Process integration of production, purification and immobilization of β

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Cite This: Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Process Integration of Production, Purification, and Immobilization of β‑Glucosidase by Constructing Glu-linker-ELP-GB System Junhui Rong,† Juan Han,‡ Yang Zhou,§ Lei Wang,† Chunmei Li,† and Yun Wang*,† †

School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang, Jiangsu Province 212013, China School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu Province 212013, China § Institute of Life Science, Jiangsu University, Zhenjiang, Jiangsu Province 212013, China Ind. Eng. Chem. Res. Downloaded from pubs.acs.org by UNIV OF LOUISIANA AT LAFAYETTE on 11/09/18. For personal use only.

‡

S Supporting Information *

ABSTRACT: In enzymatic conversion of biomass, how to degrade cellulose into fermentable glucose in an economic, efficient, and clean way has become an important subject. As for the application of cellulase in cellulose degradation, the process optimization in enzyme engineering is urgently desired. The traditional multistep purification processes lead to rising production costs and reduced activity of cellulase; meanwhile, the difficulty in reusability of cellulase has also become a big baffle in the cost-effective application of cellulase in biomass degradation. In this paper, the biocatalyst Glu-linker-ELP-GB (GLEGB) containing binary tags, elastin-like polypeptide (ELP), and graphene-binding (GB), was constructed to simplify the purification and immobilization of βglucosidase (Glu) from Coptotermes formosanus. A high recovery rate (97.2%) and purification fold (18.7) of GLEGB was obtained by only one round of inverse transition cycling (ITC) with 0.5 M (NH4)2SO4 at 25 °C in a short incubating time of 10 min. The purification performance of the one-round ITC method is superior to the commonly used Ni-NTA resin affinity method. Furthermore, the high loading amounts of GLEGB immobilized on GO (698.2 mg g−1) and C3N4 (527.3 mg g−1) were achieved by the synergistic effects of ELP and GB tags. The storage stability and thermal stability of GLEGB was significantly enhanced after immobilization. The recombinant GLEGB immobilized on GO, MGO, graphite, C3N4, C200, and C400 retained 71.4%, 69.5%, 75.1%, 61.2%, 73.5%, and 80.2% of their initial activities respectively after eight cycles. It is worth mentioning that the Km values of GLEGB immobilized on lamellar carbon materials including GO, MGO, and C3N4 are very close to free GLEGB, showing a high affinity of recombinant GLEGB to substrate. To our knowledge, this is the first report on enzyme-linker-ELP-GB system with wide application prospect in the efficient purification and immobilization of enzyme, which can achieve the goal of reducing cost and improving efficiency of biocatalyst in enzymatic conversion of biomass. purification method.6,7 Second, in view of the high cost of cellulase application, the development of enzyme immobilization technique has become the hotspot of enzyme engineering research in order to improve stability and reusability of enzyme.8−10 At present, what is lacking is an integrated method for the production, purification, and catalytic application of cellulase. Elastin-like polypeptides (ELP) are synthetic peptide-based polymer and mainly composed of repeating pentapeptide unit Val-Pro-Gly-Xaa-Gly, in which the guest residue Xaa can be any amino acid, except for Pro.11 ELP are thermally responsive polypeptides that undergo reversible aggregation above a critical temperature, known as the phase transition temperature (Tt). When the temperature is lower than Tt, ELP become soluble again. The recombinant ELP-fusion proteins maintain

1. INTRODUCTION The conversion of biomass lignocellulose into biofuel and high-quality chemicals in an efficient and clean way has become an essential energy development strategy in China. Cellulase catalysis play a vitally important role in converting cellulose into glucose that can be further used in bioethanol production.1,2 Coptotermes formosanus, an important species of wood-eating termites, can efficiently degrade lignocellulosic by using a complex cellulase catalytic system in its digestive system. β-glucosidase (Glu) is an important kind of cellulase in this cellulase catalytic system, and is necessary for the complete degradation of cellulose.3 As biocatalyst, Glu can catalyze cellulose degradation with many outstanding advantages, such as mild reaction condition, less byproduct, simple operation, high substrate specificity, and catalytic efficiency.4,5 As for the application of cellulase in biomass conversion; however, many urgent problems need to be resolved. First, the traditional multistep purification process contributes to the rising production costs and the reducing enzyme activity, generating the need to develop a simple and efficient separation and © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

