Biocatalytic production of compound K in a deep eutectic solvent based on choline chloride using a substrate fed-batch strategy

Journal Pre-proofs Biocatalytic production of compound K in a deep eutectic solvent based on choline chloride using a substrate fed-batch strategy Xin Han, Xiaoxuan Ma, Zhiguang Duan, Weina Li, Daidi Fan PII: DOI: Reference:

S0960-8524(20)30308-4 https://doi.org/10.1016/j.biortech.2020.123039 BITE 123039

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Bioresource Technology

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Please cite this article as: Han, X., Ma, X., Duan, Z., Li, W., Fan, D., Biocatalytic production of compound K in a deep eutectic solvent based on choline chloride using a substrate fed-batch strategy, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.123039

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Biocatalytic production of compound K in a deep eutectic solvent based on choline chloride using a substrate fed-batch strategy Xin Hana,b,c, Xiaoxuan Maa,b,c, Zhiguang Duana,b,c, Weina Lia,b,c,d, Daidi Fana,b,c aShaanxi

Key Laboratory of Degradable Biomedical Materials, School of Chemical

Engineering, Northwest University, 229 North Taibai Road, Xi’an, Shaanxi 710069, China bShaanxi

R&D Center of Biomaterials and Fermentation Engineering, School of

Chemical Engineering, Northwest University, 229 North Taibai Road, Xi’an, Shaanxi 710069, China cBiotech.

& Biomed. Research Institute, Northwest University, 229 North Taibai Road,

Xi’an, Shaanxi 710069, China dXi'an

Giant Biogene Co., Ltd, Xi’an, Shaanxi 710065, China

Corresponding

author.

E-mail addresses: [email protected] (W. Li), and [email protected] (D. Fan).

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Abstract This study involved the development of a β-glucosidase-catalyzed hydrolysis method based on a deep eutectic solvent (DES), choline chloride-ethylene glycol 2:1, and continuous feed technique to overcome the difficulty of high-concentration ginsenoside hydrolysis. A productivity of 142 mgL-1h-1 was achieved with the following conditions: 30 vol% DES, pH 5, 55°C, and substrate concentration of 12 mM. In the presence of DES, the affinity and catalytic efficiency of β-glucosidase to Rd increased by 49 and 64%, respectively, which promoted the continuation of hydrolysis. Moreover, conformation of β-glucosidase was mostly retained, as confirmed by spectral information. Through a combination of a substrate fed-batch technique to reduce the inhibitory effects of substrates and products, the CK conversion rate increased by 44% compared to traditional single-batch in DES-buffer. This report describes a practical method for the continuous conversion of natural compounds through biological processes and solvent engineering. Keywords: Deep eutectic solvent; Biocatalysis; Fed-batch; β-glucosidase; Ginsenoside CK

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1. Introduction Compound K (CK) is a steroid that has been shown to be the main component behind ginseng’s activity in the body. Pharmacological studies have confirmed that CK has good absorption in the body and beneficial effects, especially antitumor activities (Cui et al., 2013). Unfortunately, CK rarely occurs in nature, adding impetus to the search for efficient CK preparation methods (Rahimi et al., 2019). Because of the non-directional conditions, and environmental pollution caused by chemical conversion, the biotransformation of ginsenosides has aroused the interest of researchers. CK preparation methods using microbial enzymatic conversion have been studied, including using β-glucosidase or other glycoside hydrolases from microorganisms such as filamentous fungi and recombinant bacteria (Park et al., 2010). β-glucosidase from Paecilomyces bainier sp. 229 converted Rb1 to CK with a conversion of 84.3% (Bae et al., 2000). A recombinant β-glycosidase from Pyrococcus furiosus was shown to have a higher CK productivity: 4 mM ginsenoside Rd was converted to 3.2 mM CK in 6 h (Yoo et al., 2011). There are other reports of conversion of ginsenoside Rb2 and Rb3 into CK, such as β-glucosidase from Microbacterium esteraromaticum, which converts 0.67 mM Rb2 to 0.16 mM CK (Yang et al., 2015). Traditionally, weak enzyme catalysis and process inhibition at high substrate concentrations have been major obstacles limiting the conversion rate due to many factors, such as specific substrate and product inhibition, enzyme degradation, promiscuity of enzymes, or even restriction of intermediate products (Xu et al., 2020b). The paradigm for optimizing ginsenoside production should shift

