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Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration
Journal Pre-proofs Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration Meng-Qiu Xu, Fei-Long Li, Wen-Qian Yu, Rui-Fang Li, Ye-Wang Zhang PII: DOI: Reference:
S0141-8130(19)34639-2 https://doi.org/10.1016/j.ijbiomac.2019.09.178 BIOMAC 13459
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
20 June 2019 10 September 2019 21 September 2019
Please cite this article as: M-Q. Xu, F-L. Li, W-Q. Yu, R-F. Li, Y-W. Zhang, Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.178
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© 2019 Published by Elsevier B.V.
Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration
Meng-Qiu Xu#, Fei-Long Li#, Wen-Qian Yu, Rui-Fang Li, Ye-Wang Zhang*
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, People’s Republic of China.
#These authors contributed equally to this work. *Correspondence: Ye-Wang Zhang, School of Pharmacy, Jiangsu University, Zhenjiang, 212013, People’s Republic of China. Fax/Tel: +86-511-8503-8201; Email: [email protected]
1
Abstract Cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD+ in enzyme-catalyzed reactions. Herein, glycerol dehydrogenase (GDH) and NADH oxidase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for effective regeneration of NAD+. After optimization, the activity of combi-CLEAs and separate CLEAs mixtures were 950 and 580 U/g, respectively. And the catalytic stability of combi-CLEAs against pH and temperature was superior to the free enzyme mixture. After ten cycles of reuse, the catalytic efficiency could still retain 63.3% of its initial activity, indicating that the constructed combi-CLEAs system had excellent reusability. Also, the conversion of glycerol to 1,3-dihydroxyacetone (DHA) was improved by the constructed NAD+ regeneration system, resulting in 4.6%, which was 2.5 times of the free enzyme system. Thus, wide applications of this co-immobilization method in the production of various chiral chemicals could be expected in the industry for its high efficiency at a low cost.
Keywords Cofactor regeneration, combi-CLEAs, NADH oxidase, glycerol dehydrogenase
2
1 Introduction Many oxidoreductases depend on nicotinamide cofactors (NAD(P)+/NAD(P)H). These enzymes are useful for the synthesis of chiral chemicals which are difficult to be synthesized chemically [1-6]. However, the industrial-scale applications of these enzymes have often limited by the high requirement for these cofactors. To avoid the use of stoichiometric amounts of the expensive cofactor, an efficient regeneration method is necessary. Regeneration of reduced cofactor (NAD(P)H) was well established, but recycling oxidize counterparts is still a challenging task [7, 8]. Several strategies were proposed to regenerate oxidized cofactor, for example, chemical, electrochemical, photochemical, and enzymatic methods have been developed. Using inorganic salts (Na2S2O4 and NaBH4), H2, coenzyme analogs, the cofactor could be regenerated chemically [9, 10]. However, the low transformation efficiency, selectivity, and unavoidable waste may limit its application at the industrial-scale [6, 7, 11]. Although electrochemical and photochemical methods utilize clean and cheap solar energy to achieved cofactor regeneration, poor selectivity, bad compatibility, and low efficiency are still unavoidable defects [9, 12, 13]. Compared with these methods, enzymatic regeneration system of an ancillary redox enzyme with consumption of coenzyme has excited considerable interest because its high efficiency [14-17]. Co-expression of the primary enzyme (responsible for the primary synthesis reaction) and ancillary enzyme (for NAD(P)+) regeneration reaction) in a whole-cell system is widely used to achieve the 3
regeneration
of
oxidizing
(NAD(P)+)
cofactor
[18].
Additionally,
the
co-immobilization of two enzymes on carriers, as a controllable method, generally results in the enhanced enzyme stability against chemical degradation and thermal inactivation, and also been prepared for the recycling of NAD(P)+ [7, 11, 19-21]. These cofactor regeneration systems have been used in the preparation of valuable chemicals or pharmaceutical intermediates. Examples including co-expression or co-immobilization of NADH oxidase and glycerol dehydrogenase to convert glycerol to 1,3-dioxyacetone [7]. However, the loss of enzyme activity, low conversion rate, and the complicated preparation processes may hamper their application [22]. A carrier-free immobilization method, combined cross-linking enzyme aggregates (combi-CLEAs) has been proposed as an effective method to conventional immobilization on a solid carrier and crosslinked enzyme molecules [23, 24]. In this strategy, the soluble enzymes were precipitated by precipitators to form aggregates, and then the aggregates were linked using bi-functional agents [25]. Several advantages such as uncomplicated preparation process and higher activity retention could be achieved using the combi-CLEAs method because it is exquisitely simple and can be rapidly optimized in multiple enzymes cascade reactions. In addition, the controllable particle size, ease of recycling and volumetric productivities made this method ideally suited for industrial bio-catalysis [26, 27]. Combi-CLEAs has been applied extensively, such as efficient hydrolysis of complex lignocellulosic substances [28], synthesis of chiral alcohol [25], and production of bio-ethanol [29]. 4
In the present work, a combi-CLEAs system comprised of glycerol dehydrogenase (GDH) and H2O-forming NADH oxidase (Nox) was first developed. The prepared combi-CLEAs were further characterized and used for NAD+ regeneration to catalyze the production of DHA.
2 Materials and methods 2.1 Materials Reduced nicotinamide adenine dinucleotide, dithiothreitol and flavin adenine dinucleotide disodium salt hydrate were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Ni-NTA Prepacked chromatographic columns were bought from Sangon Biotech (Shanghai, China). 1, 3-dihydroxyacetone (DHA) was purchased from Aladdin (Shanghai, China). Isopropyl-β-D-thiogalactopyranoside (IPTG) was from Energy Chemical (Shanghai, China). Oxidized nicotinamide adenine dinucleotide (NAD+) was from Roche (Basel, Switzerland). Yeast extract, tryptone, kanamycin, glutaraldehyde, ammonium sulfate, glycerol, acetone, ethanol and acetonitrile were all obtained from Sinopharm (Shanghai, China). All the reagents were analytic or biological grade.
2.2 Preparation of soluble enzyme from recombinant E. coli E. coli BL21 (DE3) containing recombinant pET-28a-GDH or pET-28a-Nox plasmid was cultured in LB medium containing 50 μg/mL kanamycin at 37 oC 5
respectively. The recombinant protein expression was induced using 1.0 mM IPTG at 30 oC (GDH) or 15 oC (Nox) for 6 h when the OD600 value of the culture reached 0.6-0.8. The cells were harvested by centrifuging at 4 oC for 5min at 6500×g. Cells containing GDH or Nox genes were re-suspended in binding buffer 1 (20 mM pH 7.4 sodium dihydrogen phosphate, 500 mM NaCl, 20 mM imidazole) or binding buffer 2 (20 mM HEPES, pH 8.0, 10 mM imidazole), and were disrupted by sonication in an ice bath. Then the debris of cells was removed by centrifugation at 6500×g for 20 min to obtain the crude enzyme. Purification of GDH or Nox was performed on Ni-NTA super-flow column individually. The eluate from the column was used for the further preparation of combi-CLEAs. The protein contents of GDH and Nox were determined as 15 μg·mL-1 and 25 μg·mL-1 in the pre-purified solution.
2.3 Combi-CLEAs preparation Precooled different concentrations (20-100%) of organic solvents (acetone, ethanol and acetonitrile) and ammonium sulfate solution (1 mL each) were added dropwise separately to samples of free enzymes including a series concentration of GDH or Nox with shaking and kept for 30 min at 4 oC for complete precipitation of protein (Nox was pre-incubated with FAD for 5 min in advance). Glutaraldehyde was used as the cross-linker in the range of 2.5-25% and incubated at 4-50 oC under agitation for different time intervals (0.5-8.5 h). The combi-CLEAs were obtained by centrifugation at 6000×g for 3min. The pellets were washed three times with sodium
6
phosphate buffer (50mM, pH 7.0) to remove the unreacted protein and glutaraldehyde. The percent of protein yield and activity recovery were determined using Equation (1) and Equation (2), as mentioned below. The prepared combi-CLEAs were dispersed in sodium phosphate buffer (60mM, pH 7.0) and stored at 4 oC. ሺΨሻ ൌ ሺΨሻ ൌ
ሺ Ȁ ሻ ሺ Ȁ ሻ
ሺሻ ሺሻ
ൈ ͳͲͲ (1)
ൈ ͳͲͲ (2)
For comparison, separate CLEAs of GDH or Nox were prepared separately. The parameters for the preparation of GDH-CLEA and Nox-CLEA were also optimized.
