Co-Immobilization of Ketoreductase and Glucose Dehydrogenase

A two-enzyme system composed of immobilized ketoreductase (Hansenula polymorpha) and glucose dehydrogenase (Bacillus megaterium) was developed for the asymmetric reduction of keto esters to optically active hydroxy esters via immobilization in polyvinyl alcohol (PVA) gel particles. The concentration of enzymes was optimized, and the final particles were used 18 times in a row in a batch mode to achieve minimal loss of activity and complete conversion of the model substrate, β-ketoester ethyl-2-methylacetoacetate. Excellent stability was also achieved using new storage conditions of PVA particles, with 80% of activity being retained after almost 10 months.

Despite the many advantages of and recent successes in biocatalysis, the use of enzymes is often hampered by a lack of long-term operational stability due to low-substrate solubility in the aqueous reaction system, poor enzyme stability in commonly-used organic solvents, and difficulties in recovering and reusing the enzyme.Enzyme immobilization provides an attractive approach to overcoming these drawbacks [1]; however, the immobilization of KREDs remains challenging and only a few examples have been reported.For example, one system used nonporous glass as a support for the KRED and was used in a continuous gas-phase reactor [10].Another system used covalent binding of KRED on resin EC-HFA, which was applied in both batch mode and flow reactor mode [1].KRED and GDH were also separately immobilized on aldehyde-activated agarose and used in a flow reactor to perform stereoselective ketone reduction [11].Combined cross-linked enzyme aggregates (combi-CLEAs) of KRED and GDH were used for repeated conversions of ethyl 4-chloro-3-oxobutanoate (COBE).The system retained 70% of its activity after 10 cycles in aqueous system probably because of relatively weaker bond between enzyme molecules [12].
LentiKats ® is an entrapment technology that has been applied for the immobilization of whole cells and enzymes [13][14][15][16][17][18][19][20][21].Its lens-shaped capsules are made of polyvinyl alcohol gel which offers several advantages such as low matrix costs, inexpensive and simple gel preparation, low diffusion limits, etc. [13]; however, the co-immobilization of redox enzymes linked with cofactor regeneration have not been studied to date.Scale-up process of immobilization into PVA gel is also possible and was reviewed recently [22].
This article reports the optimization and application of co-entrapped KRED and GDH in a PVA gel.
Enzymes were expressed in E. coli BL21(DE3) with a combined lac and T7 promoter.The presence of enzymes (Supplementary Material Figure S1, (a) KRED: 32 kDa, (b) GDH: 35 kDa) was checked by SDS-PAGE.Enzymes were further purified, concentrated, and desalted, which was verified by protein electrophoresis (Supplementary Material Figure S2).
The activity of purified enzymes was determined spectrophotometrically by cofactor utilization separately for each enzyme.The initial specific activity of GDH was 45.7 U•mg −1 , which was 3.5-fold higher than the initial specific activity of KRED (13.06 U•mg −1 ).Based on the pH and temperature profiles of KRED and GDH (Figure 1), pH 6.5 and 37 • C were chosen as the conditions for all experiments.According to spectrophotometric measurements of enzyme temperature profiles, the optimal temperatures were 25-35 • C; however, in combined cascade reaction with both enzymes, 37 • C was observed as the optimum for product formation.For the same type of KRED, pH 7 and 30 • C were used as the optimal conditions [23], while pH 6.9-7 and 32-37 • C were used for commercial KREDs [2,3,8].
several advantages such as low matrix costs, inexpensive and simple gel preparation, low diffusion limits, etc. [13]; however, the co-immobilization of redox enzymes linked with cofactor regeneration have not been studied to date.Scale-up process of immobilization into PVA gel is also possible and was reviewed recently [22].
This article reports the optimization and application of co-entrapped KRED and GDH in a PVA gel.
Enzymes were expressed in E. coli BL21(DE3) with a combined lac and T7 promoter.The presence of enzymes (Supplementary Material Figure S1, (a) KRED: 32 kDa, (b) GDH: 35 kDa) was checked by SDS-PAGE.Enzymes were further purified, concentrated, and desalted, which was verified by protein electrophoresis (Supplementary Material Figure S2).
The activity of purified enzymes was determined spectrophotometrically by cofactor utilization separately for each enzyme.The initial specific activity of GDH was 45.7 U•mg −1 , which was 3.5-fold higher than the initial specific activity of KRED (13.06 U•mg −1 ).Based on the pH and temperature profiles of KRED and GDH (Figure 1), pH 6.5 and 37 °C were chosen as the conditions for all experiments.According to spectrophotometric measurements of enzyme temperature profiles, the optimal temperatures were 25-35 °C; however, in combined cascade reaction with both enzymes, 37 °C was observed as the optimum for product formation.For the same type of KRED, pH 7 and 30 °C were used as the optimal conditions [23], while pH 6.9-7 and 32-37 °C were used for commercial KREDs [2,3,8].