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towards dissecting each cascade into single hydrolysis units to find the rate limiting steps. The poor solubility of intermediates, such as Rd and F2, during hydrolysis of Rb1 (Liu et al., 2015) in aqueous solution, leads to a decrease in the catalytic interface between the enzyme and substrate, which is a major factor hindering rapid-reaction hydrolysis at high concentrations. Although the addition of traditional organic solvents in the biocatalytic process effectively increases the solubility of hydrophobic substrates, it does not conform to the principles of green chemistry and may even lead to product safety issues. The third-generation ionic liquid-deep eutectic solvent (DES) has many advantages as a medium that increases the solubility of the substrate, including safety, low cost, high biodegradability and biocompatibility (Pätzold et al., 2019). DES is composed of stoichiometric ratios of hydrogen bond acceptors (HBA, choline derivatives such as choline chloride and choline acetate) and hydrogen bond donors (HBD, such as natural amino acids, organic acids and sugars) (Xu et al., 2020a). DES has been successfully applied in specific enzymatic reactions, such as esterification, transesterification and hydrolysis (Gunny et al., 2015; Hümmer et al., 2018; Oh et al., 2019) and shown favorable effects on the activity, stability, and stereoselectivity (Cai and Qiu, 2019). However, no studies have reported the use of DES in ginsenoside conversion. Thus, in addition to the screening and production of β-glycosidases, further research should focus on the improving the solvent used in hydrolysis. To increase the efficiency of CK biosynthesis, designing DES to make it a safe environment for various enzymes is a key issue. The aim should be to preserve the natural structures of the enzyme in multi-

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component DES, facilitating the expression of desirable activities (Hoppe et al., 2019). Besides the heterogeneity caused by the precipitation of intermediates Rd and F2, the continuous conversion of ginsenosides is also limited by the high viscosity caused by the accumulation of large-scale substrates during the hydrolysis process (Quan et al., 2012). Although single mechanical agitation promotes mass transfer to some extent, the enzyme configuration may be affected, resulting in activity loss in the traditional batch processing with high concentrations of substrates (Guajardo et al., 2019). Thus, combining a substrate fed-batch strategy with DES to facilitate a constant low substrate concentration, high enzyme activity, and catalytic efficiency in the reaction process is necessary to increase the productivity of CK for future scale-up biocatalytic processes. In this work, the application of DES as a reaction medium in the enzymatic hydrolysis of ginsenoside Rb1 by Aspergillus terreus β-glucosidase was studied to improve the catalytic efficiency. Several DES were screened to improve the solubility of intermediates and evaluate the compatibility and stability with β-glucosidase. The kinetics of the enzymatic reaction were used to explain the mechanism by which DES improves the hydrolysis reaction, and spectral characterization was used to analyze the effect of DES on the conformation of β-glucosidase. In addition, the continuous catalytic hydrolysis reaction with fed-batch addition of the substrate achieved a conversion rate of 91.3% in this novel reaction medium. This research, therefore, provides insights into the catalysis of natural products. 2. Materials and methods

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2.1. Materials Standard ginsenoside Rb1, Rd, F2 and CK (98%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd, China. High performance liquid chromatography (HPLC) grade acetonitrile was obtained from Fisher-Scientific. p-Nitrophenol and pnitrophenyl-β-D-glucopyranoside were purchased from Solarbio (Beijing Solarbio Science & Technology co., Ltd). Standard protein was purchased from Thermo Scientific. The other chemicals used in this study were at least analytical grade. The protein concentration of the enzyme was determined using BCA kit (Sigma-Aldrich Co. USA). 2.2. Isolation, screening, and phylogenetic analysis of strains with hydrolytic activity The soil and tissue samples of notoginseng were obtained from Wenshan, China. The samples were thoroughly dissolved and diluted stepwise, and cultured on potato dextrose agar plates (PDA) and Esculin-R2A agar (β-glucosidase screening medium) at 30 ℃ for 3-5 days. Single colonies were obtained by repeated purification. The strains were identified by 18S rDNA sequence alignment. Fungal genomic DNA was extracted and purified rapidly using a bacterial genome DNA-extraction kit (Sangon Biotech, Shanghai, China) following the manufacturer’s protocol. The 18S rDNA gene was amplified by the polymerase chain reaction (PCR) using universal primers NS1 (5'GTAGTCATATGCTTGTCTC-3')

and

NS6

(5'-

GCATCACAGACCTGTTATTGCCTC-3'). The sequence analysis and homology comparison

were

performed

using

the

online

(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) (Cui et al., 2016).