2.4 Enzyme assay The activity of free GDH or Nox was determined by a photometer assay at 340 nm [30]. The reaction system (1 mL) used to determine GDH activity includes 50 mM sodium phosphate buffer, 0.2 M glycerol, 2 mM NAD+, and 10 μg GDH. The protein concentration was calculated by the Bradford method [32]. The activity of Nox and GDH was determined by incubation at 60 oC and 30 oC for 5 min respectively. One unit of enzymatic activity was defined as the amount of GDH required to increase 1 μmol NADH in each minute. The reaction mixture (1 mL) used for Nox assay containing 10 μmol FAD, 1mM dithiothreitol (DTT), 50mM citrate buffer, and 10 μg Nox. Before determining the enzyme activity, the Nox enzyme was preincubated with FAD for 5min. NADH oxidase can effectively regenerate the NAD+ consumed by GDH in both
7
free enzyme mixture and immobilized enzymes and DHA was the only detectable product of this cascade reaction. The production of DHA could be determined to calculate the catalytic activity of both free enzyme and CLEAs. The catalytic activities of free enzyme and CLEAs were estimated using a system including 50 mM sodium phosphate buffer, 0.2 M glycerol, 2 mM NAD+, 10 μM FAD, and 0.75 mg of combi-CLEAs, separate CLEAs and free enzyme mixture (0.15mg of Nox and 0.6 mg of GDH). After reaction at ambient temperature for 15 min, the reaction system was boiled for 5 min and 6000×g centrifuged for 5 min respectively. 0.5 mL supernatants were mixed with 4.5 mL of diphenylamine reagent (0.6 g diphenylamine, 6 mL sulfuric acid and 54 mL acetic acid) and were incubated in the closed tubes for 20 min with boiling water bath [33]. CLEA samples were withdrawn at different intervals, and DHA concentration was determined spectrophotometrically. One unit of enzymatic activity was defined as the amount of enzyme required to produce 1 μM DHA per minute.
2.5 Characterization of combi-CLEAs 2.5.1 Scanning electron microscope and optic microscopy analysis The morphologies of CLEAs were characterized using SEM. The combi-CLEAs and separate CLEAs were freeze-dried, lyophilized, and then coated with gold particles. The SEM images of separate CLEAs and combi-CLEAs was recorded by scanning electron microscope and was obtained by using GeminiSEM 300 (ZEISS, 8
Germany) scanning electron microscope. The size of the combi-CLEAs was characterized using the optic microscope. The combi-CLEAs before the first cycle, after five cycles and after ten cycles were harvested and resuspended in PBS. The optic images of combi-CLEAs were recorded and analyzed using ECLIPSE TS100 (Nikon, Japan).
2.5.2 Effects of temperature and pH on the activity of combi-CLEAs The relative activities of combi-CLEAs, separate CLEAs, and free enzymes were determined by measuring the amount of 1,3-dihydroxyacetone produced with 0.2 M glycerol as the substrate. The effects of pH on the activity of combi-CLEAs, separate CLEAs, and free enzymes were analyzed in the pH range of 4-10 with 50 mM buffers (pH 4-6, citrate buffer; pH 6-8, phosphate buffer; pH 8-10, Tris-HCl buffer). The effects of temperature on the activity of combi-CLEAs, CLEAs mixture, and free enzymes mixture were investigated in the range of 4-50 oC. The highest values of combi-CLEAs, separate CLEAs, or free enzyme mixture were taken to 100%.
2.5.3 Kinetic parameters The kinetic parameters of combi-CLEAs, separate CLEAs, and free enzymes was determined by measuring the initial rates of the reaction with different glycerol concentrations ranging from 0.05 to 1.0 mM in 50 mM phosphate buffer (pH 7). The kinetic parameters, including the Km and Kcat values, were calculated with nonlinear regression fitting.
9
2.5.4 Thermal stability The thermal stability of combi-CLEAs, separate CLEAs, and free enzymes mixture was examined at 45 and 55 oC respectively. At certain time intervals, the sample was withdrawn, and the residual activity of each system was calculated by comparing it with the initial activity. The thermal inactivation half-life (t1/2) was determined by comparing the remaining activity values versus incubation time and calculated based on a linear regression. The t1/2 values were calculated following this equation (k is the constant inactivation rate at the studied temperature): ͳݐൌ ʹ
݈݊ሺʹሻ ݇
2.5.5 Reusability of combi-CLEAs and separate CLEAs mixture. The reusability of this constructed two NAD+ regeneration system (the combi-CLEAs, and CLEAs mixture) was investigated in 50 mM sodium phosphate buffer, 0.2 M glycerol, 2 mM NAD+ at 30 oC. After 15 min, the biocatalysts were obtained by centrifugation at 7000×g for 3 min, washed two times with 50 mM sodium phosphate buffer (pH 7) and then re-dispersed in the fresh reaction system to determine the enzymatic activity. The residual activity of the immobilized enzymes was calculated by setting the initial activity as 100%.
10
2.6 Cofactor regeneration with combi-CLEAs 2.6.1 Effect of NAD+ concentration on the activity of enzymes The effects of NAD+ concentration on combi-CLEAs, separate CLEAs or free enzymes mixture were investigated in 50 mM, pH 7 phosphate buffer containing 10 μM FAD and 0.2 mM glycerol at ambient temperature, with NAD+ concentration varying from 0.05 to 5 μM. The concentration of DHA in the reaction was quantitated with the diphenylamine method after the reaction mixtures were boiled for 15 min.
2.6.2 Catalytic performance of combi-CLEAs, CLEAs gained and free enzymes mixture Enzymatic synthesis of DHA was performed by adding combi-CLEAs, separate CLEAs, and free enzyme mixtures into 2.5 mL catalytic system containing 50 mM potassium phosphate buffer (pH 7), 0.2 M glycerol, 2 mM NAD+, 10 μM FAD, and 0.75 mg mixed enzymes (combi-CLEAs, separate CLEAs or free enzymes mixture respectively) at ambient temperature. Aliquots were withdrawn after every 3 h, and the produced DHA was quantified by the colorimetric method at 620 nm. Triplicate measurements were made for each sample during the experiment and the corresponding standard errors were calculated.