July 27, 2018 October 22, 2018 October 30, 2018 October 30, 2018 DOI: 10.1021/acs.iecr.8b03492 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

2.2. Construction of Fusion Gene. The gene encoding βglucosidase was retrieved from the National Center for Biotechnology Information (NCBI) database as Accession No. GQ911585. The ELP gene encoding for 50 repeats of the pentapeptide Val-Pro-Gly-Val-Gly was designed and denoted as ELP (VPGVG)50. According to the existing reports, the universal connecting peptide (GGGGS)3 was used as a linker between Glu and ELP using the NheI and SacI site.20−22 The GB peptides (HNWYHWWPH) gene inserted into the 3′ of the ELP gene using the HindIII site. The synthesis of all genes and the construction of plasmids were completed by Synbio Tech (Jiangsu, China), resulting in pET-GLEGB (SI Figure S1a). In addition, expression construct pET-GLE (Glu-linkerELP) and pET-GLEH (Glu-linker-ELP-6His) were also constructed to be used as the control. The expression constructs were transformed into E. coli BL21 (DE3) cells for expression of fusion gene. 2.3. Expression of Fusion Protein. The recombinant plasmid was transformed into E. coli BL21 (DE3) cells and the transformants were selected in the presence of 50 μg mL−1 kanamycin. BL21 cell were transferred into 5 mL liquid Luria− Bertani (LB) supplemented with 50 μg mL−1 kanamycin and grown overnight as a starter culture for the following experiments. To express the GLEGB fusion protein, 200 mL of LB supplemented with 50 μg mL−1 kanamycin was inoculated with a 1:100 dilution of starter culture. The LB culture was grown at 37 °C, 180 rpm until OD600 nm reached 0.4−0.6, and then incubated in ice bath for 20 min. Then, a final concentration of 0.2 mM IPTG was added to induce fusion protein expression at 25 °C with shaking at 200 rpm for an additional 6 h. For the protocol, BL21 cells harboring pETGlu vector without ELP and GB tag and pET-28a (+) vector were used as the control and blank, respectively. After induction, cells were obtained by centrifugation (4500g for 10 min at 4 °C) and washed twice with cold tris-HCl buffer (50 mM, pH 8.0), and then pellets were stored at −80 °C for the next step. 2.4. Purification of Fusion Protein. The frozen cell pellets were thawed and suspended after adding 5 mL solution contain 4.95 mL tris-HCl buffer (50 mM, pH 8.0) and 50 μL PMSF (100 mM). Under the ice bath, the bacteria were lysed by ultrasonic cell disruption (Power 120 W, 30 min, continuous ultrasound time 10 s, ultrasonic interval time 10 s). Cell debris was removed by twice centrifugation (14 000g for 10 min at 4 °C) and the crude extract was kept at −80 °C before use. The precipitation of fusion protein was optimized at different concentrations of ammonium sulfate (0.1, 0.3, 0.5, 0.7, and1.0 M) and incubation time (1, 5, 10, 15, 20, 25, and 30 min). In order to facilitate operation and reduce the loss of enzyme activity, the precipitation operation was conducted at 25 °C, and this step was termed as hot spin. First, a certain amount of ammonium sulfate was added into 500 μL crude extract, and the solution was incubated for a certain period of time and then centrifuged at 14 000g. The cold spin process was then carried out by adding cold tris-HCl buffer (50m M, pH 8.0) to the precipitate. The obtained suspension was incubated at 4 °C and then centrifuged at 14 000g. The supernatant was moved into a new centrifuge tube. The remaining insoluble proteins were resuspended with tris-HCl buffer (50 mM, pH 8.0). The yield and purity of fusion protein were determined by enzyme activity measurement and SDSPAGE analysis, respectively.