6

BLAST

program

2.3. Enzyme production, purification, and molecular weight A. terreus was cultured in enzyme-producing medium (7.2 g/L lactose, 32.4 g/L gluten powder, 4 g/L (NH4)2SO4, 2 g/L KH2PO4, 0.6 g/L MgSO4, 0.6 g/L CaCl2, pH 5.0) at 29 ℃, 180 rpm for 5 days (Cui et al., 2016). The biomass of the fermentation system was obtained by measuring the dry cell weight (DCW) of the mycelium. After mycelium filtration, the crude enzyme solution was obtained by centrifugation (RCF = 24,000 g), salting out (60% [NH4]2SO4), and dialysis for 24 h (0.02 M sodium acetate, pH 5.0). Enzyme purification was carried out with anion exchange chromatography (DEAE Cellulose-52) (Beijing Solarbio Science & Technology co., Ltd). The crude enzyme solution was eluted gradually with 0.05- 0.25 M NaCl in acetate buffer (0.02 M, pH 5.0), and the fractions were examined for the hydrolysis activity of pNPG. The fractions with the high hydrolytic activity underwent for further molecular weight evaluation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), using 5% (w/v) stacking polyacrylamide gel and 12% (w/v) separating gel. Protein bands were stained with Coomassie bright blue R-250. 2.4. Preparation of DES DES was thermally synthesized as reported elsewhere (Cheng and Zhang, 2017). Choline chloride (ChCl) and hydrogen-bonded donors (glycerol [G], ethylene glycol [EG], urea [U]) at different molar ratios, were mixed in a round-bottom flask and stirred in a water bath at 80 °C (300 r/min), until a uniform, colorless, transparent liquid formed. The DES solution was cooled to room temperature before use.

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2.5. Biotransformation system for ginsenosides 2.5.1. Bioconversion of Rb1 to CK The conversion reaction of ginsenoside was performed in a screw cap-sealed 10 mL bottle containing β-glucosidase and ginsenoside Rb1. The hydrolysate was quantitatively tested after dilution with methanol. The conversion and productivity of the final product CK were calculated as follows: 𝐶𝐾 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛(%) = 𝐶𝐾 (𝑚𝑀)/𝑅𝑏1 (𝑚𝑀) 𝐶𝐾 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝐶𝐾 (𝑚𝑔) 𝑉 (𝐿)𝑇 (ℎ) V and T represent the reaction system volume and reaction time, respectively. 2.5.2. Kinetic analysis of ginsenoside hydrolysis Kinetic parameters for ginsenosides hydrolysis were obtained by reacting with a series of concentrations of ginsenosides Rb1, Rd, and F2 (3, 5, 10, 15, 25 and 30 mM) at 55°C in acetate buffer (0.02 M, pH 5) and DES-buffer within 5-10min. The conversion rate was obtained quantitatively by HPLC. The values of Michaelis constant (Km) and maximum reaction rate (Vmax) were calculated from the Lineweaver-Burk plots, and the catalytic rate constant (kcat) was calculated from Km and Vmax (Wang et al., 2020). 2.5.3. Production of CK by fed-batch procedure The fed-batch operation was performed in a reactor equipped with mechanical stirring (300 r/min), a temperature controller (55 ℃), and a peristaltic pump with feed tube. Acetate buffer (0.02 M, pH 5.0) containing 30 vol% DES was used as the reaction medium. The feed flow rate of Rb1 (2.4 M Rb1/190 mL DES-buffer) was set to 0.2