Free-Enzyme Biotransformations
Biotransformations were performed to verify the activity of purified KRED in combination with NADPH regeneration with GDH (Section 3.6).Ethyl-2-methylacetoacetate, which is an α-alkyl-β-keto ester, was selected as a model substrate for these purposes.This group of substrates are used in relatively low concentrations (25-50 mM) due to their low solubility in water and their potential ability to inhibit enzymes [3].
Free enzymes were used at different ratios for the complete conversion of the substrate (Supplementary Material, Figure S3, S4).According to the results summarized in Table 1, a five-fold higher concentration of KRED increased the initial activity approximately five and half times and total conversion of the substrate was achieved within 90 min.In contrast, a four-fold increase in the

Free-Enzyme Biotransformations
Biotransformations were performed to verify the activity of purified KRED in combination with NADPH regeneration with GDH (Section 3.6).Ethyl-2-methylacetoacetate, which is an α-alkyl-β-keto ester, was selected as a model substrate for these purposes.This group of substrates are used in relatively low concentrations (25-50 mM) due to their low solubility in water and their potential ability to inhibit enzymes [3].
Free enzymes were used at different ratios for the complete conversion of the substrate (Supplementary Material, Figures S3 and S4).According to the results summarized in Table 1, a five-fold higher concentration of KRED increased the initial activity approximately five and half times and total conversion of the substrate was achieved within 90 min.In contrast, a four-fold increase in the GDH concentration had no significant effect on initial activity.As expected, KRED was the rate-limiting enzyme and GDH was added in excess in biotransformations.GDH was previously used at concentrations equal to [3] or three-quarters lower than KRED [23], which is similar to the experiments reported above.One study reported the effect of GDH concentration on reaction rate in the kinetic resolution of 11-oxo-5,6,7,8,9,10-hexahydro-6,9-methanobenzocyclooctene and an increasing rate was only observed until 0.15 g•dm −3 , while the KRED concentration was 0.2 g•dm −3 [8].This represents a KRED:GDH ratio of 1.33:1.In general, to overcome the limitation of NADPH access in the reaction, GDH should always be in excess and the enzyme ratio should be optimized.