the reversible phase transition properties of ELP which provides a novel method known as inverse transition cycling (ITC) for protein purification, separating based on precipitation behavior.12−14 As a new type of nonchromatographic protein purification technology, ITC has the advantages of high recovery rate and simplicity of purification operation for target protein. Several rounds of ITC will always be performed to obtain a desired purity. Immobilization of enzyme onto insoluble support such as various nanoparticles offers an effective solution to improve stability and reusability of enzyme, making an economically feasible process. Among various immobilization strategies, noncovalent adsorption and covalent coupling are most frequently used.15,16 The immobilization of enzyme using a covalent coupling method is superior to a physical adsorption method in binding strength. The covalent coupling method is superior to physical adsorption method in its strong interaction between enzyme and support, nevertheless, a great challenge still remains is the loss of enzyme activity during the immobilization procedure. The immobilization of enzyme by physical adsorption has obvious advantages in simple and mild immobilization procedures.15,17 However, the adsorbed enzyme is easily detached from solid support due to the weak adsorption force. So it is still a great challenge to develop a novel immobilization method which can simultaneously achieve simple immobilization process, high enzyme activity, and cyclic utilization. How to enhance adsorption affinity is a key issue for adsorption method. The graphene-binding peptide (GB), HisAsn-Trp-Tyr-His-Trp-Trp-Pro-His, has been found to have strong affinity toward the hydrophobic reduced graphene oxide (rGO) surfaces due to its rich in hydrophobic aromatic amino acid residues, such as tryptophan (Trp).18,19 There are few reports so far on GB polypeptide, and the immobilization of recombinant GB-fusion enzyme on hydrophobic surfaces via GB-tag has not been reported. In this work, a novel multifunctional binary tag, ELP&GB, was developed for the purification and immobilization of Glu. In order not to affect the activity of the target enzyme, a linker was inserted between Glu and ELP-GB tags, constructing Glulinker-ELP-GB (GLEGB). The rapid separation and purification of the recombinant fusion protein (GLEGB) was achieved by on-step ITC process based on the temperature-responsiveness of ELP-tag, meanwhile, the immobilization of GLEGB on hydrophobic carbon-based materials was enhanced by the synergistic effect of GB and ELP tags. Our strategy significantly simplifies the purification and immobilization operation by expression of Glu with binary GB&ELP tags, which is crucial to large-scale application of biocatalyst in biomass conversion.

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. Escherichia coli BL21 (DE3) and pET-28a expression vector were used to produce the fusion protein. The β-glucosidase gene used for cloning was derived from the cDNA of Coptotermes formosanus. All restriction endonucleases, broad molecular maker were purchased from TaKaRa (Dalian, China). High Affinity NiNTA Resin was bought from Genscript (Nanjing, China). Activated charcoal (200, 400 mesh) was purchased from Shanghai Macklin Biochemical Co., Ltd. Graphite were bought from Aladdin Industrial Corporation. All other reagents were of analytical grade and used without any further treatment. B

DOI: 10.1021/acs.iecr.8b03492 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Scheme 1. Schematic Representations of the Purification of ELP&GB-Tags Fusion Protein by Inverse Transition Cycling Method and Enzyme Immobilization on Carbon Materials

recovery rate(%) =

purified enzyme activity × 100 crude enzyme activity

purification fold =

specific activity of purified enzyme specific activity of crude enzyme

(pH 8.0) to wash and remove the unbound and not firmly adsorbed enzyme. Considering the efficiency of industrial production, the optimal immobilization time was studied. A series of equal amounts enzymes and activated charcoal were mixed and incubated for different times under the same conditions. The protein concentration in the supernatant was measured before and after immobilization. The concentration of residual enzyme in the adsorption solution was determined with a BCA Protein Assay Kit (Thermo Scientific (China) Co., Ltd.) using bovine serum albumin (BSA) to generate the calibration curve protein concentration vs absorbance at 560 nm. The loading amount of enzyme can be calculated by the following equation:

2.5. Immobilization of Enzyme. We used inexpensive, readily available carbon-based materials (graphene oxide (GO), Magnetic graphene oxide (MGO), C3N4, 200 mesh activated carbon (C200), 400 mesh activated carbon (C400)) as immobilized material. The immobilized enzyme was prepared by adding 50 μL support material (1 mg mL−1) to a series of purified GLEGB solutions at different concentrations, followed by incubation (25 °C, 200 rpm) in an incubator shaker for a certain period of time. After incubation, the obtained immobilized enzyme was separated by centrifugation. Then, the supernatant was carefully decanted without loss of any conjugate. Next, the immobilized enzyme was resuspended with fresh 50 mM tris-HCl buffer solution

Q=

C0V − C tV (mg g −1) m

In this equation, C0 expresses the initial concentration of protein solution; Ct expresses the equilibrium and washed C

DOI: 10.1021/acs.iecr.8b03492 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

formosanus contains 488 amino acids. The gene encoding the protein has 1488 bp. The length of genes encoding linker, ELP and GB were 45, 750, and 27 bp, respectively. For validating the recombinant plasmid, the corresponding DNA digestion tests were performed. The restriction endonucleases for DNA digestion and corresponding restriction fragments were shown in Supporting Information (SI) Figure S1c. As shown in SI Figure S1b, the result of enzyme digestion was the same as that of the theory. Furthermore, the results of DNA sequencing coincide completely with that of the theoretical sequence, and no mutation sites were found. 3.2. Purification and Immobilization of Fusion Protein. The main process of fusion protein purification by ITC and enzyme immobilization were shown in Scheme 1. The separation and purification of fusion protein by ITC was mainly divided into two steps: (1) “hot spin”, in which the fusion protein was separated by adding an appropriate amount of salt to the crude extract solution and incubating for a few minutes at moderate temperature; (2) “cold spin”, in which the fusion protein was redissolved in precooling tris-HCl buffer at low temperature. The purity of the purified fusion protein was determined by SDS-PAGE electrophoresis, and the number of ITC round was determined according to the purity of the protein. The purified enzyme solution and the carbon materials were mixed and incubated for a short time, then the quick and easy immobilization of enzyme was achieved. 3.2.1. Purification of Fusion Protein. Although thermally triggered phase-separation behavior of ELP can enable the simple separation and purification of their fusion proteins, their phase transition temperature is always so high that a series of salts are needed to induce aggregation and precipitation at a relative low temperature without inactivation and denaturation of fusion proteins. The influence of different ions on Tt of ELP abides by Hofmeister series, for anion: SO42− > HPO42− > CH3COO − > Cl − > Br − > NO3− > ClO4 − > SCN −, for cation: NH4 + > K + > Na+ > Mg2+ > Ca2+ > Mn2+ > Cu2+.23 Taking both the above series and the solution pH values into account, (NH4)2SO4 was selected as precipitant and the effect of different concentration of (NH4)2SO4 (0.1, 0.3, 0.5, 0.7, 1.0 M) on purification was investigated. The purity of GLEGB obtained by ITC purification process with different (NH4)2SO4 concentrations was analyzed and the results were shown Figure 1. The molecular weights of native Glu and GLEGB are about 53 and 78 kDa respectively, which is consistent with the results of Lane 2 and 3 in Figure 1. The efficient phase-separation of GLEGB was not achieved with the concentration of (NH4)2SO4 below 0.1 M, so there was no any stripe in Lane 0.1 M. Simultaneously, we can see that there are clear fusion protein stripes from Lane 0.3 to 1.0 M and an obvious increase in impurity protein stripes can be observed with the concentration of (NH4)2SO4 above 0.7 M after first round of ITC. So it can be seen from Figure 2a that the purification fold of fusion protein GLEGB reach the maximum value of 18.7 at a moderate salt concentration of 0.5 M. With the further increase of (NH4)2SO4 concentration from 0.7 to 1.0 M, the purification fold decreases gradually. This is attributed to the strong salting-out effect induced by the high concentration of (NH4 )2 SO 4, which would cause the coprecipitation of impurity protein in the process of hot spin. The recovery rate of fusion protein was basically unchanged and also reached the maximum value of 97.2% at the (NH4)2SO4 concentration of 0.5 M. Thus, 0.5 M

