Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate

Journal Pre-proof Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate

Xingyu Lu, Qian Zhong, Jian Liu, Fulin Yang, Chenghui Lu, Huan Xiong, Sha Li, Yibo Zhu, Lingtian Wu PII:

S0141-8130(19)38064-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.215

Reference:

BIOMAC 13996

To appear in:

International Journal of Biological Macromolecules

Received date:

6 October 2019

Revised date:

25 November 2019

Accepted date:

26 November 2019

Please cite this article as: X. Lu, Q. Zhong, J. Liu, et al., Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate, International Journal of Biological Macromolecules(2019), https://doi.org/10.1016/ j.ijbiomac.2019.11.215

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© 2019 Published by Elsevier.

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Efficient

expression

of

chondroitinase

ABC

I

for

specific

disaccharides detection of chondroitin sulfate Xingyu Lua,1, Qian Zhonga,1, Jian Liua, Fulin Yanga, Chenghui Lua, Huan Xionga, Sha Lic, Yibo Zhua, Lingtian Wua,b,* a

College of Biological and Food Engineering, Changshu Institute of Technology, 99

b

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South Third Ring Road, Changshu 215500, China. College of Food Science and Light Industry, Nanjing Tech University, 30 Puzhu

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South Road, Nanjing 211816, China.

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* Corresponding Author. Tel/Fax: +86-512-52251562;

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E-mail address: [email protected] (Lingtian Wu)

1

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Abstract Chondroitinase ABC I (ChSase ABC I) is a key enzyme of chondroitin sulfate (CS) degradation and widely used for CS detection in the medicine filed. However, the recombinant ChSase ABC I was weakly expressed in Escherichia coli because the forms of it was mostly inclusion bodies. In this study, a signal peptide (pelB) was

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used for the soluble form expression of ChSase ABC I in E. coli. Then the culture condition for ChSase ABC I expression was optimized through response surface

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methodology. Results revealed that the expression level of ChSase ABC I in a 7.5 L

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fermentor (29.03·mL−1) was approximately 1.65-fold higher than that of the shake

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flask level (17.55·mL−1). The enzymatic properties and kinetic constants of

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recombinant ChSase ABC I were also studied. Recombinant ChSase ABC I was also

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used to detect the specific disaccharides content of CS from different sources. This study not only eliminates the problem of the enzyme expressed as an inclusion body,

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but also solves the current problem of expensive ChSase ABC. In a word, it would be an ideal strategy for ChSase ABC high-efficiency expression and a great method to detect specific disaccharides of CS in biomedical field.

Keywords: Souble expression; Chondroitinase ABC I; Signal peptide; Chondroitin sulfate.

2

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1 Introduction Glycosaminoglycans (GAGs) are special group of carbohydrates, mainly distributed in extracellular matrices and cell surfaces and implicated in certain biological processes, such as cell proliferation, signal transmission, and inflammation mediation [1-3]. Chondroitin sulfate (CS, a glycosaminoglycan) extracted from animal cartilages plays

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an important role in the complement system. It consists of repeated disaccharide units (D-glucuronic acid and D-N-acetylgalactosamine), linking together through alternating

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β-1,3 and β-1,4-glycosidic bonds in the CS linear polysaccharide structure [4-7]. This

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unique carbohydrate has several pharmacological applications, such as rheumatism and

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arthritis treatment, and atherosclerosis prevention. CS can also be used as raw materials

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of cosmetics, food, and health products [8-12].

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Chondroitinase ABC I (ChSase ABC I, EC 4.2.2.4) is one of the tool enzymes to study glycosaminoglycan [4, 13, 14]. It can be used to enhance the adhesion strength

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between chondrocytes and cartilages and to dissolve the nucleus pulposus or ruptured fiber rings in an intervertebral disk [15, 16]. For example, ChSase ABC I is applied to treat the injury of fibrocartilage, showing the fibrocartilage tensile stiffness increases by 7.3 times compared to that without enzyme treatments [15]. The effects of ChSase ABC I on spinal cord injuries have confirmed that it can promote the regeneration of neuron axon [17]. Moreover, ChSase ABC I could decompose natural CS into low molecular weight CS, which can promote cartilage regeneration and signal transmission [4]. Several strains, such as Proteus vulgaris and Flavobacterium heparinum, can produce ChSase ABC I [18-20]. The ChSase ABC I from different strains has been expressed in Escherichia coli and was widely applied in medical research [21]. However, most of the 3

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recombinant ChSase ABC I in E. coli are weakly expressed, or they appear as inclusion bodies [22]. The introduction of fusion tags, such as glutathione transferase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and maltose-binging protein (MBP), can improve the ChSase ABC I expression level [22, 23]. However, the fusion tags also have some negative effects on the characteristics of ChSase ABC I. Hence, the

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advantages of these approaches should be explored to mediate the production and characteristics of ChSase ABC I.

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Using signal peptides to guide heterologous proteins transport across the inner

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membrane to the periplasm with the Sec translocase apparatus can improve the

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expression of soluble enzymes and avoid difficulties in the refolding of inclusion bodies

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[24, 25]. In this work, the ChSase ABC I gene was ligated to the plasmid pET-22b(+)

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(digested with NcoI and XhoI) and pET-22b(+)-ΔpelB, respectively, in order to obtain a strain that can highly express soluble ChSase ABC I. To obtain the over-production of

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ChSase ABC I, the medium was optimized in Erlenmeyer flasks using statistical techniques. Moreover, the biochemical properties and kinetic constants of ChSase ABC I were researched. Finally, the recombinant ChSase ABC I was used to detect the specific disaccharides rate of CS from different sources. 2 Materials and methods 2.1 Materials. DNA standard marker, Phanta® Super-Fidelity DNA polymerase, restriction enzymes (NcoI and XhoI), ClonExpress II One Step Cloning Kit, and premixed protein marker were

purchased

from

Vazyme

Biotech

Co.,

Ltd.