Optimization of Co-Immobilization
Various factors of LentiKats ® need to be verified as the co-immobilization of two or more enzymes using this technology has not been described to date.As for the free enzymes, the most important factor is determining the optimal concentration and ratio of both enzymes to achieve total substrate conversion in a reasonable time.Five different ratios and concentrations of KRED and GDH were used for co-immobilizations (Table 2).Based on the free-enzyme experiments described above, KRED was added in excess as it is rate limiting enzyme.The graph in Figure 2 displays the dependence of initial specific activity on the concentrations of enzymes entrapped in particles.A four-fold lower amount of KRED and a constant concentration of GDH led to a three-fold decrease in activity while only a 50% conversion rate was achieved.Half of the amount of KRED had no influence on initial specific activity.The concentration of GDH in batch four was limiting and caused a significant decrease in activity, while a two-fold higher amount made no additional improvements.Therefore, 0.19 mg•g −1 LKs of GDH was optimum.The major impact of KRED concentration within particles was proven in the free-enzyme reactions.To prevent the possible inactivation of KRED during co-immobilization and the repetitive use of particles for all further experiments, a KRED:GDH weight ratio of 25:1 was applied (batch number 1, Table 2).The ratio depends on the enzyme activities; for example, in previously reported immobilized KRED and GDH cascades, the optimal KRED:GDH ratio was 1:25 [11], which is opposite to the results reported here.Furthermore, the concentrations of KRED (Table 2) used were very low in comparison with the 100 g•dm −3 of KRED used in the immobilization on EC-HFA resin [1].

Repeated Biotransformations
To verify the stability of selected co-immobilized KRED and GDH (Table 2, batch number 1), particles were used for 18 repeated batch bioconversions in nonstop mode.Three parallel reactions were stopped at different stages of the conversion (50, 60, and 90 min) to see how the presence of the product could influence the level of bioconversion and the initial specific activity (Figure 3).The nonconstant trend of activity during repeated bioconversions can be caused by different residual reaction components after each washing of particles.There were observed similar effect in work with PVA particles before [19].The activity of the 90-min reaction dropped at the same speed as the others despite particles being exposed to the highest product concentrations, therefore inhibition was excluded.The level of total conversion was still >95%, which is an advantage for product separation and downstream processing.
Conversely, the 50-min reaction had the highest activity decline (36%), which may have been caused by the substrate remaining in the particles after each round.The total level of conversion decreased faster than in the other reactions, which also signifies possible inhibition by the accumulated substrate.
The 60-min biotransformations achieved the most consistent results.The overall activity decline was only 16% after 18 biotransformations, while the total conversion reached 90% for all rounds.Termination of the biotransformation at the exact moment of substrate utilization seemed to be optimal for stability of the co-immobilized enzymes' activity.
Despite the least activity decrease being achieved with the 60-min reaction, a better method would be to let the reaction run to the total conversion as product inhibition was not observed and the subsequent product separation is easier without any residual substrate.
In comparison, only a few KRED immobilizations have been described; for example, covalent binding of KRED on EC-HFA resin with a self-isopropanol regeneration cycle showed no activity decrease during 10 repetitions [1], and covalent binding of KRED on aldehyde-activated agarose together with immobilized GDH in a flow reactor for 15 days also showed no activity decrease [11].
Co-immobilized enzymes demonstrated possible reusability with 80% retained activity and >95% conversion, which is an enormous advantage and yield increasing mainly in biotransformations with small concentrations of inhibiting ketone substrates (25-100 mM).Enantiomeric ratio of chiral products was not changed after co-immobilization of enzymes (Supplementary Material Figures S11  and S12).Complete time graphs of the bioconversions are provided in the Supplementary Material, Figure S8, S9, S10.