11
3. Result and discussion 3.1
Optimization
of
the
preparation
of
GDH/Nox
combi-CLEAs Different concentrations of precipitants, including acetone, ethanol, acetonitrile, and ammonium sulfate were tested for precipitation of GDH and Nox. The volume of the precipitating agent was lower than that of the enzyme solution. Thus, to better precipitate the protein molecules, precipitating agents were added dropwise separately to samples of free enzymes. Fig. 1A and 1B indicate that saturated ammonium sulfate allows almost the entire precipitation of GDH (98.75%) and Nox (92.66%) into aggregates and high activity recovery (127.14% for GDH) and 136.84% for Nox) could be obtained. More than 100% activity recovery called “hyper activation” could be obtained when the concentration of saturated ammonium sulfate was higher than 60% because the precipitation conditions may induce the enzyme molecules to adopt a more active conformation [34, 35]. Also, the precipitated proteins have larger surfaces because these protein molecules are fine-grained and have many cavities whereas the coarse-grained proteins in low concentration of saturated ammonium sulfate have smaller surfaces and show lower activity recovery [35]. Different amounts of NADH oxidase and glycerol dehydrogenase were tested to achieve the best catalytic efficiency. Fig. 2A shows that when the GDH/Nox ratio was increased from 2:1 to 5:1, but the activity of both combi-CLEAs and the CLEA mixtures were
12
slightly increased. And the maximal apparent rate of combi-CLEAs was observed when the ratio of GDH and Nox reach to 5:1. The results indicate that the GDH is a rate-limiting enzyme in the NAD+ regeneration reaction because of its relatively low activity and also suggested that NADH oxidase could catalyze the regeneration of NAD+ molecules which are used as the co-substrate of GDH. Generally, the particle size of CLEAs dependent on the aggregation agent type, enzyme/cross-linker ratio, as well as pH conditions. Among these factors, the enzyme/cross-linker ratio plays a significant role in affecting particle size compared with other factors. It is evident that more proteins precipitated at a higher cross-linker concentration solution and formed bigger CLEAs [34]. However, the catalytic efficiency of combi-CLEAs with large particles was lower than that of small particle CLEAs because the latter has a more significant surface area, but the formation of large clusters leads to the unavoidable internal mass transfer limitations [23, 34]. Thus, to obtain the combi-CLEAs with the highest catalytic efficiency, the optimal enzyme/glutaraldehyde ratio was determined. Fig. 2B clearly shows that a higher activity recovery could be obtained by slightly increasing glutaraldehyde concentration up to 5% (mol/mol) glutaraldehyde. However, the recovery of activity decreased obviously when a higher concentration of glutaraldehyde was added as a cross-linking agent because more amounts of glutaraldehyde are able to make more proteins cross-linked and the particle size of CLEAs will increase obviously [34, 35, 36]. This result suggested that the excessive glutaraldehyde may have negative effects 13
on the activity of CLEAs because the inner enzymes of the CLEAs could not form a complex with substrates and cofactor when increasing the CLEAs diameter significantly (over 60 μm) [34, 36]. The cross-linking temperature and time were also optimized to achieve the highest activity recovery. The results showed that the optimal temperature for the preparation of combi-CLEAS is 10
o
C (Fig. 2C). The activity recovery in
combi-CLEAs was decreased significantly to 52% when the cross-linking temperature increased to 50 oC, which might due to the thermal inactivation of enzymes [37]. In another way, the activity recovery of combi-CLEAs increased to achieve the optimal value in 2 h and decreased obviously over this point (Fig. 2D). This might be because glutaraldehyde is a bi-functional reagent which has could react with other functional groups such as active cysteine. Thus, it could get access to the buried motif of the protein and may react with the critical amino acid residues of the enzyme, and thus the active sites could be destroyed because of the excessive cross-linking [24, 38]. The cross-linking process of mixed GDH-CLEAs and Nox-CLEAs (the ratio was 4:1) was also performed with saturated ammonium sulfate and 5% glutaraldehyde at 10 oC for 1 h. The results showed that the obtained apparent rate CLEAs was 580 ± 0.12
U/g.
But
the
maximum
activity
recovery
of
combi-CLEAs
after
co-immobilization at a different time and the temperature was up to 950 ± 0.04 U/g. Thus, it can be observed that the enzyme activity of the combi-CLEAs was 1.6 times that of the CLEAs mixture. Cofactor in situ regeneration using the combi-CLEAs 14
method is expected to have a higher overall enzyme reaction rate compared to the free counterpart [39]. This may be because the adjacent dispersion and proximity of two enzymes might lead to fast uptake of a cofactor for the catalytic reaction by the co-immobilization of the two proteins [40, 41].
3.2 Biochemical characterization of combi-CLEAs 3.2.1 Morphology of combi-CLEAs and CLEAs mixture by SEM The Combi-CLEAs and CLEAs mixture were also freeze-dried for scanning electron microscopy (SEM) observation. Fig. 3A shows that the combi-CLEAs have a less structured form, which is different from the separate CLEAs displaying the typical and spherical aggregate appearance (Fig. 3B) [26]. Although the combi-CLEAs with spherical appearance contain fewer enzyme molecules than the typical “ball” appearance of separate CLEAs, the proximity of each enzyme molecules may increase the cofactor uptake rate in the reaction system and increase the NAD+ regeneration efficiency.
3.2.2 Optimal reaction conditions of GDH/Nox combi-CLEAs The activity of the combi-CLEAs was investigated at different pH values (4-10), and the activity of enzymes was calculated by setting the activity of combi-CLEAs as 100%. Fig. 4A showed that the optimal pH of combi-CLEAs was shifted from 9.0 to a neutral condition compared with free enzyme mixture. At pH 7.0, the activities of
15
combi-CLEAs and CLEAs mixture were 3.6 and 1.7 times higher than that of the free enzyme mixtures. And the combi-CLEAs had 54.3% of relative activity while free enzyme mixtures only kept 42.2% of relative activity at pH 10.0. This might be because that the active glutaraldehyde molecules may react with the surface amino groups of enzymes in combi-CLEAs. Therefore, more negative charges could be transferred by the acidic groups of enzyme surface to the enzyme and change the optimal pH values [26]. Also, the optimal temperature of the free enzyme mixture was 30 oC, but the CLEAs mixture and combi-CLEAs both showed the highest activity at 40 oC, respectively (Fig. 4B). This suggested that the immobilization process increases the optimal temperature by approximately 10 oC. The relative activities of combi-CLEAs and CLEAs mixtures were 1.6 and 1.4 times higher than that of free enzyme mixtures at 40 oC (the activity of combi-CLEAs at 40 oC as 100%.). This enhanced activity might due to that the formed covalent bonds between protein molecules caused by glutaraldehyde might decrease the conformational flexibility of the enzymes and protect them from distortion or damage by heat exchange [42, 43].
3.2.3 Kinetic characterization of GDH/Nox combi-CLEAs The apparent kinetic parameters of constructed combi-CLEAs, separate CLEAs, and free enzymes mixture referring to the overall heterogeneous process were determined. As shown in Fig. 5 and Table 1, the Km values of combi-CLEA (18.45 μM) or CLEAs mixture (15.3 μM) were higher than that of free enzyme mixture (14.7 μM) after immobilization. This seems to result from that these enzymes are gathered in 16
compared to the free enzyme mixtures and cause the mass transport limitations in reactions. Moreover, the super-molecular structure assembled by micro-molecular of glutaraldehyde and the active sites of enzymes got obscured [28, 29, 44]. As shown in table 1, combi-CLEAs had 1.64 times and 1.11 times higher Kcat values than that of CLEAs mixture and free enzyme mixture. The possible explanation was that the adjacent dispersion of two enzymes may lead to the quick consumption of NAD+ [40, 45].
3.2.4 Stability and reusability of GDH/Nox combi-CLEAs The thermal stability of the combi-CLEAs, free enzymes and separate CLEAs were evaluated by incubating them for different time intervals at 55 oC. In Fig. 6A, compared with the half time (t1/2) of the free enzyme (4.5 h), the separate CLEAs and combi-CLEAs showed higher t1/2 values of 6.0 and 6.5 h, respectively. It suggested that separate CLEAs and combi-CLEAs have an enhancement in the stability of enzymes at 55 oC. And the better thermal stability of co-immobilized proteins also demonstrated that co-immobilization of enzymes using the combi-CLEAs method could prevent the enzymes from the inactivation because more energy was required to destroy the active motif than free enzymes. The reusability of CLEAs mixture or combi-CLEAs was explored up to 10 cycles, and the results were shown in Fig. 6B. The catalytic activity of combi-CLEAs decreased to 82% at the fifth cycles and 71% at the eighth cycles, but the corresponding values of separate CLEAs were 74% and 66%, which may result from 17
the deactivation of the enzyme during the catalysis and the mechanical damage or loss of the centrifugation. To further explore the reasons behind the decreased activity, optic microscope was performed to analyze the size of CLEAs particles during the reuse process. Fig. 7A displays that the diameter of combi-CLEA particle before the first reuse cycle was approximately 50 μm and Fig. 7B show that the diameters of CLEA particle decreased slightly after five cycles reuse. However, an obvious decline of the CLEA particle could be observed after ten cycles reuse (30 μm) due to the repeated centrifugation and resuspension applied (Fig. 7C) and its catalytic activity reduced to only 64% of the initial activity simultaneously, which suggested that the reduction of CLEAs size may lead to the decrease of its activity. This is probably because the squeezing of combi-CLEA particles and the reduced CLEAs size elevate mass transfer limitations for substrates and cofactors. The results suggested that the treatment presumably squeezes the combi-CLEA particle and makes it closes together [46-48]. The aforementioned advantages make the combi-CLEAs hold excellent operational stability and reusability.