(Nanjing,

China).

KOD-Plus-Mutagenesis Kit was purchased from TOYOBO CO., LTD. (Shanghai, 4

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China). The pET-22b(+) vector was preserved in our laboratory. CS from different sources and CS A were purchased from Yantai Dongcheng Pharmaceutical Group Co., Ltd. United States Pharmacopoeia CS and other reagents were obtained from Aladdin Chemical Co., Ltd. Proteus vulgaris ATCC33420, a ChSase ABC I producing strain, was purchased from BeNa Culture Collection.

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2.2 Construction of the recombinant strains. The ChSase ABC I gene (KR708541.1) was amplified from the genomic DNA of P.

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vulgaris ATCC33420 using P1 and P2 (Table S1). The pET-22b(+) vector was digested

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with NcoI and XhoI, then the linearized vector and the PCR fragments were ligated by

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ClonExpress II One Step Cloning Kit to generate the pET-22b(+)-ChSase ABC I, which

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was transformed into E. coli DH5α. A positive colony carrying pET-22b(+)-ChSase

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ABC I plasmid was cultured in 5 mL of Luria-Bertani (LB) medium containing 100 µg/mL ampicillin and incubated at 37°C for 12 h. The pET-22b(+)-ChSase ABC I was

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identified by enzyme digestion (NcoI and XhoI). A signal peptide (periplasmic leader, pelB) was in the N-terminal of ChSase ABC I gene and a His6 tag was in the C-terminal. To research the effects of signal peptide on the expression level of ChSase ABC I, the linearized pET-22b(+)-ΔpelB-ChSase ABC I without pelB was amplified from the pET-22b(+)-ChSase ABC I by using P3 and P4 (Table S1). Then the circular pET-22b(+)-ΔpelB-ChSase ABC I was constructed using KOD-Plus-Mutagenesis Kit. Furthermore, two plasmids were sent to Synbio Biotech Co. Ltd. (Suzhou, China) for DNA sequencing. The resulting two plasmids were transformed into competent E. coli BL21 (DE3), respectively. SDS−PAGE and enzyme assay were used to illuminate the effect of the pelB for enzyme expression. The rapid plate method was also used to 5

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determine whether the enzyme was soluble production [26]. The recombinant ChSase ABC I activity was tested with the method previously reported using CS A as its substrate [27]. One unit (U) of ChSase ABC I was defined as the amount of enzyme required to form 1 μmol 4,5-unsaturated uronic acid per minute. 2.3 Culture optimization

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The culture medium for recombinant E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I was optimized to improve the ChSase ABC I expression level. Factors including CS,

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peptone, yeast extract power (YEP), K2HPO4·3H2O, MgSO4·7H2O, CaCl2 and cane

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molasses (CM) in the fermentation of recombinant E. coli were examined. The level of

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each factor was firstly estimated by the single factor experiment. The Plackett–Burman

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(PB) design is one of the most efficient methods for the medium component

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optimization, which is usually used to estimate the effect of variables assuming whether there are interactions among the constituents [28]. Table 1 shows the selected levels of

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each factor used in our experimental design. According to the results of the PB design, the optimum levels of these factors were examined by the well-known steepest ascent method. After the steepest ascent experiments, CS, YEP, and MgSO4·7H2O were selected as the independent variables and further optimized through response surface methodology (RSM) for ChSase ABC I production. Box-Behnken Design (BBD) was used in this study and a set of total 17 experiments were listed in the Table 2. All batch fermentations were performed in 500 mL Erlenmeyer flasks with 80 mL medium and repeated three times. 2.4 Protein overproduction and purification 2.4.1 Protein overproduction in shake flask level 6

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LB medium containing 100 µg/mL ampicillin was used for seed culture of recombinant strains and ChSase ABC I production. Recombinant strains were cultured at 37°C, 180 rpm for 12 h. Then the seed culture (3%, v/v) was transferred to the 500 mL flask containing 80 mL of fresh LB medium at 37°C. Subsequently, a final concentration of 0.6 mM IPTG was added when the OD660 reached 0.8. The culture was further grown at

2.4.2 Protein overproduction in a 7.5 L fermentor

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20°C for additional 10 h to overproduce the ChSase ABC I.

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The fermentation of recombinant E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I was

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investigated in a 7.5 L fermentor. The seed culture (3%, v/v) was transferred to the 7.5

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L fermentor containing 4.5 L fermentation medium and then cultured at 37°C. The

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stirring speed was 400 rpm and the volume of ventilation was 1.1 vvm. Thus, ammonia

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solution was used to control pH at 7.0 ± 0.1. Subsequently, a final concentration of 0.6 mM IPTG was added into the fermentor when the OD660 reached 0.8. The culture was