Repeated Biotransformations
To verify the stability of selected co-immobilized KRED and GDH (Table 2, batch number 1), particles were used for 18 repeated batch bioconversions in nonstop mode.Three parallel reactions were stopped at different stages of the conversion (50, 60, and 90 min) to see how the presence of the product could influence the level of bioconversion and the initial specific activity (Figure 3).The non-constant trend of activity during repeated bioconversions can be caused by different residual reaction components after each washing of particles.There were observed similar effect in work with PVA particles before [19].The activity of the 90-min reaction dropped at the same speed as the others despite particles being exposed to the highest product concentrations, therefore inhibition was excluded.The level of total conversion was still >95%, which is an advantage for product separation and downstream processing.
Conversely, the 50-min reaction had the highest activity decline (36%), which may have been caused by the substrate remaining in the particles after each round.The total level of conversion decreased faster than in the other reactions, which also signifies possible inhibition by the accumulated substrate.
The 60-min biotransformations achieved the most consistent results.The overall activity decline was only 16% after 18 biotransformations, while the total conversion reached 90% for all rounds.Termination of the biotransformation at the exact moment of substrate utilization seemed to be optimal for stability of the co-immobilized enzymes' activity.
Despite the least activity decrease being achieved with the 60-min reaction, a better method would be to let the reaction run to the total conversion as product inhibition was not observed and the subsequent product separation is easier without any residual substrate.
In comparison, only a few KRED immobilizations have been described; for example, covalent binding of KRED on EC-HFA resin with a self-isopropanol regeneration cycle showed no activity decrease during 10 repetitions [1], and covalent binding of KRED on aldehyde-activated agarose together with immobilized GDH in a flow reactor for 15 days also showed no activity decrease [11].
Co-immobilized enzymes demonstrated possible reusability with 80% retained activity and >95% conversion, which is an enormous advantage and yield increasing mainly in biotransformations with small concentrations of inhibiting ketone substrates (25-100 mM).Enantiomeric ratio of chiral products was not changed after co-immobilization of enzymes (Supplementary Material Figures S11  and S12).Complete time graphs of the bioconversions are provided in the Supplementary Material, Figures S8-S10.

Storage of Particles
The storage of immobilized enzymes is one of the crucial parameters in applied immobilized biocatalysis.Samples from batch number 1 (Table 2) were stored at 4 °C in 0.1 M potassium phosphate buffer (pH 6.5) or at −80 °C without any buffer or solvent.After each biotransformation, particles were washed twice with buffer and stored.Storage at 4 °C caused a significant decrease in relative activity after 90 h (Figure 4) and is therefore not suitable for these co-immobilized enzymes.Storage at 4 °C was chosen based on the previously reported application for immobilized invertase [19].The particles retained 78% of activity after eight months.Complete time graphs of the bioconversions are provided in the Supplementary Material, Figure S5, S6, S7).The particles stored at −80 °C surprisingly retained 87% of their relative activity after nine-anda-half months (Figure 5).This was the first-time the storage conditions of PVA particles had been reported and it showed a huge improvement compared with storage at 4 °C where particles lost their entire activity after four days.Moreover, the properties of the PVA particles remained unchanged.

Storage of Particles
The storage of immobilized enzymes is one of the crucial parameters in applied immobilized biocatalysis.Samples from batch number 1 (Table 2) were stored at 4 • C in 0.1 M potassium phosphate buffer (pH 6.5) or at −80 • C without any buffer or solvent.After each biotransformation, particles were washed twice with buffer and stored.Storage at 4 • C caused a significant decrease in relative activity after 90 h (Figure 4) and is therefore not suitable for these co-immobilized enzymes.Storage at 4 • C was chosen based on the previously reported application for immobilized invertase [19].The particles retained 78% of activity after eight months.Complete time graphs of the bioconversions are provided in the Supplementary Material, Figures S5-S7).

Storage of Particles
The storage of immobilized enzymes is one of the crucial parameters in applied immobilized biocatalysis.Samples from batch number 1 (Table 2) were stored at 4 °C in 0.1 M potassium phosphate buffer (pH 6.5) or at −80 °C without any buffer or solvent.After each biotransformation, particles were washed twice with buffer and stored.Storage at 4 °C caused a significant decrease in relative activity after 90 h (Figure 4) and is therefore not suitable for these co-immobilized enzymes.Storage at 4 °C was chosen based on the previously reported application for immobilized invertase [19].The particles retained 78% of activity after eight months.Complete time graphs of the bioconversions are provided in the Supplementary Material, Figure S5, S6, S7).The particles stored at −80 °C surprisingly retained 87% of their relative activity after nine-anda-half months (Figure 5).This was the first-time the storage conditions of PVA particles had been reported and it showed a huge improvement compared with storage at 4 °C where particles lost their entire activity after four days.Moreover, the properties of the PVA particles remained unchanged.The particles stored at −80 • C surprisingly retained 87% of their relative activity after nine-and-a-half months (Figure 5).This was the first-time the storage conditions of PVA particles had been reported and it showed a huge improvement compared with storage at 4 • C where particles lost their entire activity after four days.Moreover, the properties of the PVA particles remained unchanged.