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mL/min (Guajardo et al., 2019); feed was stopped for 30 min every 3 h, and most of the CK was removed by centrifugation. The initial system was a 10 mL DES-buffer reaction system in which 1 mM Rb1 and 800 mg β-glucosidase were dissolved. 2.6. Analysis of enzymolysis products HPLC (SSI, USA) was used to quantify ginsenosides at 203 nm with a C18 column. The mobile phase was water (A) and acetonitrile (B), and the injected volume was 20 μL. The structure of the enzymatic product was analyzed using nuclear magnetic resonance (NMR) spectrometry. The products were dissolved in pyridine-d5, and the spectra of 1HNMR and

13C-NMR

were assessed using the Bruker Avance NEO 600 MHz NMR

spectrometer (Bruker Corporation) (Zheng et al., 2019). β-glucosidase activity was assayed by colorimetry using p-nitrophenyl-β-Dglucopyranoside (pNPG) as the substrate. One unit (U mL-1) of β-glucosidase activity was defined as the amount of enzyme required to hydrolyze 1 μL pNPG per min under standard conditions (Park et al., 2017). 2.7. Structural characterization of β-glucosidase After being dispersed throughout the acetate buffer or DES-buffer for 48 h, changes in the secondary and tertiary structure of β-glucosidase in the presence of DES were determined through spectroscopic analysis, as follows. FTIR was performed on a Varian 670 IR spectrometer with a scanning wavelength range of 400-3600 nm. Peakfit software was used to analyze the secondary structural elements based on the information of the amide I region (Liu et al., 2019).

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Circular Dichroism (CD) was recorded on a JASCO J-815 CD spectrophotometer (Jasco Inc., Tokyo, Japan). We used a protein concentration of 0.2 mg/mL for farultraviolet and 1.0 mg/mL for near-ultraviolet, time per point of 0.5 s, pathlength of 0.5 mm and wavelengths ranging from 320-190 nm (Yu et al., 2017). Fluorescence studies were performed on a Hitachi F4600 fluorescence spectrometer (Hitachi Hi-Tech co., Ltd., Japan), based on the tryptophan fluorescence excitation of βglucosidase structure (Yu et al., 2017). The measurement conditions were an excitation wavelength of 280 nm and emission wavelength of 300-400 nm. 2.8. Statistical analysis All experiments were carried out in triplicate, and the results are expressed as mean ± standard deviation. 3. Results and discussion 3.1. Preparation, purification and characterization of β-glucosidase 3.1.1. Isolation and phylogenetic analysis of A. terreus strain with hydrolytic activity A total of 37 strains were isolated from tissue and root soil of notoginseng by means of progressive dilution and tissue separation, of which 14 strains produced β-glucosidase. A strain named X-51 exhibited a dark reddish colony region on an Esculin-R2A agar plate and showed the strongest hydrolysis of glucoside bonds activity. Phylogenetic analysis based on 18S rDNA gene sequencing showed that X-51 belonged to Aspergillus, and X51 was most closely related to A. terreus MF678562. The nucleotide sequence of strain X-51 has been submitted to GenBank with the accession number of. MN945288.

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3.1.2. Fermentation, purification and characterization of β-glucosidase β-glucosidase showed a continuous synthesis mode, and the relationship between the biological activity of β-glucosidase and the DCW is shown in Fig. 1a. The synthesis of β-glucosidase began with cell growth, but the enzyme continued to synthesize for some time, reaching the highest cumulative activity of the enzyme at 120 h as the cell growth reached a stable plateau, thereby providing a crude enzyme solution. The enzyme activity of each fraction separated on the DEAE-Cellulose-52 column was measured to identify the main active component. The fraction eluted with 0.05 M NaCl showed the highest pNPG hydrolytic activity. A summary of purification steps and process parameters is listed in Table 1. The specific activity of β-glucosidase in the fermented supernatant was 2.43 U/mg. The crude β-glucosidase was purified 1.57-fold after (NH4)2SO4 precipitation and desalting. The β-glucosidase was purified 6.02-fold with a 23.06% recovery, and the specific activity of β-glucosidase reached 14.62 U/mg through DEAE-Cellulose-52 separation. The molecular weight (MW) of the β-glucosidase was further estimated by PAGE and the result is shown in Fig. 1b. The enzyme protein appeared as almost a single band, with a molecular weight of approximately 121 kDa. There are several reports available on β-glucosidase production from filamentous fungi such as Aspergillus niger, A. oryzae, decumbens, Paecilomyces sp., etc (Singhania et al., 2013), in which Aspergillus fermentation produced higher titers and active enzymes, so that crude enzymes could be obtained without requiring too many complex procedures (Günata and Vallier, 1999). 3.1.3. Pathway of ginsenoside Rb1 hydrolysis by β-glucosidase