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further grown at 20°C for additional 24 h. Samples were taken every 2 hours to determine the ChSase ABC I activity, biomass, and residual sugar. 2.4.3 ChSase ABC I purification To purify ChSase ABC I, the recombinant cells were washed with buffer A [10% (v/v) glycerol, 50 mM Tris-HCl pH 8.0] three times. In order to obtain crude enzyme liquid, cell disruption was carried out by high pressure homogeniser at 4°C, and the cell lysate was centrifuged at 12,000 × g for 0.5 h (4°C) to remove cell debris. The crude enzyme liquid was applied onto a column loaded with Ni-NTA superflow resin (GE Healthcare Corp.). Buffer B (buffer A with 500 mM NaCl) was used to equilibrate the column. The unbound proteins were washed out from the column by buffer B with 50 mM imidazole. 7

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Then, recombinant ChSase ABC I was eluted from the column by buffer B with 300 mM imidazole. 2.5 Biochemical properties of ChSase ABC I To determine the optimum pH of ChSase ABC I, the enzyme assays were incubated at 37°C from pH 5.5 to 9.5 in citric acid-phosphate buffer. The pH stability of the enzymes

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was evaluated at different pH values (5.5 to 9.5) at 4°C for 6 h. Optimum temperature was studied at 15°C to 55°C. Thermal stability of ChSase ABC I was determined from

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15°C to 55°C for 6 h. Kinetic parameters of ChSase ABC I were obtained by calculating

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the rate of the catalytic reaction with an increasing concentration (10–100 μM) of CS A.

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using Lineweaver–Burk equation.

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The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated

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2.6 Detection of the specific disaccharides of CS The reaction mixture was composed of 100 μL CS sample solution (2.5 mg/mL, w/v)

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[or USP CS standard solution (2.4 mg/mL, w/v)] and 400 μL of ChSase ABC I solution (0.2 U/mL). The CS sample was dissolved with water, and the ChSase ABC I was diluted with buffer C [2.5 mM trisaminomethane and 3 mM sodium acetate (1:1), adjusted with 1 M HCl to pH of 8.0]. The reaction was cultured at 35°C for 3 h, which was ended by boiling for 10 min, and then centrifuged at 12,000 × g for 10 min to remove denatured enzymes. All the samples were filtered with 0.22 μm membrane filters for High Performance Liquid Chromatography (HPLC) analysis. The HPLC system was equipped with a Hypersil-SAX column (10 μm, 4.6 mm × 250 mm, Thermo, USA) to confirm the disaccharides components. The column was gradient eluted at 35°C with the mobile phase at a flow rate of 1.0 mL/min. The mobile phase A is the 8

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double distilled water, adjusted to pH 3.5 with HCl; the mobile phase B is 1 M NaCl, adjusted to pH 3.5 with HCl. ∑ 𝑟𝑣

Specific disaccharides content (%) =

×

∑ 𝑟𝑠

𝐶𝑠 𝐶𝑣

× 100%

(1)

Σrv, sum of the peak areas of β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine (ΔDi-0S), β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-4-sulfated (ΔDi-4S); β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-6-sulfated (ΔDi-6S) from the

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sample solution; Σrs, sum of the peak areas of ΔDi-6S, ΔDi-4S, and ΔDi-0S from the

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concentration in the sample solution (mg/mL).

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standard sample; Cs, CS concentration in the standard solution (mg/mL); Cv, CS

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3 Results and Discussion

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3.1 Recombinant strains construction

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In previous studies, ChSase ABC I had been produced in E. coli strain. However, the low ChSase ABC I expression level restricted its application in industry, and the

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time-consuming process of inclusion body renaturation would no doubt increase the production costs. Therefore, it was very significant to explore the ways to improve the ChSase ABC I expression level. It was reported that signal peptides could guide heterologous proteins transport across the inner membrane to the periplasm with the Sec translocase apparatus can improve the expression of soluble enzymes and avoid difficulties in the refolding of inclusion bodies [24, 25]. Hence, we introduced a signal peptide (pelB) for the ChSase ABC I expression and folding in this work. The ChSase ABC I gene (3033 bp, containing the homologous arms) was successfully cloned from the genomic DNA of P. vulgaris ATCC33420. Fig. 1A showed that the purified ChSase ABC I gene was ligated into the linearized vector pET-22b(+) (5431 bp, digestion with 9

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NcoI and XhoI) to construct pET-22b(+)-ChSase ABC I. The correct plasmid pET-22b(+)-ChSase ABC I was confirmed by restriction enzyme digestion and DNA sequencing. Nucleic acid electrophoresis gel (Fig. 1B) showed the successful ligation of ChSase ABC I gene to the corresponding vectors. The pET-22b (+)-ΔpelB-ChSase ABC I, constructed using KOD-Plus-Mutagenesis Kit, was confirmed by DNA sequencing.

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The pET-22b(+)-ΔpelB-ChSase ABC I and pET-22b(+)-ChSase ABC I were transformed into the competent E. coli BL21 (DE3) cells, respectively.

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It was reported that nondegraded CS conjugates with bovine albumin fraction V, as

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confirmed by milky white precipitates [26]. Obviously, three clear zones in the plate

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(Fig. 2A), suggesting that the target protein realized soluble expression and possessed

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activity. The estimated recombinant ChSase ABC I molecular mass was about 100 kDa

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(Fig. 2B), which was consistent with previous findings [21-23]. The whole cell bands show that two recombinant strains have the similar expression level of ChSase ABC I.