Storage of Particles
The storage of immobilized enzymes is one of the crucial parameters in applied immobilized biocatalysis.Samples from batch number 1 (Table 2) were stored at 4 °C in 0.1 M potassium phosphate buffer (pH 6.5) or at −80 °C without any buffer or solvent.After each biotransformation, particles were washed twice with buffer and stored.Storage at 4 °C caused a significant decrease in relative activity after 90 h (Figure 4) and is therefore not suitable for these co-immobilized enzymes.Storage at 4 °C was chosen based on the previously reported application for immobilized invertase [19].The particles retained 78% of activity after eight months.Complete time graphs of the bioconversions are provided in the Supplementary Material, Figure S5, S6, S7).The particles stored at −80 °C surprisingly retained 87% of their relative activity after nine-anda-half months (Figure 5).This was the first-time the storage conditions of PVA particles had been reported and it showed a huge improvement compared with storage at 4 °C where particles lost their entire activity after four days.Moreover, the properties of the PVA particles remained unchanged.

Cloning
Codon-optimized genes encoding KRED from Hansenula polymorpha [23] and GDH from Bacillus megaterium were obtained from Generay Biotech Co., Ltd.(Shanghai, China).Genes were inserted into a pET28b vector system, and these plasmids were transformed into Escherichia coli BL21(DE3) cells.

Preparation and Purification of KRED and GDH
An inoculum of E. coli BL21(DE3) expressing KRED or GDH was prepared from a single colony.Glass tubes containing 3 mL of LB medium were incubated overnight at 37 • C and 200 rpm.After approximately 12 h, a 500 mL flask containing 100 mL of LB medium was inoculated with 1% (v/v) inoculation culture and cultivated at 37 • C and 200 rpm.Isopropyl β-D-thiogalactopyranosid was added at a final concentration 0.5 mM when the cell density at OD600 reached 0.5-0.6.
After cultivation at 30 • C for 3 h, the induced cells were harvested by centrifugation (7197× g, 15 min, 4 • C).The sediment was subsequently resuspended in 20 mL of 0.1 M potassium phosphate buffer (KRED: pH 6.5; GDH: pH 7).The cell suspension was disrupted in a continual cell disruptor (CF Range, Constant Systems Ltd., Daventry, UK) in two cycles at 4 • C and 20 kPSI.After disruption, 70 µL of protease inhibitor (Complete EDTA-free, Roche, Berlin, Germany) per 20 mL of cell lysate was added.The cell debris was removed by centrifugation (7197× g, 30 min, 4 • C).

Enzyme Assays
KRED assays (1 mL) contained 0.1 M potassium phosphate buffer (pH 6.5), 35 mM ethyl 2-methylacetoacetate and 16.53 µg•mL −1 of purified enzyme.The reaction was started with 0.5 mM NADPH and its decrease was measured spectrophotometrically at 340 nm.GDH assays (1 mL) contained 0.1 M potassium phosphate buffer (pH 7), 100 mM glucose, and 3.72 µg•mL −1 of purified enzyme.The reaction was started with 0.5 mM NADP+.One unit of an enzyme was defined as the amount of KRED catalyzing the oxidation of 1 µmol NADPH/min or the amount of GDH catalyzing the reduction of 1 µmol NADP + /min at a pH of 6.5 (KRED) or 7 (GDH) and 25 • C. Experiments were duplicated.
The concentrations of purified enzymes were measured spectrophotometrically at 280 nm using an Eppendorf µCuvette ® G1.0 (Eppendorf, Hauppauge, NY, USA).The initial specific activity was expressed in units/mg of protein used in the reaction.