3.3 Cofactor regeneration with combi-CLEAs The NAD+ regenerative performance of prepared combi-CLEAs was further verified for long-time continuous operation. Fig. 8A shows that the TTN of combi-CLEAs, separate CLEAs and free enzyme mixtures were 2137, 1849 and 1897, respectively (0.05 μM NAD+, 10 μM FAD, 0.2 mM glycerol and 15 min,). These
18
results suggested that co-immobilization has higher utilization efficiency for NAD+. Fig. 8B shows that the DHA production by combi-CLEAs, separate CLEAs, and free enzymes mixture reached to 9.1, 3.6, and 4.7 mM, respectively and also shows that the yield of DHA of combi-CLEAs is higher than that of separate CLEAs and free enzymes in 30 h. This might be due to that the combi-CLEAs method enhanced the approach of the enzyme molecules and may reduce the uptake time of the cofactors (NAD+/NADH) between the two enzymes. Thus far, the enzymatic synthesis of DHA has been reported in several types of research. For example, immobilized GDH (without Nox) produced 3.5 mM DHA with a low conversion of 1.8% [49]. A cofactor regeneration system makes the reaction more economical and feasible. For example, GDH and Nox were immobilized as an NAD+ regeneration system with nanoparticles respectively to produce DHA. After 10 h, The production of DHA reached up to 3.5 mM, and the 3.6 % conversion efficiency with 100 mM glycerol was achieved. Rocha-Martin et al. immobilized GDH and Nox in nanoparticles and gain the production of 3.0 mM DHA with 20 mM glycerol consumption after 8.5 h reaction [50]. In addition, nanoparticle-supported GDH and xylose reductase (XR) also have been prepared for simultaneous bio-convention of DHA and the production and convention rate of the bio-catalysis system reached 8.2 mM [51] (Table 1). In this study, co-immobilization of GDH and Nox by combi-CLEAs method obtained 9.1 mM DHA and 4.6% conversion efficiency after 30 h. Although the production of DHA was limited using glycerol dehydrogenase 19
because of the severe product inhibition which could be reduced by continuous product extraction or removing the specific residues in order to decrease the binding affinity between the product and the enzyme [50, 52]. In this study, the yield of DHA was relatively higher and the DHA was produced by a more commercial and simple process compared to several previous reports, which indicated its potential in the industrial production of DHA.
4 Conclusions In summary, combi-CLEAs consisting of glycerol dehydrogenase and NADH oxidase were firstly prepared to achieve the highly effective NAD+ regeneration. Compared with CLEAs mixture and free enzyme mixture. The higher thermal stability, pH tolerance, reusability, and long-term operational stability of combi-CLEAs suggest its potential in practical application. Besides, the combi-CLEAs has a higher TTN of 2137, indicating that the high-speed regeneration of NAD+ was successfully achieved. By which the conversion of glycerol catalyzed by combi-CLEAs reached 4.6% after 30 mins which were 2.5 times higher than that of free enzyme mixtures. Thus, the prepared combi-CLEAs of glycerol dehydrogenase and NADH oxidase are promising biocatalyst for the development of NAD+ regeneration for the synthesis of DHA.
Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 21376110) 20
Conflict of interest The authors declare no conflicts of interest.
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immobilized glycerol dehydrogenases with higher stability and lower product inhibition, Bioresour. Technol. 170 (2014) 445–453. [51] Y. Zhang, F. Gao, S.-P. Zhang, Z.-G. Su, G.-H. Ma, P. Wang, Simultaneous production of 1,3-dihydroxyacetone and xylitol from glycerol and xylose using a nanoparticle-supported multi-enzyme system with in situ cofactor regeneration, Bioresour. Technol. 102 (2011) 1837–1843. [52] D. Weuster-Botz, D. Hekmat, R. Puskeiler, E. Franco-Lara, Enabling technologies: fermentation and downstream processing, in: R. Ulber, D. Sell (Eds.), White Biotechnology, Springer Berlin Heidelberg, Berlin, Heidelberg, 2007: pp. 205–247.
29
Table 1. Apparent kinetic parameters of combi-CLEAs, separate CLEAs, and free enzymes. Enzymes
Km (μM)
Kcat (S-1)
Kcat / Km (S-1gμM-1)
Free enzymes
14.70
21.34
1.45
Separate CLEAs
15.33
31.65
2.06
Combi-CLEAs
18.45
34.96
1.90
30
Figure legends Fig. 1 Precipitation of GDH and Nox. Effects of precipitants type and concentration on the precipitation rate and activity recovery of GDH (A and B), and Nox (C and D).
31
32
Fig. 2 Effects of (A) GDH/Nox ratio; (B) protein/glutaraldehyde ratio; (C) temperature and (D) immobilization time on the activities of combi-CLEAs (red) and
Relative activity (%)
separate CLEAs (green).
100
A
80 60 40 20 0
2
3
4
5
6
7
8
Relative activity (%)
GDH/Nox ratio (mol/mol) 100
B
80 60 40 20 0
:1 0 :1 3:1 0 :1 8:1 7 :1 .5:1 4 :1 .5:1 5 :1 0 4 2 1 1 4 5
Protein/glutaraldehyde (mol/mol)
33
Relative activity (%)
100
C
80 60 40 20 0 0
5 10 15 20 25 30 35 40 45 50 55
Relative activity (%)
Temperature (oC)
100
D
80 60 40 20 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (h)
34
Fig. 3 SEM images of combi-CLEAs (A) and CLEAs mixture (B).
35
Fig. 4 Characterization of combi-CLEAs, CLEAs mixture and free enzyme mixture. Effects of pH and temperature on activities of combi-CLEAs, separate CLEAs mixture, and free enzyme mixture. (Red: combi-CLEAs; Green: CLEAs mixture; Blue: free enzymes mixture).
Relative activity (%)
100
A
75 50 25 0
4
5
6
7
8
9
10
pH
Relative activity (%)
100
B
75 50 25 0
0 5 10 15 20 25 30 35 40 45 50 55
Temperature (oC)
36
Fig. 5 Effects of glycerol concentration on the activity of combi-CLEAs, CLEAs mixture and free enzyme mixture (Red: combi-CLEAs; Green: CLEAs mixture; Blue: free enzymes mixture).
Activity (U·mg-1)
1.0 0.8 0.6 0.4 0.2 0.0 0
250
500
750
Glycerol (μmol)
37
1000
Fig. 6. Thermal stability (A) of combi-CLEAs, CLEAs mixture and free the mixture at 55 oC (Red: combi-CLEAs, green: separate CLEAs mixture and blue: free enzyme). Recyclability (B) of combi-CLEAs and CLEAs mixture (Green: combi-CLEAs and
Relative activity (%)
red: CLEAs mixture).
100
A
90 80 70 60 50 0 20 40 60 80 100120140160180200
Time (min)
Residual activity (%)
100
B
80 60 40 20 0 0
1
2
3
4
5
6
Cycles
38
7
8
9 10
Fig. 7. Optic microscope images of combi-CLEAs before the first cycle (A), after five cycles of use (B), and after ten cycles of use (C).
39
Fig. 8. Catalytic performance of combi-CLEAs. (A) Total turnover number (TTN) values of combi-CLEAs, CLEAs mixture and free enzymes mixture. (B) Production of DHA catalyzed by combi-CLEAs, CLEAs mixture and free enzyme mixture. (Red: combi-CLEAs, green: separate CLEAs and blue: free enzymes).