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However, the disruption supernatant bands exhibited the expression level of E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I was greater than that of E. coli BL21 (DE3)-pET-22b(+)-ΔpelB-ChSase ABC I. This phenomenon suggesting that the presence of signal peptides could facilitate ChSase ABC I folding and eliminate ChSase ABC I in the form of inclusion bodies. 3.3 Culture optimization. In order to further increase the ChSase ABC I expression level, the medium components of recombinant E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I were optimized. The single-factor experiments (data not shown) suggested that the estimated preliminary concentrations of CS, K2HPO4·3H2O, cane molasses (CM), peptone, YEP, 10

Journal Pre-proof MgSO4·7H2O, and CaCl2 were 5.5, 3.0, 8.0, 5.5, 6.5, 1.0, and 1.8 g·L−1, respectively. A PB experimental design was used to evaluate the factors that played important roles in enzyme expression. On the basis of these results, we selected high and low values of each variable as depicted in Table 1. The 12 experiments with two levels for each factor corresponding to enzyme production are illustrated in Table S2. To obtain the optimum

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response, we derived a fitted first-order model for the ChSase ABC I expression from the PB experiments as follows:

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× X5) – (0.12 × X6) + (0.49 × X7).

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Enzyme activity = 11.13 – (0.67 × X1) + (0.36 × X2) – (0.11 × X3) + (0.35 × X4) + (1.03 (2)

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R2 of the linear regression model was 0.9666, and R2 (Adj) was 0.9081, indicating that

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the model was reasonable for the PB design. The effects of the seven factors and the

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statistical analysis of PB experiment results are shown in Table 1. P-values of CS (X1), YEP (X5), and MgSO4·7H2O (X7) are far less than 0.05, suggesting that CS, YEP, and

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MgSO4·7H2O are the most important among the examined variables. The coefficients of X5 and X7 are positive (+1.28 and +0.61, respectively), whereas the coefficient of X1 is negative (−0.83), implying that increasing the concentrations of YEP and MgSO4·7H2O while decreasing the concentration of CS can enhance the ChSase ABC I expression level. The path of the steepest ascent is an effective experiment to move gradually along the path of the steepest increase to the optimum range. In other words, the direction of the maximum increase is the farthest in the response [29]. The design of the steepest ascent experiments and the corresponding results are displayed in Table S3. The enzyme expression level was the highest when the selected concentrations of CS, YEP, and 11

Journal Pre-proof MgSO4·7H2O were 5.00, 7.00, and 1.10 g·L−1, respectively. The levels of the three factors were near the range of the maximum ChSase ABC I expression response. To further examine the optimal concentrations of CS, YEP, and MgSO4·7H2O, we optimized the three critical factors by conducting the BBD experiment (Table 2). ANOVA of BBD experiments was performed to examine three factors effects on

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ChSase ABC I expression (Table 3). The second-order polynomial equation was derived from the multiple regression analysis of the experimental data as follows:

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Enzyme activity = 17.49 + (0.82 × X1) – (0.20 × X5) – (0.68 × X7) – (0.14 × X1 × X5) –

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(0.18 × X1 × X7) + (0.43 × X5 × X7) – (1.13 × X12) – (1.58 × X52) – (1.37 × X72).

(3)

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In general, a regression model with R2 above 0.90 is considered to have a high

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correlation. Here, R2 was 0.9860, indicating that this model could explain 98.60% of the

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substrate variation in response to ChSase ABC I production. Adj R2 and F-value were 0.9681 and 54.96, respectively, and these values revealed the significance of the model.

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Consequently, the regression model could be reasonably utilized to predict the ChSase ABC I expression within the range of the studied variables. “Probe > F” was used to confirm the significance of factors, indicating the strength of the interaction between independent factors. ANOVA suggested that the model terms of X1, X7, X12, X52, and X72 were significant because each “Probe > F” was less than 0.05. The 3D response surface curves visualized the relationship between the expression ChSase ABC I level and the corresponding variables (Fig. 3). Significant interactions were observed between CS and YEP and between CS and MgSO4·7H2O because the contour plots were elliptical. By contrast, the interaction between MgSO4·7H2O and YEP was negligible when the contour plot of the response surface was circular. The predicted 12

Journal Pre-proof maximum ChSase ABC I activity was 17.49 U·mL−1 when the optimal values of the variables were 5.39 g·L−1 CS, 6.88 g·L−1 YEP, and 1.04 g·L−1 MgSO4·7H2O, respectively. The experimental value was 17.55 ± 0.68 U·mL−1, which was in agreement with the model prediction showing the accuracy of the experiments. On the basis of the ANOVA test results, we could conclude that RSM with an appropriate

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design provided important information about the main culture composition combinations that could be applied to improve ChSase ABC I production.

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3.4 ChSase ABC I expression in a 7.5 L fermentor

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Fermentation was carried out in a 7.5 L fermentor with the optimum culture medium

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components, and the initial total sugar concentration of the medium was approximately

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10.25 ± 0.24 g·L−1. The OD660 and ChSase ABC I activity were explored during

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fermentation (Fig. 4). 17.8 of the OD660 and 2.4 ± 0.07 U·mL−1 ChSase ABC I activity were obtained when the sugar was almost consumed at 16 h. To reach a high enzyme

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expression level, we added extra CM to the medium for continuous bacterial growth and enzyme expression. Subsequently, as the biomass (OD660) increased, the enzyme activity exponentially increased and peaked at 29.03 ± 0.46 U·mL−1 at 38 h. This value was 1.65-fold higher than that of the shake flask level (17.55 U·mL−1). Specifically, the activity of ChSase ABC I-His (17.6 U·mL−1 fermentation liquor) was 5.5 times as much as the activity of MBP-ChSase ABC I (3.18 U·mL−1 fermentation liquor), which was also attributed to the optimization of the culture medium. The recombinant strain grew well with CM as a major carbon source for the enzyme expression, suggesting that the vitamins and amino acids found in CM positively affected E. coli physiology. In addition, CM could form an automatic induction system in enzyme production by 13