Co-Immobilization
LentiKats ® technology was used for co-immobilization.The PVA gel was composed of polyvinyl alcohol (PVA, 10% w/v), polyethylene glycol (6% w/v), and water.The amount of water varied according to the concentration of the added enzyme (Table 2).This mixture was heated at 90 • C for 30 min before being cooled at 40 • C under continuous stirring.Enzymes were added to the mixture in a dropwise manner and, after approximately 5 min, the particles were prepared by dripping onto a polyethylene plate using a syringe.The PVA drops were subsequently dried in an air flow cabinet at 35-40 • C for 45 min and hardened in 0.1 M sodium sulfate.The prepared particles were washed with a sterile 0.1 M potassium phosphate buffer (pH 6.5) and used directly for biocatalysis.

Biotransformation
Ethyl 2-methylacetoacetate, was used as a β-keto ester substrate for the KRED biotransformations.The GDH regeneration system (Figure 6) was used to regenerate NADPH.The reaction mixture contained 0.1 M potassium phosphate buffer (pH 6.5), 35 mM ethyl 2-methylacetoacetate, 100 mM glucose, 1 mM NADP + , and free or 20% (w/v) co-immobilized enzyme.Free enzymes were used in different concentrations and ratios as reported in Table 1.The amount of buffer added depended on the volume of enzyme used.In repeated batch biotransformations, the particles were washed twice in 0.1 M potassium phosphate buffer (pH 6.5) after each biotransformation.Reactions were carried out in Eppendorf tubes in a total volume of 1 mL at 37 • C with shaking at 550 (free enzymes) or 700 rpm (co-immobilized enzymes).Experiments were duplicated.

Co-Immobilization
LentiKats ® technology was used for co-immobilization.The PVA gel was composed of polyvinyl alcohol (PVA, 10% w/v), polyethylene glycol (6% w/v), and water.The amount of water varied according to the concentration of the added enzyme (Table 2).This mixture was heated at 90 °C for 30 min before being cooled at 40 °C under continuous stirring.Enzymes were added to the mixture in a dropwise manner and, after approximately 5 min, the particles were prepared by dripping onto a polyethylene plate using a syringe.The PVA drops were subsequently dried in an air flow cabinet at 35-40 °C for 45 min and hardened in 0.1 M sodium sulfate.The prepared particles were washed with a sterile 0.1 M potassium phosphate buffer (pH 6.5) and used directly for biocatalysis.

Biotransformation
Ethyl 2-methylacetoacetate, was used as a β-keto ester substrate for the KRED biotransformations.The GDH regeneration system (Figure 6) was used to regenerate NADPH.The reaction mixture contained 0.1 M potassium phosphate buffer (pH 6.5), 35 mM ethyl 2methylacetoacetate, 100 mM glucose, 1 mM NADP + , and free or 20% (w/v) co-immobilized enzyme.Free enzymes were used in different concentrations and ratios as reported in Table 1.The amount of buffer added depended on the volume of enzyme used.In repeated batch biotransformations, the particles were washed twice in 0.1 M potassium phosphate buffer (pH 6.5) after each biotransformation.Reactions were carried out in Eppendorf tubes in a total volume of 1 mL at 37 °C with shaking at 550 (free enzymes) or 700 rpm (co-immobilized enzymes).Experiments were duplicated.