2000 A
1000 500 0 0
10
DHA (mM)
TTN
1500
4
2
0
2
4
0
2
4
NAD+ concentration (μM) B
8 6 4 2 0 0
5
10
15
20
Time (h)
40
25
30
Highlights
¾ Combi-CLEAs of glycerol dehydrogenase and NADH oxidase were prepared for in situ NAD+ regeneration. ¾ Adjacent dispersion of two enzymes may lead to the quick uptake of NAD+ and achieve the high-speed NAD+ regeneration. ¾ The conversion of glycerol in combi-CLEAs reaches 4.60 %, which is 2.5 folds of the free enzyme (1.85%).
41
Graphical abstract
42
S0141-8130(19)34639-2 https://doi.org/10.1016/j.ijbiomac.2019.09.178 BIOMAC 13459
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
20 June 2019 10 September 2019 21 September 2019
Please cite this article as: M-Q. Xu, F-L. Li, W-Q. Yu, R-F. Li, Y-W. Zhang, Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.178
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© 2019 Published by Elsevier B.V.
Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration
Meng-Qiu Xu#, Fei-Long Li#, Wen-Qian Yu, Rui-Fang Li, Ye-Wang Zhang*
School of Pharmacy, Jiangsu University, Zhenjiang, 212013, People’s Republic of China.
#These authors contributed equally to this work. *Correspondence: Ye-Wang Zhang, School of Pharmacy, Jiangsu University, Zhenjiang, 212013, People’s Republic of China. Fax/Tel: +86-511-8503-8201; Email: [email protected]
1
Abstract Cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD+ in enzyme-catalyzed reactions. Herein, glycerol dehydrogenase (GDH) and NADH oxidase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for effective regeneration of NAD+. After optimization, the activity of combi-CLEAs and separate CLEAs mixtures were 950 and 580 U/g, respectively. And the catalytic stability of combi-CLEAs against pH and temperature was superior to the free enzyme mixture. After ten cycles of reuse, the catalytic efficiency could still retain 63.3% of its initial activity, indicating that the constructed combi-CLEAs system had excellent reusability. Also, the conversion of glycerol to 1,3-dihydroxyacetone (DHA) was improved by the constructed NAD+ regeneration system, resulting in 4.6%, which was 2.5 times of the free enzyme system. Thus, wide applications of this co-immobilization method in the production of various chiral chemicals could be expected in the industry for its high efficiency at a low cost.
Keywords Cofactor regeneration, combi-CLEAs, NADH oxidase, glycerol dehydrogenase
2
1 Introduction Many oxidoreductases depend on nicotinamide cofactors (NAD(P)+/NAD(P)H). These enzymes are useful for the synthesis of chiral chemicals which are difficult to be synthesized chemically [1-6]. However, the industrial-scale applications of these enzymes have often limited by the high requirement for these cofactors. To avoid the use of stoichiometric amounts of the expensive cofactor, an efficient regeneration method is necessary. Regeneration of reduced cofactor (NAD(P)H) was well established, but recycling oxidize counterparts is still a challenging task [7, 8]. Several strategies were proposed to regenerate oxidized cofactor, for example, chemical, electrochemical, photochemical, and enzymatic methods have been developed. Using inorganic salts (Na2S2O4 and NaBH4), H2, coenzyme analogs, the cofactor could be regenerated chemically [9, 10]. However, the low transformation efficiency, selectivity, and unavoidable waste may limit its application at the industrial-scale [6, 7, 11]. Although electrochemical and photochemical methods utilize clean and cheap solar energy to achieved cofactor regeneration, poor selectivity, bad compatibility, and low efficiency are still unavoidable defects [9, 12, 13]. Compared with these methods, enzymatic regeneration system of an ancillary redox enzyme with consumption of coenzyme has excited considerable interest because its high efficiency [14-17]. Co-expression of the primary enzyme (responsible for the primary synthesis reaction) and ancillary enzyme (for NAD(P)+) regeneration reaction) in a whole-cell system is widely used to achieve the 3
regeneration
of
oxidizing
(NAD(P)+)
cofactor
[18].
Additionally,
the
co-immobilization of two enzymes on carriers, as a controllable method, generally results in the enhanced enzyme stability against chemical degradation and thermal inactivation, and also been prepared for the recycling of NAD(P)+ [7, 11, 19-21]. These cofactor regeneration systems have been used in the preparation of valuable chemicals or pharmaceutical intermediates. Examples including co-expression or co-immobilization of NADH oxidase and glycerol dehydrogenase to convert glycerol to 1,3-dioxyacetone [7]. However, the loss of enzyme activity, low conversion rate, and the complicated preparation processes may hamper their application [22]. A carrier-free immobilization method, combined cross-linking enzyme aggregates (combi-CLEAs) has been proposed as an effective method to conventional immobilization on a solid carrier and crosslinked enzyme molecules [23, 24]. In this strategy, the soluble enzymes were precipitated by precipitators to form aggregates, and then the aggregates were linked using bi-functional agents [25]. Several advantages such as uncomplicated preparation process and higher activity retention could be achieved using the combi-CLEAs method because it is exquisitely simple and can be rapidly optimized in multiple enzymes cascade reactions. In addition, the controllable particle size, ease of recycling and volumetric productivities made this method ideally suited for industrial bio-catalysis [26, 27]. Combi-CLEAs has been applied extensively, such as efficient hydrolysis of complex lignocellulosic substances [28], synthesis of chiral alcohol [25], and production of bio-ethanol [29]. 4
In the present work, a combi-CLEAs system comprised of glycerol dehydrogenase (GDH) and H2O-forming NADH oxidase (Nox) was first developed. The prepared combi-CLEAs were further characterized and used for NAD+ regeneration to catalyze the production of DHA.
2 Materials and methods 2.1 Materials Reduced nicotinamide adenine dinucleotide, dithiothreitol and flavin adenine dinucleotide disodium salt hydrate were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Ni-NTA Prepacked chromatographic columns were bought from Sangon Biotech (Shanghai, China). 1, 3-dihydroxyacetone (DHA) was purchased from Aladdin (Shanghai, China). Isopropyl-β-D-thiogalactopyranoside (IPTG) was from Energy Chemical (Shanghai, China). Oxidized nicotinamide adenine dinucleotide (NAD+) was from Roche (Basel, Switzerland). Yeast extract, tryptone, kanamycin, glutaraldehyde, ammonium sulfate, glycerol, acetone, ethanol and acetonitrile were all obtained from Sinopharm (Shanghai, China). All the reagents were analytic or biological grade.
2.2 Preparation of soluble enzyme from recombinant E. coli E. coli BL21 (DE3) containing recombinant pET-28a-GDH or pET-28a-Nox plasmid was cultured in LB medium containing 50 μg/mL kanamycin at 37 oC 5
respectively. The recombinant protein expression was induced using 1.0 mM IPTG at 30 oC (GDH) or 15 oC (Nox) for 6 h when the OD600 value of the culture reached 0.6-0.8. The cells were harvested by centrifuging at 4 oC for 5min at 6500×g. Cells containing GDH or Nox genes were re-suspended in binding buffer 1 (20 mM pH 7.4 sodium dihydrogen phosphate, 500 mM NaCl, 20 mM imidazole) or binding buffer 2 (20 mM HEPES, pH 8.0, 10 mM imidazole), and were disrupted by sonication in an ice bath. Then the debris of cells was removed by centrifugation at 6500×g for 20 min to obtain the crude enzyme. Purification of GDH or Nox was performed on Ni-NTA super-flow column individually. The eluate from the column was used for the further preparation of combi-CLEAs. The protein contents of GDH and Nox were determined as 15 μg·mL-1 and 25 μg·mL-1 in the pre-purified solution.