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recombinant E. coli. Galactose, the hydrolysis product of CM (trisaccharide, 1.2%, w/w), could effectively induce lactose operon for the recombinant protein synthesis [30]. Hence, a byproduct of the sugar industry (CM) could be used as a low-cost raw material for ChSase ABC I production by E. coli-pET-22b(+)-ChSase ABC I. It was reported that utilization of CM by engineered E. coli for sucrose isomerase and carbonyl reductase

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expression also had a better effect [31, 32]. 3.5 Biochemical properties of ChSase ABC I

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The temperature and pH optimization are important steps for increasing the activity of

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enzyme molecules [34, 35]. The ChSase ABC I activity was tested at different pH levels

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(5.5 to 9.5). The highest activity of ChSase ABC I was observed at pH 8.0 shown in Fig.

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5A. The ChSase ABC I activity was more than 80% in the pH range of 6.8 to 8.7, but its

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activity decreased remarkably when pH was lower than 6.5 or higher than 9.0, suggesting that ChSase ABC I was stable over a broad pH range. Fig. 5B shows that the enzyme

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activity increased to 35°C and decreased when the temperature exceeded 40°C. Its activity was more than 90% in the temperature range of 25°C–40°C, and the optimum temperature was 35°C. To calculate Vmax and Km of ChSase ABC I, we used Michaelis– Menten model and the Lineweaver–Burk equation. Lineweaver–Burk plots of ChSase ABC I are illustrated in Fig. S1. The Km, Vmax and kcat of ChSase ABC I-His was 6.8 μmol·L−1, 5.6 μmol·L−1·s−1, and 1212.1 μmol·L−1, respectively. Table 4 shows the effects of different tags on ChSase ABC I characteristics. Chen reported that His-tag located at the N-terminal of ChSase ABC I shifts the optimum pH toward acidic conditions but does not influence the optimum temperature of ChSase ABC I [36]. The optimum

temperature

and

pH

of

ChSase 14

ABC

I

expressed

in

E.

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coli-pET-22b(+)-ChSase ABC I were consistent with ChSase ABC I from P. vulgaris [33]. It was confirmed that His-tag located at C-terminal of ChSase ABC I had little influence on the optimum pH and temperature. Besides, MBP-ChSase ABC I and GAPDH-ChSase ABC I yielded high Vmax (18.7 and 13.0 μmol·L−1·s−1, respectively). Meanwhile, GAPDH-ChSase ABC I and MBP-ChSase have the highly catalytic

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temperature, suggesting that fusion tags could increase the rigidity and thermal stability of the enzyme. However, the kcat of them were lower than ChSase ABC I (1092.1

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μmol·L-1), and the kcat of ChSase ABC I-His (1212.1 μmol·L−1) was slightly greater

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than that of ChSase ABC I. It was implied that the existence of the fusion tags

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weakened the ChSase ABC I catalytic efficiency and fusing with a C-terminal His6-tag

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had little influence on CS combining with ChSase ABC I and ChSase ABC I catalyzing

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CS decomposition. Thus, Km of MBP-ChSase ABC I was higher than that of the other forms of ChSase ABC I, revealing that MBP decreased the affinity between enzymes

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and substrates. Obviously, the specific activity of ChSase ABC I was lower than that of enzyme without fusion tag, because the fusion tag had a strong impact on the combination of CS and ChSase ABC I resulting in enzyme activity reduction. These results implied that the introduction of fusion tags solved the problems of inclusion bodies but elicited several adverse effects on the biochemical properties of enzyme molecules. Fortunately, specific activity of ChSase ABC I-His (306.5 U·mg-1) was close to ChSase ABC I (400.0 U·mg-1) and was 1.70 times higher than specific activity of His-ChSase ABC I (180.1 U·mg-1), suggesting that fusing with a C-terminal His6-tag has a little negative influence on enzyme activity comparing to other fusion tag. From the above discussion, a conclusion could be obtained that a best of both worlds 15

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approach was designed for mediating the expression level and the specific activity of ChSase ABC I in this work. 3.6 Detection of the specific disaccharides of CS It was reported that the ratio of ΔDi-4S/ΔDi-6S of CS from different animals is not identical [37]. The compositions of the degradation products of CS were detected by HPLC [28, 38]. Fig. 6 shows three main peaks corresponding to three kinds of

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disaccharides, respectively. They are ΔDi-0S, ΔDi-4S, and ΔDi-6S in turn. Table 5

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shown that ΔDi-4S/ΔDi-6S ratio of CS from shark, porcine, chicken, and bovine

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cartilage were corresponding with the results of literature, respectively. The specific

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disaccharides rate of CS shark was lower than CS from other sources, this is because the

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degradation products of CS shark also contains other disaccharides, such as

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β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-2,6-sulfated (ΔDi-2,6diS), β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-2,6-sulfated (ΔDi-4,6diS), and

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β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-2,4-sulfated (ΔDi-2,4diS). Thus, it can be seen that ChSase ABC I-His produced by recombinant E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I has great effect on CS degradation. It was reported that one unit of commercialized ChSase ABC I costs about $ 257.33. In other words, it costs approximately $10.29 to test the specific disaccharides of each CS sample. In this work, a novel strategy was proposed and applied to the cheap expression of ChSase ABC I, which not only eliminates ChSase ABC I in the form of inclusion bodies but also solves the current problem of expensive ChSase ABC I. 4 Conclusion In this work, a signal peptide (pelB) was used to improve the expression level of 16

