is to give the customer a variety of viable enzyme carrier candidates. This minimizes the risk of stalling enzyme reactor development due to cost or supply chain issues that often arise when purchasing large amounts of material. In this screening case study, five out of forty-three enzyme carrier candidates worked well enough in this experiment to be considered for further development such as application in a pilot scale continuous reactor.

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Materials:

Immobilized activity assay

1.) Remove soluble enzyme

Wash 50 mg carrier samples by placing a pipette tip at the bottom of samples and fully draining liquid. Add 1 mL of reaction buffer and remove again. Repeat washing five times to fully remove soluble enzymes.

2.) Add 1.5X reaction buffer.

Add 0.917 mL of 1.5X reaction buffer. This allows for 0.133 mL of buffer that is left within the pores of the carrier (determined previously by drying an extra sample)

3.) Cool the samples

Place carriers with buffer and lactose solution separately into 4^oC for at least 30 minutes to cool.

4.) Begin the reaction

Start a timer for 20 minutes. Quickly add 0.45 mL of 0.5 M lactose to each sample. Go in order and keep track of the time it takes to start all the reactions (should be < 2 minutes). Place the reactions in 4^oC refrigerator with shaking.

5.) Collect glucose samples

Label 1.5 mL microcentrifuge tubes for each sample. Put 90 uL of reaction buffer in each tube. After 20 minutes take 10 uL from the supernatant of each lactase reaction and wash into 90 uL tubes with pipette. Take samples in order and match the pace of starting the reaction (Step 4).

6.) (optional) Measure glucose accumulation over time

If more time points are desired put the lactase reactions back in 4^oC with shaking. Repeat step 5 over time as desired.

7.) Measure glucose concentration from samples

a) Warm Glucose Liquicolor enzymatic glucose detection reagent to 37^oC in a water bath.

b) Preheat an incubator to 37^oC.

c) Mix the 100 uL samples of 10X diluted glucose product by shaking vigorously. Pipette 5 uL from each sample into an empty 96 well plate. It is a good idea to do two wells per sample for duplicate reads.

d) Also pipette 5 uL of glucose standard solutions into the 96-well plate.

e) Pour the glucose detection reagent into a trough. Transfer 200 uL from the trough into the wells with glucose samples quickly using a multi-channel pipette.

f) Place the 96-well plate into 37^oC incubator for 5 minutes while the red color develops.

g) Read the absorbance of the samples at 500 nm using a platereader. Average duplicate sample readings together. Calculate a straight line from glucose standard samples. Use the line to calculate glucose concentration of samples.

8.) Calculate lactase activity in umol / min / mg

NOTE – lactase hydrolyzes lactose producing one glucose per lactose.

Find the micromoles of glucose by multiplying the concentration (in mM) by the reaction volume (in mL). Divide by minutes that the reaction continued and milligrams of enzyme to get the activity. For example, in this lactase experiment if the glucose concentration is 32 mM then activity = 32 * 1.5 / 1 / 20 or 2.4 units of activity.