2.3 Combi-CLEAs preparation Precooled different concentrations (20-100%) of organic solvents (acetone, ethanol and acetonitrile) and ammonium sulfate solution (1 mL each) were added dropwise separately to samples of free enzymes including a series concentration of GDH or Nox with shaking and kept for 30 min at 4 oC for complete precipitation of protein (Nox was pre-incubated with FAD for 5 min in advance). Glutaraldehyde was used as the cross-linker in the range of 2.5-25% and incubated at 4-50 oC under agitation for different time intervals (0.5-8.5 h). The combi-CLEAs were obtained by centrifugation at 6000×g for 3min. The pellets were washed three times with sodium
6
phosphate buffer (50mM, pH 7.0) to remove the unreacted protein and glutaraldehyde. The percent of protein yield and activity recovery were determined using Equation (1) and Equation (2), as mentioned below. The prepared combi-CLEAs were dispersed in sodium phosphate buffer (60mM, pH 7.0) and stored at 4 oC. ሺΨሻ ൌ ሺΨሻ ൌ
ሺ Ȁ ሻ ሺ Ȁ ሻ
ሺሻ ሺሻ
ൈ ͳͲͲ (1)
ൈ ͳͲͲ (2)
For comparison, separate CLEAs of GDH or Nox were prepared separately. The parameters for the preparation of GDH-CLEA and Nox-CLEA were also optimized.
2.4 Enzyme assay The activity of free GDH or Nox was determined by a photometer assay at 340 nm [30]. The reaction system (1 mL) used to determine GDH activity includes 50 mM sodium phosphate buffer, 0.2 M glycerol, 2 mM NAD+, and 10 μg GDH. The protein concentration was calculated by the Bradford method [32]. The activity of Nox and GDH was determined by incubation at 60 oC and 30 oC for 5 min respectively. One unit of enzymatic activity was defined as the amount of GDH required to increase 1 μmol NADH in each minute. The reaction mixture (1 mL) used for Nox assay containing 10 μmol FAD, 1mM dithiothreitol (DTT), 50mM citrate buffer, and 10 μg Nox. Before determining the enzyme activity, the Nox enzyme was preincubated with FAD for 5min. NADH oxidase can effectively regenerate the NAD+ consumed by GDH in both
7
free enzyme mixture and immobilized enzymes and DHA was the only detectable product of this cascade reaction. The production of DHA could be determined to calculate the catalytic activity of both free enzyme and CLEAs. The catalytic activities of free enzyme and CLEAs were estimated using a system including 50 mM sodium phosphate buffer, 0.2 M glycerol, 2 mM NAD+, 10 μM FAD, and 0.75 mg of combi-CLEAs, separate CLEAs and free enzyme mixture (0.15mg of Nox and 0.6 mg of GDH). After reaction at ambient temperature for 15 min, the reaction system was boiled for 5 min and 6000×g centrifuged for 5 min respectively. 0.5 mL supernatants were mixed with 4.5 mL of diphenylamine reagent (0.6 g diphenylamine, 6 mL sulfuric acid and 54 mL acetic acid) and were incubated in the closed tubes for 20 min with boiling water bath [33]. CLEA samples were withdrawn at different intervals, and DHA concentration was determined spectrophotometrically. One unit of enzymatic activity was defined as the amount of enzyme required to produce 1 μM DHA per minute.
2.5 Characterization of combi-CLEAs 2.5.1 Scanning electron microscope and optic microscopy analysis The morphologies of CLEAs were characterized using SEM. The combi-CLEAs and separate CLEAs were freeze-dried, lyophilized, and then coated with gold particles. The SEM images of separate CLEAs and combi-CLEAs was recorded by scanning electron microscope and was obtained by using GeminiSEM 300 (ZEISS, 8
Germany) scanning electron microscope. The size of the combi-CLEAs was characterized using the optic microscope. The combi-CLEAs before the first cycle, after five cycles and after ten cycles were harvested and resuspended in PBS. The optic images of combi-CLEAs were recorded and analyzed using ECLIPSE TS100 (Nikon, Japan).
2.5.2 Effects of temperature and pH on the activity of combi-CLEAs The relative activities of combi-CLEAs, separate CLEAs, and free enzymes were determined by measuring the amount of 1,3-dihydroxyacetone produced with 0.2 M glycerol as the substrate. The effects of pH on the activity of combi-CLEAs, separate CLEAs, and free enzymes were analyzed in the pH range of 4-10 with 50 mM buffers (pH 4-6, citrate buffer; pH 6-8, phosphate buffer; pH 8-10, Tris-HCl buffer). The effects of temperature on the activity of combi-CLEAs, CLEAs mixture, and free enzymes mixture were investigated in the range of 4-50 oC. The highest values of combi-CLEAs, separate CLEAs, or free enzyme mixture were taken to 100%.
2.5.3 Kinetic parameters The kinetic parameters of combi-CLEAs, separate CLEAs, and free enzymes was determined by measuring the initial rates of the reaction with different glycerol concentrations ranging from 0.05 to 1.0 mM in 50 mM phosphate buffer (pH 7). The kinetic parameters, including the Km and Kcat values, were calculated with nonlinear regression fitting.
9
2.5.4 Thermal stability The thermal stability of combi-CLEAs, separate CLEAs, and free enzymes mixture was examined at 45 and 55 oC respectively. At certain time intervals, the sample was withdrawn, and the residual activity of each system was calculated by comparing it with the initial activity. The thermal inactivation half-life (t1/2) was determined by comparing the remaining activity values versus incubation time and calculated based on a linear regression. The t1/2 values were calculated following this equation (k is the constant inactivation rate at the studied temperature): ͳݐൌ ʹ
݈݊ሺʹሻ ݇
2.5.5 Reusability of combi-CLEAs and separate CLEAs mixture. The reusability of this constructed two NAD+ regeneration system (the combi-CLEAs, and CLEAs mixture) was investigated in 50 mM sodium phosphate buffer, 0.2 M glycerol, 2 mM NAD+ at 30 oC. After 15 min, the biocatalysts were obtained by centrifugation at 7000×g for 3 min, washed two times with 50 mM sodium phosphate buffer (pH 7) and then re-dispersed in the fresh reaction system to determine the enzymatic activity. The residual activity of the immobilized enzymes was calculated by setting the initial activity as 100%.
10
2.6 Cofactor regeneration with combi-CLEAs 2.6.1 Effect of NAD+ concentration on the activity of enzymes The effects of NAD+ concentration on combi-CLEAs, separate CLEAs or free enzymes mixture were investigated in 50 mM, pH 7 phosphate buffer containing 10 μM FAD and 0.2 mM glycerol at ambient temperature, with NAD+ concentration varying from 0.05 to 5 μM. The concentration of DHA in the reaction was quantitated with the diphenylamine method after the reaction mixtures were boiled for 15 min.
2.6.2 Catalytic performance of combi-CLEAs, CLEAs gained and free enzymes mixture Enzymatic synthesis of DHA was performed by adding combi-CLEAs, separate CLEAs, and free enzyme mixtures into 2.5 mL catalytic system containing 50 mM potassium phosphate buffer (pH 7), 0.2 M glycerol, 2 mM NAD+, 10 μM FAD, and 0.75 mg mixed enzymes (combi-CLEAs, separate CLEAs or free enzymes mixture respectively) at ambient temperature. Aliquots were withdrawn after every 3 h, and the produced DHA was quantified by the colorimetric method at 620 nm. Triplicate measurements were made for each sample during the experiment and the corresponding standard errors were calculated.