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ChSase ABC I in E. coli BL21 (DE3). After the fermentation condition optimization, the maximum ChSase ABC I activity was 29.03 U·mL−1 in a 7.5 L fermentor. The result of the biochemical properties and the detection of the specific disaccharides indicated that fusing with a C-terminal His6-tag had little influence on ChSase ABC I properties. Moreover, CS degradation experiments revealed ChSase ABC I-His can be used to

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detect the specific disaccharides content of CS completely. In conclusion, a novel approach was designed for mediating the expression level and the specific activity of

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ChSase ABC I and it also would be a great method to detect specific disaccharides

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Acknowledgement

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content of CS in biomedical field.

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This work was supported by the National Natural Science Foundation of China

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(21908011), Natural Science Foundation of Jiangsu Province (BK20191028), Natural Science Foundation of the Higher Education Institutions of Jiangsu Province

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(18KJB530001), the Science and Technology Development Project of Changshu (CN201912), the Scientific Research Foundation of Changshu Institute of Technology (KYZ2017107Z) and the Agricultural Science and Technology Innovation Project of Suzhou (SNG2018041 and SNG201927).

17

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References [1] K. Sugahara, T. Mikami, T. Uyama, S. Mizuguchi, K. Nomura, and H. Kitagawa, Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate, Curr. Opin. Struc. Biol. 13 (2003), 612-620. [2] X. F. Bao, S. Nishimura, T. Mikami, S. Yamada, N. Itoh, and K. Sugahara, Chondroitin sulfate/dermatan sulfate hybrid chains from embryonic pig brain, which contain a higher proportion of L-iduronic acid than those from adult pig

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[13] J. Fu, Z. Jiang, J. Chang, B. Han, W. Liu, and Y. Peng, Purification,

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[15] R. M. Lauder, Chondroitin sulphate: A complex molecule with potential impacts on a wide range of biological systems, Complement. Ther. Med. 17 (2009), 56-62. [16] H. J. Lee, S. Bian, I. Jakovcevski, B. Wu, A. Irintchev, and M. Schachner, Delayed applications of L1 and chondroitinase ABC promote recovery after spinal cord injury, J. Neurotraum. 29 (2012), 1850-1863. [17] W. Gu, S. Fu, Y. Wang, Y. Li, H. Lü, X. Xu, and P. Lu, Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway, BMC Neurosci. 10 (2009), 128. [18] T. Yamagata, H. Saito, O. Habuchi, and S. Suzuki, Purification and properties of bacterial chondroitinases and chondrosulfatases., J. Biol. Chem. 243 (1968), 1523-35. [19] M. Kitamikado and Y. Z. Lee, Chondroitinase-producing bacteria in natural 19

Journal Pre-proof habitats, Applied microbiology. 29 (1975), 414-21. [20] S. Linn, T. Chan, L. Lipeski, and A. A. Salyers, Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron, J. Bacteriol. 156 (1983), 859-866. [21] N. Sato, M. Shimada, H. Nakajima, H. Oda, and S. Kimura, Cloning and expression in Escherichia coli of the gene encoding the Proteus vulgaris chondroitin ABC lyase, Appl. Microbiol. Biot. 41 (1994), 39-46. [22] Z. Chen, Y. Li and Q. Yuan, Expression, purification and thermostability of

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MBP-chondroitinase ABC I from Proteus vulgaris, Int. J. Biol. Macromol. 72

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[23] Y. Li, Z. Chen, Z. Zhou, and Q. Yuan, Expression, purification and characterization

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Expres. Purif. 128 (2016), 36-41.

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[24] E. H. Manting and A. Driessen, Escherichia coli translocase: the unravelling of a

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molecular machine, Mol. Microbiol. 37 (2000), 226-238.

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[25] H. P. Sorensen and K. K. Mortensen, Advanced genetic strategies for recombinant protein expression in Escherichia coli, J. Biotechnol. 115 (2005), 113-128.

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[26] R. F. Smith and N. P. Willett, Rapid plate method for screening hyaluronidase and chondroitin sulfatase-producing microorganisms, Applied microbiology. 16 (1968), 1434-1436.

[27] L. B. E. Shields, Y. P. Zhang, D. A. Burke, R. Gray, and C. B. Shields, Benefit of chondroitinase ABC on sensory axon regeneration in a laceration model of spinal cord injury in the rat, Surgical Neurology. 69 (2008), 568-577. [28] K. Pojasek, Z. Shriver, P. Kiley, G. Venkataraman, and R. Sasisekharan, Recombinant

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Chondroitinase AC and Chondroitinase B from Flavobacterium heparinum, Biochem. Bioph. Res. Co. 286 (2001), 343-351. [29] J. Gao, H. Xu, Q. Li, X. Feng, and S. Li, Optimization of medium for one-step fermentation of inulin extract from Jerusalem artichoke tubers using Paenibacillus polymyxa ZJ-9 to produce R,R-2,3-butanediol, Bioresource Technol. 101 (2010), 7076-7082. 20

Journal Pre-proof [30] P. Calik and H. Levent, Effects of pretreated beet molasses on benzaldehyde lyase production by recombinant Escherichia coli BL21(DE3)pLySs, J. Appl. Microbiol. 107 (2009), 1536-1541. [31] Q. Ye, X. Li, M. Yan, H. Cao, L. Xu, Y. Zhang, Y. Chen, J. Xiong, P. Ouyang, and H. Ying, High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium, Appl. Microbiol. Biot. 87 (2010), 517-525. [32] S. Li, H. Xu, J. G. Yu, Y. Y. Wang, X. H. Feng, and P. K. Ouyang, Enhancing

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isomerase: culture medium optimization containing agricultural wastes and cell immobilization, Bioproc. Biosyst. Eng. 36 (2013), 1395–1405.