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The hydrolysis pathway of ginsenoside Rb1 to CK was confirmed by HPLC. It was apparent that β-glucosidase from A. terreus converted the ginsenoside Rb1 into ginsenosides Rd, F2 and CK. The changes in content of ginsenosides Rb1, Rd, F2, and CK within 8 h of β-glucosidase-catalyzed Rb1 hydrolysis are shown in Fig. 2. The structure of the final product was confirmed by NMR spectrometry. The information for 13C-NMR

and 1H-NMR indicated that the product was CK, consistent with previous

reports (Tanaka and Kasai, 1984). The results indicate that the β-glucosidase from A. terreus first converted Rb1 into Rd by hydrolysis of 20-O-β-D-(1-6)-glucopyranoside, and then converted Rd into F2 and CK by continuous hydrolysis of 3-O-β-D-(1-2)glucopyranoside. 3.2 Screening of optimum DES for CK production Improvement and optimization of the system by changing the solvent was an effective method of accelerating hydrolysis reaction. The HBD, differences in hydrogen bond network strength, and the internal environment, significantly affected the properties (such as polarity, viscosity and surface tension) of the ChCl-based DES solvent (Zhang et al., 2012). The main factors effecting β-glucosidase catalytic performance, i.e., HBD type (including EG, G and urea U), and molar ratio of HBD to ChCl, were screened and analyzed. The highest relative activity was found to approach the result in acetate buffer, and β-glucosidase activity was higher in polyol-based DES than in U-based DES. However, there was no clear trend in terms of thermostability, with the maximum halflife observed in ChCl:EG 2:1 and ChCl:G 1:2 (Fig. 3a). Therefore, three DESs (ChCl:

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EG 2:1; ChCl: G 1:2 and ChCl: U 1:1) were selected to evaluate the solubility of the intermediates in them. The equilibrium solubility of Rd increased with increasing temperature, and solubility was much greater in all DES-buffers than in acetate buffer (Fig. 3b). The equilibrium solubility of Rd at 70°C reached 733.9 mg/L in 30 vol% ChCl: EG (2:1), but only 307.1 mg/L in pure buffer. Similarly, the solubility of F2 in 30 vol% ChCl: EG (2: 1) was 2.32 times that in acetate buffer. It is obvious that DES, as a cosolvent, significantly improves the solubility of intermediates. The effects of DES on the glycosidase hydrolysis system were further evaluated with regards to DES concentration. As DES (ranging from 0 to 60%) in acetate buffer caused activity loss, a DES concentration above 30% produced a more obvious effect, in particular, it decreased linearly with increasing ChCl: U (Fig. 3c). However, activity loss was not obvious in ChCl: EG and ChCl: G concentration under 30%. Compared to the high viscosity of the U-based DES, polyol-based DES forms a low-viscosity transparent liquid. Certain hydroxyl groups retained in the molecules (EG:2; G:3) help DES to form stronger hydrogen bonds. In addition, the influence of buffer content on enzyme activity may be attributed to this effect: The large amount of water reduces the viscosity of the system caused by DES, which helps maintain the structural flexibility of the enzyme's active site. However, the active site of the enzyme surrounded by DES may be masked or changed if excessive DES is added (Wu et al., 2019; Xu et al., 2018). After all, most enzymes, including β-glucosidase, need an adequate aqueous environment to maintain their conformation and the structural integrity of their active centers. As described above,