11
3. Result and discussion 3.1
Optimization
of
the
preparation
of
GDH/Nox
combi-CLEAs Different concentrations of precipitants, including acetone, ethanol, acetonitrile, and ammonium sulfate were tested for precipitation of GDH and Nox. The volume of the precipitating agent was lower than that of the enzyme solution. Thus, to better precipitate the protein molecules, precipitating agents were added dropwise separately to samples of free enzymes. Fig. 1A and 1B indicate that saturated ammonium sulfate allows almost the entire precipitation of GDH (98.75%) and Nox (92.66%) into aggregates and high activity recovery (127.14% for GDH) and 136.84% for Nox) could be obtained. More than 100% activity recovery called “hyper activation” could be obtained when the concentration of saturated ammonium sulfate was higher than 60% because the precipitation conditions may induce the enzyme molecules to adopt a more active conformation [34, 35]. Also, the precipitated proteins have larger surfaces because these protein molecules are fine-grained and have many cavities whereas the coarse-grained proteins in low concentration of saturated ammonium sulfate have smaller surfaces and show lower activity recovery [35]. Different amounts of NADH oxidase and glycerol dehydrogenase were tested to achieve the best catalytic efficiency. Fig. 2A shows that when the GDH/Nox ratio was increased from 2:1 to 5:1, but the activity of both combi-CLEAs and the CLEA mixtures were
12
slightly increased. And the maximal apparent rate of combi-CLEAs was observed when the ratio of GDH and Nox reach to 5:1. The results indicate that the GDH is a rate-limiting enzyme in the NAD+ regeneration reaction because of its relatively low activity and also suggested that NADH oxidase could catalyze the regeneration of NAD+ molecules which are used as the co-substrate of GDH. Generally, the particle size of CLEAs dependent on the aggregation agent type, enzyme/cross-linker ratio, as well as pH conditions. Among these factors, the enzyme/cross-linker ratio plays a significant role in affecting particle size compared with other factors. It is evident that more proteins precipitated at a higher cross-linker concentration solution and formed bigger CLEAs [34]. However, the catalytic efficiency of combi-CLEAs with large particles was lower than that of small particle CLEAs because the latter has a more significant surface area, but the formation of large clusters leads to the unavoidable internal mass transfer limitations [23, 34]. Thus, to obtain the combi-CLEAs with the highest catalytic efficiency, the optimal enzyme/glutaraldehyde ratio was determined. Fig. 2B clearly shows that a higher activity recovery could be obtained by slightly increasing glutaraldehyde concentration up to 5% (mol/mol) glutaraldehyde. However, the recovery of activity decreased obviously when a higher concentration of glutaraldehyde was added as a cross-linking agent because more amounts of glutaraldehyde are able to make more proteins cross-linked and the particle size of CLEAs will increase obviously [34, 35, 36]. This result suggested that the excessive glutaraldehyde may have negative effects 13
on the activity of CLEAs because the inner enzymes of the CLEAs could not form a complex with substrates and cofactor when increasing the CLEAs diameter significantly (over 60 μm) [34, 36]. The cross-linking temperature and time were also optimized to achieve the highest activity recovery. The results showed that the optimal temperature for the preparation of combi-CLEAS is 10
o
C (Fig. 2C). The activity recovery in
combi-CLEAs was decreased significantly to 52% when the cross-linking temperature increased to 50 oC, which might due to the thermal inactivation of enzymes [37]. In another way, the activity recovery of combi-CLEAs increased to achieve the optimal value in 2 h and decreased obviously over this point (Fig. 2D). This might be because glutaraldehyde is a bi-functional reagent which has could react with other functional groups such as active cysteine. Thus, it could get access to the buried motif of the protein and may react with the critical amino acid residues of the enzyme, and thus the active sites could be destroyed because of the excessive cross-linking [24, 38]. The cross-linking process of mixed GDH-CLEAs and Nox-CLEAs (the ratio was 4:1) was also performed with saturated ammonium sulfate and 5% glutaraldehyde at 10 oC for 1 h. The results showed that the obtained apparent rate CLEAs was 580 ± 0.12
U/g.
But
the
maximum
activity
recovery
of
combi-CLEAs
after
co-immobilization at a different time and the temperature was up to 950 ± 0.04 U/g. Thus, it can be observed that the enzyme activity of the combi-CLEAs was 1.6 times that of the CLEAs mixture. Cofactor in situ regeneration using the combi-CLEAs 14
method is expected to have a higher overall enzyme reaction rate compared to the free counterpart [39]. This may be because the adjacent dispersion and proximity of two enzymes might lead to fast uptake of a cofactor for the catalytic reaction by the co-immobilization of the two proteins [40, 41].
3.2 Biochemical characterization of combi-CLEAs 3.2.1 Morphology of combi-CLEAs and CLEAs mixture by SEM The Combi-CLEAs and CLEAs mixture were also freeze-dried for scanning electron microscopy (SEM) observation. Fig. 3A shows that the combi-CLEAs have a less structured form, which is different from the separate CLEAs displaying the typical and spherical aggregate appearance (Fig. 3B) [26]. Although the combi-CLEAs with spherical appearance contain fewer enzyme molecules than the typical “ball” appearance of separate CLEAs, the proximity of each enzyme molecules may increase the cofactor uptake rate in the reaction system and increase the NAD+ regeneration efficiency.
3.2.2 Optimal reaction conditions of GDH/Nox combi-CLEAs The activity of the combi-CLEAs was investigated at different pH values (4-10), and the activity of enzymes was calculated by setting the activity of combi-CLEAs as 100%. Fig. 4A showed that the optimal pH of combi-CLEAs was shifted from 9.0 to a neutral condition compared with free enzyme mixture. At pH 7.0, the activities of
15
combi-CLEAs and CLEAs mixture were 3.6 and 1.7 times higher than that of the free enzyme mixtures. And the combi-CLEAs had 54.3% of relative activity while free enzyme mixtures only kept 42.2% of relative activity at pH 10.0. This might be because that the active glutaraldehyde molecules may react with the surface amino groups of enzymes in combi-CLEAs. Therefore, more negative charges could be transferred by the acidic groups of enzyme surface to the enzyme and change the optimal pH values [26]. Also, the optimal temperature of the free enzyme mixture was 30 oC, but the CLEAs mixture and combi-CLEAs both showed the highest activity at 40 oC, respectively (Fig. 4B). This suggested that the immobilization process increases the optimal temperature by approximately 10 oC. The relative activities of combi-CLEAs and CLEAs mixtures were 1.6 and 1.4 times higher than that of free enzyme mixtures at 40 oC (the activity of combi-CLEAs at 40 oC as 100%.). This enhanced activity might due to that the formed covalent bonds between protein molecules caused by glutaraldehyde might decrease the conformational flexibility of the enzymes and protect them from distortion or damage by heat exchange [42, 43].
3.2.3 Kinetic characterization of GDH/Nox combi-CLEAs The apparent kinetic parameters of constructed combi-CLEAs, separate CLEAs, and free enzymes mixture referring to the overall heterogeneous process were determined. As shown in Fig. 5 and Table 1, the Km values of combi-CLEA (18.45 μM) or CLEAs mixture (15.3 μM) were higher than that of free enzyme mixture (14.7 μM) after immobilization. This seems to result from that these enzymes are gathered in 16
compared to the free enzyme mixtures and cause the mass transport limitations in reactions. Moreover, the super-molecular structure assembled by micro-molecular of glutaraldehyde and the active sites of enzymes got obscured [28, 29, 44]. As shown in table 1, combi-CLEAs had 1.64 times and 1.11 times higher Kcat values than that of CLEAs mixture and free enzyme mixture. The possible explanation was that the adjacent dispersion of two enzymes may lead to the quick consumption of NAD+ [40, 45].
3.2.4 Stability and reusability of GDH/Nox combi-CLEAs The thermal stability of the combi-CLEAs, free enzymes and separate CLEAs were evaluated by incubating them for different time intervals at 55 oC. In Fig. 6A, compared with the half time (t1/2) of the free enzyme (4.5 h), the separate CLEAs and combi-CLEAs showed higher t1/2 values of 6.0 and 6.5 h, respectively. It suggested that separate CLEAs and combi-CLEAs have an enhancement in the stability of enzymes at 55 oC. And the better thermal stability of co-immobilized proteins also demonstrated that co-immobilization of enzymes using the combi-CLEAs method could prevent the enzymes from the inactivation because more energy was required to destroy the active motif than free enzymes. The reusability of CLEAs mixture or combi-CLEAs was explored up to 10 cycles, and the results were shown in Fig. 6B. The catalytic activity of combi-CLEAs decreased to 82% at the fifth cycles and 71% at the eighth cycles, but the corresponding values of separate CLEAs were 74% and 66%, which may result from 17
the deactivation of the enzyme during the catalysis and the mechanical damage or loss of the centrifugation. To further explore the reasons behind the decreased activity, optic microscope was performed to analyze the size of CLEAs particles during the reuse process. Fig. 7A displays that the diameter of combi-CLEA particle before the first reuse cycle was approximately 50 μm and Fig. 7B show that the diameters of CLEA particle decreased slightly after five cycles reuse. However, an obvious decline of the CLEA particle could be observed after ten cycles reuse (30 μm) due to the repeated centrifugation and resuspension applied (Fig. 7C) and its catalytic activity reduced to only 64% of the initial activity simultaneously, which suggested that the reduction of CLEAs size may lead to the decrease of its activity. This is probably because the squeezing of combi-CLEA particles and the reduced CLEAs size elevate mass transfer limitations for substrates and cofactors. The results suggested that the treatment presumably squeezes the combi-CLEA particle and makes it closes together [46-48]. The aforementioned advantages make the combi-CLEAs hold excellent operational stability and reusability.