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immobilizing sucrose isomerase on ε-poly-L-lysine modified mesoporous TiO2,

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dioxide as a novel carrier for enzyme immobilization, Biosensors and Bioelectronics. 80 (2016), 59-66.

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[35] Z. Chen, Y. Li and Q. Yuan, Study the effect of His-tag on chondroitinase ABC I based on characterization of enzyme, Int. J. Biol. Macromol. 78 (2015), 96-101. [36] A. Hamai, N. Hashimoto, H. Mochizuki, F. Kato, Y. Makiguchi, K. Horie, and S. Suzuki, Two distinct chondroitin sulfate ABC lyases an endoeliminase yielding tetrasaccharides and an exoeliminase preferentially acting on oligosaccharides, J. Biol. Chem. 272 (1997), 9123-9130. [37] N. Volpi, Analytical aspects of pharmaceutical grade chondroitin sulfates, J. Pharm. Sci.-US. 96 (2007), 3168-3180. [38] V. Prabhakar, I. Capila, V. Soundararajan, R. Raman, and R. Sasisekharan, Recombinant Expression, Purification, and Biochemical Characterization of Chondroitinase ABC II from Proteus vulgaris, J. Biol. Chem. 284 (2009), 974-982.

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Journal Pre-proof Author Statement Qian Zhong: Conceptualization, Methodology, Software. Xingyu Lu: Data curation, Writing- Original draft preparation. Jian Liua and Fulin Yang: Visualization, Investigation. Chenghui Lu: Writing- Reviewing and Editing. Huan Xiong: Data curation, Writing- Original draft preparation. Sha Li and Yibo Zhu: Software, Validation.

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Lingtian Wu: Project administration and Supervision.

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Fig. 1 (A) Construction of recombinant pET-22b(+)-ChSase ABC I plasmid; (B) Lane 1,

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DNA 5000 marker; Lane 2, the PCR products of ChSase ABC I gene; Lane 3, the

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restriction analysis of pET-22b(+)-ChSase ABC I with XhoI; Lane 4, the restriction

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analysis of pET-22b(+)-ChSase ABC I with XhoI and NcoI; Lane 5, DNA 15000 marker.

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Fig. 2 (A) The result of recombinant ChSase ABC I activity on plate culture; (B) SDS-PAGE analysis of recombinant ChSase ABC I. Lane 1, whole cell of E. coli BL21 (DE3)-pET-22b(+)-ΔpelB-ChSase ABC I; Lane 2, whole cell of E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I; Lane 3, the supernatant of E. coli BL21 (DE3)-pET-22b(+)-ΔpelB-ChSase ABC I disruption; Lane 4, the supernatant of E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I disruption; Lane 5, protein molecular weight marker.

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Fig. 3 Response surface curves for ChSase ABC I production by E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I. (A) Function of YEP and CS concentration, when MgSO4·7H2O was maintained at 1.1 g·L-1; (B) function of MgSO4·7H2O and and CS concentration, when YEP concentration was maintained at 7.0 g·L-1; (C) function of MgSO4·7H2O and YEP concentration, when CS concentration was maintained at 5.0 g·L-1.

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Fig. 4 Fermentation of E. coli BL21 (DE3)-pET-22b(+)-ChSase ABC I in a 7.5 L

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fermentor. (■) represents the total sugar concentration in the fermentation broth, (●)

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represents the cell biomass (OD660) and (○) represents the ChSase ABC I activity.

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Fig. 5 (A) Effect of pH on the activity of ChSase ABC I and pH stability of

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ChSase ABC I; (B) Effect of temperature on the activity of ChSase ABC I and

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thermal stability of ChSase ABC I. The highest activity was defined as 100%.

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Fig. 6 The HPLC analysis of disaccharides content of chondroitin sulfate from different sources. (A) CS from shark cartilage; (B) CS from porcine cartilage; (C) CS from chicken cartilage; (D) CS from bovine cartilage; 1 presents β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine (ΔDi-0S); 2 presents β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-6-sulfated (ΔDi-6S); 3 presents β-dehydrated glucuronic acid-1,3-N-acetylgalactosamine-4-sulfated (ΔDi-4S).

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Table 1 Experimental design and responses of Plackett-Burman design on the ChSase ABC I production. Variable

CS KH2PO4·3H2O CM Peptone YEP CaCl2 MgSO4·7H2O

X1 X2 X3 X4 X5 X6 X7

High level (g·L-1) 6.50 3.50 9.50 6.50 7.50 2.40 1.20

Coefficient

SEa

F-value

P-value

-0.83 0.46 -0.13 0.44 1.28 -0.15 0.61

0.17 0.17 0.17 0.17 0.17 0.17 0.17

25.36 7.60 0.64 7.11 60.34 0.87 13.73

0.0073** 0.051 0.47 0.056 0.0015** 0.40 0.021**

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Standard error; ** indicate highly significant.