13

ChCl: EG (2:1) buffer resulted in less activity loss than the other 2 DESs, with thermostability higher than that achieved with acetate buffer (Fig. 3a), as well as having good solvent viscosity and substrate solubility and retaining suitable conditions for βglucosidase activity (Mokhtarpour et al., 2020). Therefore, it can be concluded that ChCl: EG (2: 1) is more suitable as a co-solvent for the enzymatic hydrolysis system compared with ChCl: G (1: 2) and ChCl: U (1: 1). Furthermore, CK conversion was maximal with an initial Rb1 concentration of 6 mM for 48 h in 30 vol% ChCl: EG (2:1) buffer (89.1%), increased CK conversion by 29.1% compared with acetate buffer (Fig. 3d). However, although 10 vol% ChCl: EG (2: 1) activated β-glucosidase slightly, the CK conversion rate was only improved by 4.5%. The hydrogen bond between HBA and HBD tends to weaken non-linearly as a result of dilution with excess water, resulting in the DES component becoming decomposed and hydrated (Gabriele et al., 2019). In other words, 10 vol% DES-buffer is close to pure buffer in terms of properties, which explains why 10 vol% DES is not the optimal choice as a solvent. When more than 30 vol% of ChCl: EG was added to the reaction system, the conversion of CK gradually decreased, even to 58.4% (60 vol% ChCl: EG), which was lower than the yield in buffer (69.5%). This can be attributed to the increased mass transfer restriction and enzyme inactivation caused by high concentrations of DES. Therefore, 30 vol% DES-buffer is the medium with the highest conversion of CK and was selected for further experiments. Traditional polar organic solvents with strong dissolving properties are considered

14

to be enzyme denaturants, as they break the intramolecular hydrogen bonds of the proteins; for example, A. niger β-glucosidase quickly lost 40% of its activity in 10% methanol, making the reaction impossible (Xu et al., 2018). Whereas, the quaternary ammonium salt in DES forms hydrogen bonds with hydrogen-bond donors (especially those with hydroxyl functional groups), which changes the strength of O-H bonds. The hydrogen bond interactions were obvious in ChCl: EG 2:1, and this strong hydrogen bonding provides excellent solubility properties for insoluble organic compounds and exhibits high biocompatibility for β-glucosidase (Shafie et al., 2019), thereby further promoting the conversion of ginsenosides. 3.3. Characterization of the effect of ChCl/EG 2:1 medium on CK production 3.3.1 Biotransformation optimization and analysis based on pH and temperature The effects of reaction temperature and pH on CK production by β-glucosidase in acetate buffer and DES (ChCl: EG 2: 1) buffer were fairly comparable. CK increased with an increase in pH from 3.0 to 5.0 and reached the maximum value of close to 100% in 48 h with 6 mM Rb1.Whereas conversion decreased as the pH increased to 7.0, and almost no activity was shown when the pH was less than 3.0 or greater than 7.0 (Fig. 4a). This β-glucosidase was eosinophilic, with a pH tolerance of between 3 and 7. Furthermore, the environmental ionic strength affected the charge properties of the substrate and the structural characteristics of the protein, affecting the binding efficiency of the substrate to the active site (Bey et al., 2016). The conversion of CK gradually increased as the temperature increased from 40 to

15

55 ℃, reaching its maximum at 55 ℃, then decreased to almost no activity as the temperature continued to rise to 70 ℃ (Fig. 4b). The activity of β-glucosidase increased with increasing temperature, as β-glucosidase began to denature and inactivate when the temperature rose higher than 60 °C due to changes in the structure and active site. However, the corresponding DES viscosity decreased, thereby minimizing mass transfer problems (Durand et al., 2013). Therefore, the maximum CK conversion of close to 100% was obtained at 55 ℃ with the DES-buffer system, which is more sensitive to temperature. 3.3.2 Kinetics of ginsenoside hydrolysis The kinetic parameters of β-glucosidase in acetate buffer and DES-buffer were determined according to the Michaelis-Menten kinetic model to further understand the effect of DES (ChCl: EG 2:1) on the catalytic mechanism. The kinetic parameters for ginsenosides Rb1, Rd and F2 are presented in Table 2. In both the acetate buffer and the DES-buffer system, the Km, kcat and kcat /Km values follow the order Rd> Rb1> F2, Rb1 > F2 > Rd, and Rb1 > F2 > Rd, respectively, which implies that DES does not change the substrate affinity priority of the β-glucosidase. In particular, affinity of A. terreus βglucosidase to Rd and F2 both significantly increased by 49.5 and 28.1% (as Km decreased to 9.86 and 5.24 mM), respectively; the catalytic efficiency for ginsenoside Rd and F2 showed 1.64 and 1.33 fold improvement (as kcat / Km increased to 1.25 and 3.16) in the DES-buffer, respectively. Generally, compared with Rb1, the significant improvements in the affinity and catalytic efficiency of β-glucosidase for Rd and F2 may have been though a reduction in the impact of the rate-limiting reaction by the increased solubility