Analytics
During the biotransformations, 10 μL of sample was taken at regular intervals and 0.5 mL of ethyl acetate was added to the extract substrate and product.After vortexing, the sample was centrifuged (13,300× g, 1 min) to facilitate phase separation.The organic phase was transferred to a glass vial for analysis.The bioconversion of keto ester to hydroxy ester was monitored by gas chromatography-flame ionization detector (GC-FID) (Agilent 6890N, Santa Clara, CA, USA) with a DB-5 capillary column (Agilent J&W, 30 m × 0.25 mm × 0.25 μm) and H2 as the carrier gas with a flow of 1 mL•min −1 .The volume of injection was 1 μL with split 50:1.The oven temperature was initially 90 °C for 5.5 min, but increased to 280 °C with a gradient of 30 °C/min and was held for 3 min.
The percentage conversion was evaluated as the peak area of alcohol product/(peak area of ketone substrate + peak area of alcohol product) × 100.One unit of KRED was the amount of enzyme that catalyzed the production of 1 μmol•min −1 of hydroxy ester (Figure 6).The middle volume of the reaction mixture was included in the calculation of co-immobilized KRED activity.The initial specific activity of free (or co-immobilized) KRED was defined as μmol of hydroxy ester (Figure 6) produced/min by 1 mg of KRED (or 1 g of particles).

Analytics
During the biotransformations, 10 µL of sample was taken at regular intervals and 0.5 mL of ethyl acetate was added to the extract substrate and product.After vortexing, the sample was centrifuged (13,300× g, 1 min) to facilitate phase separation.The organic phase was transferred to a glass vial for analysis.The bioconversion of keto ester to hydroxy ester was monitored by gas chromatography-flame ionization detector (GC-FID) (Agilent 6890N, Santa Clara, CA, USA) with a DB-5 capillary column (Agilent J&W, 30 m × 0.25 mm × 0.25 µm) and H 2 as the carrier gas with a flow of 1 mL•min −1 .The volume of injection was 1 µL with split 50:1.The oven temperature was initially 90 • C for 5.5 min, but increased to 280 • C with a gradient of 30 • C/min and was held for 3 min.
The percentage conversion was evaluated as the peak area of alcohol product/(peak area of ketone substrate + peak area of alcohol product) × 100.One unit of KRED was the amount of enzyme that catalyzed the production of 1 µmol•min −1 of hydroxy ester (Figure 6).The middle volume of the reaction mixture was included in the calculation of co-immobilized KRED activity.The initial specific activity of free (or co-immobilized) KRED was defined as µmol of hydroxy ester (Figure 6) produced/min by 1 mg of KRED (or 1 g of particles).

Figure 1 .
Figure 1.pH and temperature profile of KRED and GDH.

Figure 1 .
Figure 1.pH and temperature profile of KRED and GDH.

Figure 2 .
Figure 2. Influence of KRED and GDH concentrations in co-immobilization on the initial specific activity in biotransformations.

Figure 2 .
Figure 2. Influence of KRED and GDH concentrations in co-immobilization on the initial specific activity in biotransformations.

Catalysts 2018, 8 , 10 Figure 3 .
Figure 3. Initial specific activity and final bioconversion in repeated biotransformations of three parallel reactions of co-immobilized KRED and GDH.

Figure 4 .
Figure 4. Relative activity of co-immobilized KRED and GDH during storage at 4 °C.

Figure 3 .
Figure 3. Initial specific activity and final bioconversion in repeated biotransformations of three parallel reactions of co-immobilized KRED and GDH.

Catalysts 2018, 8 , 10 Figure 3 .
Figure 3. Initial specific activity and final bioconversion in repeated biotransformations of three parallel reactions of co-immobilized KRED and GDH.

Figure 4 .
Figure 4. Relative activity of co-immobilized KRED and GDH during storage at 4 °C.

Figure 4 .
Figure 4. Relative activity of co-immobilized KRED and GDH during storage at 4 • C.

10 Figure 3 .
Figure 3. Initial specific activity and final bioconversion in repeated biotransformations of three parallel reactions of co-immobilized KRED and GDH.

Figure 4 .
Figure 4. Relative activity of co-immobilized KRED and GDH during storage at 4 °C.

Table 2 .
Concentration of enzymes loaded into particles.