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Low level (g·L-1) 4.50 2.50 6.50 4.50 5.50 1.40 0.80

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Table 2 Experimental design and results of Box-Behnken Design. ChSase ABC I activity (U·mL-1)

X5

X7

Observed

Predicted

1

4.00 (-1)

6.00 (-1)

1.10 (0)

13.75 ± 0.48

14.03

2

6.00 (1)

6.00 (-1)

1.10 (0)

15.94 ± 0.52

15.95

3

4.00 (-1)

8.00 (1)

1.10 (0)

13.92 ± 0.68

13.91

4

6.00 (1)

8.00 (1)

1.10 (0)

15.53 ± 0.22

15.26

5

4.00 (-1)

7.00 (0)

0.90 (-1)

14.80 ± 0.34

14.67

6

6.00 (1)

7.00 (0)

0.90 (-1)

16.53 ± 0.51

16.67

7

4.00 (-1)

7.00 (0)

1.30 (1)

13.81 ± 0.78

13.67

8

6.00 (1)

7.00 (0)

1.30 (1)

14.82 ± 0.28

14.95

9

5.00 (0)

6.00 (-1)

0.90 (-1)

16.00 ± 0.24

15.86

10

5.00 (0)

8.00 (1)

0.90 (-1)

14.45 ± 0.36

14.59

11

5.00 (0)

6.00 (-1)

1.30 (1)

13.77 ± 0.63

13.63

12

5.00 (0)

8.00 (1)

1.30 (1)

13.95 ± 0.44

14.10

13

5.00 (0)

7.00(0)

1.10 (0)

17.33 ± 0.56

17.49

14

5.00 (0)

7.00 (0)

1.10 (0)

17.37 ± 0.48

17.49

15

5.00 (0)

7.00 (0)

1.10 (0)

17.88 ± 0.71

17.49

16

5.00 (0)

7.00 (0)

1.10 (0)

17.58 ± 0.60

17.49

17

5.00 (0)

7.00 (0)

1.10 (0)

17.33 ± 0.41

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X1, X5 and X7 are the coded values of the test variables CS, YEP and MgSO4·7H2O

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concentration (g·L-1), respectively.

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Table 3 Regression analysis of the central composite design.

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Source SSa DFb MSc F-Value Probe > F Model 36.87 9 4.10 54.96 < 0.00010** X1 5.35 1 5.35 71.78 < 0.00010** X5 0.32 1 0.32 4.32 0.077 X7 3.70 1 3.70 49.60 0.00020** X1X5 0.083 1 0.083 1.11 0.33 X1X7 0.13 1 0.13 1.76 0.23 X5X7 0.75 1 0.75 10.13 0.015 2 X1 5.39 1 5.39 72.31 < 0.00010** X52 10.47 1 10.47 140.42 < 0.00010** X72 7.95 1 7.95 106.62 < 0.00010** Residual 0.525 7 0.075 Lack of Fit 0.30 3 0.10 1.78 0.29 Pure Error 0.22 4 0.056 Cor Total 37.40 16 a b c ** Sum of Squares; Degree of freedom; Mean Square; indicate highly significant; X1, X5 and X7 are the coded values of the test variables CS, YEP and MgSO4·7H2O concentration (g·L-1), respectively.

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Journal Pre-proof Table 4 Biochemical properties of ChSase ABC I in different forms. Activity Enzymes

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Specific activitya

Optimum

Vmax

Km

kcat (s-1)

Reference

4.0

1092.1

[33]

13.0

3.0

393.0

[23]

18.7

73.1

586.7

(U·mL )

(U·mg )

Temp

pH

(μmol·L ·s )

(μmol·L-1)

ChSase ABC I

NGc

400.0

37

8.0

0.3

GAPDH-ChSase ABC I

NG

131.0

40

7.5

MBP-ChSase ABC I

3.2

76.0

50

7.7

His-ChSase ABC I ChSase ABC I-His

NG 17. 6

d

-1

-1

180.1

37

7.5

2.4

318.4

306.5

35

8.0

5.6

6.8

6.97×10

1212.1

[22] -2

[36] This work

after Ni column purification; the enzyme was expressed in the optimized culture; c not given; d activity: U·mL-1 fermentation liquor. ChSase ABC I represents the chondroitinase ABC I without fusion tag; GAPDH-ChSase ABC I represents the chondroitinase ABC I with glyceraldehyde-3-phosphate dehydrogenase; MBP-ChSase ABC I represents the chondroitinase ABC I with maltose-binging protein; His-ChSase ABC I represents the chondroitinase ABC I with a N-terminal His6-tag, and ChSase ABC I-His represents the chondroitinase ABC I with a C-terminal His6-tag.

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Journal Pre-proof Table 5 The ΔDi-4S/ΔDi-6S ratio, specific disaccharides content, and CS content of

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samples purified from shark, porcine, chicken, and bovine cartilages ΔDi-4S/ΔDi-6S ratio Specific Samples Nicola reported[37] This work disaccharides (%) CS Shark 0.45–0.70 0.63 83.01 CS Porcine 4.50–7.00 5.01 92.32 CS Chicken 3.00–4.00 3.67 93.28 CS Bovine 1.50–2.00 1.71 95.12

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Highlights 

Signal peptide (pelB) was used to Chondroitinase ABC I (ChSase ABC I) expression.



Cane molasses as a major carbon source for ChSase ABC I high-efficiency expression.



The problem of ChSase ABC I expressed as inclusion body was solved

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ChSase ABC I-His was used to detect specific disaccharides of chondroitin sulfate

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successfully.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6