concentration of protein solution; V denotes the volume of protein solution; m is the quality of the support material. 2.6. Stability of Fusion Protein. The stability of free and immobilized enzymes was investigated, which included reusability, thermal and storage stability. The thermal stability of immobilized and free enzymes was studied by testing their enzyme activity after keeping at 25−70 °C for 30 min. The storage stability of immobilized and free enzymes was studied by detecting their enzyme activity every 3 days for 30 days keeping at 4 °C. The reusability of the immobilized enzyme (Glu, GLE, and GLEGB) in the hydrolysis of p-NPG test was assessed under optimized reaction condition. After each cycle, the immobilized enzyme was collected and washed with trisHCl buffer solution (50 mM, pH 8.0). The activity of the immobilized enzyme after first cycle was defined as the control and recognized as a relative activity of 100%. The reusability assay was repeated for eight cycles. 2.7. Enzyme Activity Assays. The activity of Glu was determined the amount of catalytic product p-nitrophenol (pNP) decomposed from substrate p-nitrophenyl-β-D-glucopyranoside (p-NPG). The mixture of 40 μL p-NPG (50 mM) and 450 μL acetic acid-sodium acetate (NaAc-HAc) buffer (50 mM, pH 5.5) was preheated in a water bath at 45 °C for 10 min. Ten μL enzyme solution was added into the preheated mixture, and the reaction was terminated after 10 min by adding 500 μL Na2CO3 (1 M). The absorbance of reaction solution at 410 nm was measured by spectrophotometer and the enzyme activity was calculated. The specific activity (U) of enzyme is defined as the amount of products produced by the catalytic reaction during 1 min. The formula for calculation is as follows: U=

Cp ‐ NP × Vreaction treaction

In this equation, Cp‑NP expresses the concentration of catalytic product p-NP; Vreaction expresses the volume of enzyme solution; treaction denotes the reaction time of substrate and enzyme. 2.8. Measurement of Kinetic Parameters. Kinetic parameters (Km and Vmax) of free and immobilized enzymes were determined by measuring the enzyme activity with pNPG as substrate under various concentrations ranging from 1 to 10 mM. Km and Vmax were calculated by using the classical Lineweaver−Burk plot equation: K 1 1 1 = m × + V Vmax [S ] Vmax

In the equation, V is the initial reactive rate (mM min−1), Vmax is the maximal reaction rate (mM min−1), [S] is the initial p-NPG concentration, and Km is the Michaelis-Menten constant (mM). 2.9. Circular Dichroism (CD) Analysis of Enzymes. CD spectroscopy analysis was performed at 25 °C using an instrument J-815 (JASCO) and a quartz cell with 0.1 mm optical path length. Glu and purified GLEGB were diluted to the same concentration of 0.1 mg mL−1 with tris-HCl buffer solution (50 mM, pH 8.0), and the buffer solution was served as blank control.

3. RESULTS AND DISCUSSION 3.1. Construction and Expression of Fusion Protein GLEGB. Glu, CfGlu1B, along to GH1 family from Coptotermes D

DOI: 10.1021/acs.iecr.8b03492 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

of ITC, the recovery rate of enzyme activity reached 97.2%, and the purification fold was greatly improved to 18.7 as listed in Table 1. It was calculated from Figure 1 (Lane 0.5 M) that the purity of GLEGB reached more than 90%, which also proved the excellent purification performance of one-round ITC method. Compared with a single ITC operation, the two round of ITC did not significantly increase the purity of GLEGB, but led to an obvious decrease in the recovery rate of GLEGB. The high purity and recovery rate of GLEGB could be obtained by only one round of ITC with 0.5 M (NH4)2SO4 at 25 °C in a short incubating time of 10 min (Figure 2b). Moreover, in order to compare the purification performance of the ITC method based on ELP-tag with the commonly used Ni-NTA resin affinity purification method, the analysis of the purification fold of GLEGB, Glu-linker-ELP-6His (GLEH), Glu-linker-ELP (GLE), and Glu-linker-6His (GLH) was performed. By comparing the purification fold of GLEGB (18.7) and GLE (17.9), it can be seen that the GB-tag has no significant effect on the purification performance of ITC method. Meanwhile, the purification fold of GLEGB and GLE is much higher than that of GLEH (7.7) and GLH (8.4) respectively, so it can be concluded that the purification performance of the one-round ITC method is superior to the commonly used Ni-NTA resin affinity method and many published literatures.24−26 The results show the prospective of applying the thermosensitive polypeptides-based ITC method for the separation and purification of ELP-tag containing recombinant proteins. To explore the secondary structural changes of Glu in the presence of ELP and GB tags, the CD spectra of Glu and