3.3 Cofactor regeneration with combi-CLEAs The NAD+ regenerative performance of prepared combi-CLEAs was further verified for long-time continuous operation. Fig. 8A shows that the TTN of combi-CLEAs, separate CLEAs and free enzyme mixtures were 2137, 1849 and 1897, respectively (0.05 μM NAD+, 10 μM FAD, 0.2 mM glycerol and 15 min,). These
18
results suggested that co-immobilization has higher utilization efficiency for NAD+. Fig. 8B shows that the DHA production by combi-CLEAs, separate CLEAs, and free enzymes mixture reached to 9.1, 3.6, and 4.7 mM, respectively and also shows that the yield of DHA of combi-CLEAs is higher than that of separate CLEAs and free enzymes in 30 h. This might be due to that the combi-CLEAs method enhanced the approach of the enzyme molecules and may reduce the uptake time of the cofactors (NAD+/NADH) between the two enzymes. Thus far, the enzymatic synthesis of DHA has been reported in several types of research. For example, immobilized GDH (without Nox) produced 3.5 mM DHA with a low conversion of 1.8% [49]. A cofactor regeneration system makes the reaction more economical and feasible. For example, GDH and Nox were immobilized as an NAD+ regeneration system with nanoparticles respectively to produce DHA. After 10 h, The production of DHA reached up to 3.5 mM, and the 3.6 % conversion efficiency with 100 mM glycerol was achieved. Rocha-Martin et al. immobilized GDH and Nox in nanoparticles and gain the production of 3.0 mM DHA with 20 mM glycerol consumption after 8.5 h reaction [50]. In addition, nanoparticle-supported GDH and xylose reductase (XR) also have been prepared for simultaneous bio-convention of DHA and the production and convention rate of the bio-catalysis system reached 8.2 mM [51] (Table 1). In this study, co-immobilization of GDH and Nox by combi-CLEAs method obtained 9.1 mM DHA and 4.6% conversion efficiency after 30 h. Although the production of DHA was limited using glycerol dehydrogenase 19
because of the severe product inhibition which could be reduced by continuous product extraction or removing the specific residues in order to decrease the binding affinity between the product and the enzyme [50, 52]. In this study, the yield of DHA was relatively higher and the DHA was produced by a more commercial and simple process compared to several previous reports, which indicated its potential in the industrial production of DHA.
4 Conclusions In summary, combi-CLEAs consisting of glycerol dehydrogenase and NADH oxidase were firstly prepared to achieve the highly effective NAD+ regeneration. Compared with CLEAs mixture and free enzyme mixture. The higher thermal stability, pH tolerance, reusability, and long-term operational stability of combi-CLEAs suggest its potential in practical application. Besides, the combi-CLEAs has a higher TTN of 2137, indicating that the high-speed regeneration of NAD+ was successfully achieved. By which the conversion of glycerol catalyzed by combi-CLEAs reached 4.6% after 30 mins which were 2.5 times higher than that of free enzyme mixtures. Thus, the prepared combi-CLEAs of glycerol dehydrogenase and NADH oxidase are promising biocatalyst for the development of NAD+ regeneration for the synthesis of DHA.
Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 21376110) 20
Conflict of interest The authors declare no conflicts of interest.
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29
Table 1. Apparent kinetic parameters of combi-CLEAs, separate CLEAs, and free enzymes. Enzymes
Km (μM)
Kcat (S-1)
Kcat / Km (S-1gμM-1)
Free enzymes
14.70
21.34
1.45
Separate CLEAs
15.33
31.65
2.06
Combi-CLEAs
18.45
34.96
1.90
30
Figure legends Fig. 1 Precipitation of GDH and Nox. Effects of precipitants type and concentration on the precipitation rate and activity recovery of GDH (A and B), and Nox (C and D).
31
32
Fig. 2 Effects of (A) GDH/Nox ratio; (B) protein/glutaraldehyde ratio; (C) temperature and (D) immobilization time on the activities of combi-CLEAs (red) and
Relative activity (%)
separate CLEAs (green).
100
A
80 60 40 20 0
2
3
4
5
6
7
8
Relative activity (%)
GDH/Nox ratio (mol/mol) 100
B
80 60 40 20 0
:1 0 :1 3:1 0 :1 8:1 7 :1 .5:1 4 :1 .5:1 5 :1 0 4 2 1 1 4 5
Protein/glutaraldehyde (mol/mol)
33
Relative activity (%)
100
C
80 60 40 20 0 0
5 10 15 20 25 30 35 40 45 50 55
Relative activity (%)
Temperature (oC)
100
D
80 60 40 20 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (h)
34
Fig. 3 SEM images of combi-CLEAs (A) and CLEAs mixture (B).
35
Fig. 4 Characterization of combi-CLEAs, CLEAs mixture and free enzyme mixture. Effects of pH and temperature on activities of combi-CLEAs, separate CLEAs mixture, and free enzyme mixture. (Red: combi-CLEAs; Green: CLEAs mixture; Blue: free enzymes mixture).
Relative activity (%)
100
A
75 50 25 0
4
5
6
7
8
9
10
pH
Relative activity (%)
100
B
75 50 25 0
0 5 10 15 20 25 30 35 40 45 50 55
Temperature (oC)
36
Fig. 5 Effects of glycerol concentration on the activity of combi-CLEAs, CLEAs mixture and free enzyme mixture (Red: combi-CLEAs; Green: CLEAs mixture; Blue: free enzymes mixture).
Activity (U·mg-1)
1.0 0.8 0.6 0.4 0.2 0.0 0
250
500
750
Glycerol (μmol)
37
1000
Fig. 6. Thermal stability (A) of combi-CLEAs, CLEAs mixture and free the mixture at 55 oC (Red: combi-CLEAs, green: separate CLEAs mixture and blue: free enzyme). Recyclability (B) of combi-CLEAs and CLEAs mixture (Green: combi-CLEAs and
Relative activity (%)
red: CLEAs mixture).
100
A
90 80 70 60 50 0 20 40 60 80 100120140160180200
Time (min)
Residual activity (%)
100
B
80 60 40 20 0 0
1
2
3
4
5
6
Cycles
38
7
8
9 10
Fig. 7. Optic microscope images of combi-CLEAs before the first cycle (A), after five cycles of use (B), and after ten cycles of use (C).
39
Fig. 8. Catalytic performance of combi-CLEAs. (A) Total turnover number (TTN) values of combi-CLEAs, CLEAs mixture and free enzymes mixture. (B) Production of DHA catalyzed by combi-CLEAs, CLEAs mixture and free enzyme mixture. (Red: combi-CLEAs, green: separate CLEAs and blue: free enzymes).
2000 A
1000 500 0 0
10
DHA (mM)
TTN
1500
4
2
0
2
4
0
2
4
NAD+ concentration (μM) B
8 6 4 2 0 0
5
10
15
20
Time (h)
40
25
30
Highlights
¾ Combi-CLEAs of glycerol dehydrogenase and NADH oxidase were prepared for in situ NAD+ regeneration. ¾ Adjacent dispersion of two enzymes may lead to the quick uptake of NAD+ and achieve the high-speed NAD+ regeneration. ¾ The conversion of glycerol in combi-CLEAs reaches 4.60 %, which is 2.5 folds of the free enzyme (1.85%).
41
Graphical abstract
42