Figure 1. SDS-PAGE gel of fusion proteins purified by ITC method Lanes from left to right: Lane M, Protein molecular weight marker; Lane 1, cleared lysates containing empty pET-28a vector; Lane 2, cleared lysates containing Glu; Lane 3, cleared lysates containing GLEGB; Lane 4, products of cleared lysates of Glu after one round of ITC operation using 0.5 M of (NH4)2SO4; Lane 0.1−1.0 M, products of cleared lysates of GLEGB after one round of ITC operation using different concentrations of (NH4)2SO4; Lane R2, the purified product after two round of ITC (0.5 M (NH4)2SO4, 25 °C); Lane Ni, the obtained GLEH by Ni-NTA resin affinity purification method.

ammonium sulfate was selected as the optimum precipitant for the following purification of fusion protein. The effect of the number of ITC operation on the purification of GLEGB was also studied. After the first round

Figure 2. Effect of different (NH4)2SO4 concentration (a) and incubating time (b) on purification. (c) The circular dichroism spectrum of Glu and GLEGB. E

DOI: 10.1021/acs.iecr.8b03492 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research Table 1. Purification of Glucosidase Purified by Different Methods target protein GLEGB crude extract GLEGB GLEGB GLEH GLE GLH β-glucosidase β-glucosidase β-galactosidase-ELP

purification method

specific activity (U mg−1)

recovery rate (100%)

purification fold

3.3 61.7 63.0 45.4 60.5 47.3 25.5

100 97.2 78.3 80.6 96.1 82.3 7.9 21.0 92.4

1.0 18.7 19.1 7.7 17.9 8.4 5.9 9.0 1.9

1 × ITC 2 × ITC high affinity Ni-NTA resin 1 × ITC high affinity Ni-NTA Resin ammonium sulfate precipitation sepharose NTA-Ni2+ ITC

236.8

references this this this this this this 24 25 26

work work work work work work

Figure 3. Effects of initial enzyme concentration on loading amount of enzyme. GO (a), MGO (b), graphite (c), C3N4 (d), C200 (e), C400 (f). All experiments were performed at 25 °C with shaking at 200 rpm using same amount of support materials at optimum pH 5.5 in acetate buffer (50 mM).

and GB tags have no drastic effect on the secondary structure of Glu, which may be attributed to the linker peptide. 3.2.2. Immobilization of Fusion Protein on Carbon Materials. Different carbon materials (GO, MGO, graphite, C3N4, C200, C400) were used for the immobilization of enzymes at their different initial concentrations. GO, MGO, and C3N4 were synthesized according to the reported method

GLEGB was recorded from 190 to 250 nm and displayed in Figure 2c. As a common characteristic of all β-glucosidases (≥40% α-helices), the typical α-helical structure of the enzyme, two negative peaks at 208 and 222 nm were observed, while a positive peak at around 196 nm was assigned to β-sheet structure.27,28 Compared with Glu, the secondary structure of GLEGB has no significant change. This shows that the ELP F

DOI: 10.1021/acs.iecr.8b03492 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research Table 2. Equilibrium Time of Enzyme Immobilization by Different Methods target enzymes

supported materials

methods

immobilization temperature (°C)

immobilization time (min)

references

GLEGB β-glucosidase β-glucosidase lipase

carbon materials multiwalled nanotubes smectite clays Fe3O4@MOF

physical adsorption physical adsorption covalent binding covalent binding

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