16

of Rd in DES, which increased the rate of interface between the substrate and βglucosidase molecule and enhanced the mass transfer (Yao et al., 2018). The addition of DES eased the rate-limiting step of Rd hydrolysis to F2, which benefited the entire hydrolysis reaction. 3.3.3 Spectrum analysis of β-glucosidase structure The effects of 30 vol% ChCl: EG (2:1) on the structure of β-glucosidase were analyzed by FTIR, CD, and FS. In the FTIR spectrum, the amide I (1600-1700 cm-1) band caused by C=O tensile vibration is the region most sensitive to the protein secondary structure, secondary structural elements were calculated according to the amide I band (Liu et al., 2019). The α-helix, β-sheet, and random coil accounted for 12.7, 25.7, and 33.3% in the acetate buffer, respectively, compared to 9.7, 23.1, and 35.1% in the DESbuffer. In the far-ultraviolet CD spectra range of 190–250 nm, the two curves show similar shapes with a positive band centered at 192 nm and two negative bands centered at 208 and 222 nm, which correspond to the characteristic ellipticity of α-helix, β-turn and βsheet, respectively (Li et al., 2016). The results show that the α-helix, β-sheet, β-turn, parallel and random coil accounted for 16.5, 29.6, 19.7, 5.5, and 30.2% in acetate buffer, respectively, while 13.7, 28.6, 20.5, 4.8, and 30.7% in DES-buffer, respectively. The above observations are consistent with FTIR spectra analysis, confirming that almost no conformation changes occurred because of molecular interactions between β-glucosidase and the DES compared with pure buffer, except for the slight decrease of 3% in the αhelix, which facilitates substrate binding. In addition, there is a minimum value around

17

280 nm (near-UV CD spectra) assigned to disulfide bonds and tyrosine (Tyr) residues, while the maximum value at 255 nm may be the characteristic peak of disulfide bonds (Li et al., 2011). The tertiary structure of the enzyme did not involve the rearrangement of disulfide bonds or three-dimensional structural changes of aromatic amino acids. Generally, the endogenous fluorescence of proteins comes mainly from tryptophan (Trp) and Tyr (Yu et al., 2017). The emission spectrum in our study did not undergo a red or blue shift, but the slight increase in fluorescence intensity means that the tertiary structure of β-glucosidase is more stable in the DES-buffer. The amino acid residues were still embedded in the hydrophobic cavity of the protein and were not exposed at the molecular surface. The dense and strong intermolecular hydrogen bonding network between the DES components limits solvent diffusion into the protein chain (Monhemi et al., 2014), maintaining the natural conformation to promote enzyme function. Therefore, the residual activity of β-glucosidase in 30 vol% ChCl: EG (2:1) permitted long and complex reactions with minimal modification of the protein structure. 3.4. Process optimization for CK production using a substrate fed-batch strategy To overcome the difficulty of CK production at high substrate concentrations, the inhibitory effects of the initial substrate, intermediate products and final products on the conversion process were examined. As shown in Fig. 5a, the maximum levels of CK reached 6.0 mM at 8 mM Rb1, with an Rb1 conversion of 74.5%. However, substrate inhibition was triggered at 4 mM Rb1 (when Rb1 conversion dropped below 100%), then gradually increased until a significant inhibitory effect began at 8 mM Rb1, until Rb1

18

conversion dropped below 60%. The decrease in CK yield at > 8 mM Rb1 may also be due to the high viscosity of the Rb1-rich system. However, compared with the acetatebuffer system, the DES-buffer system moderately relaxed the inhibitory effect of the substrate, with the CK concentration increasing by 10-20% due to the increase in the mass transfer rate and reaction interface between substrate and enzyme (Sule et al., 2015). While the inhibitory effect of the final CK product was more obvious than for the intermediates Rd and F2 (Fig. 5b). F2 was easier to transform into CK than Rd. F2 led to the least inhibition among the added compounds. Specifically, the product inhibition effect was not obvious until 2 mM was reached. However, at concentrations of 2-4 mM, CK production increased (as conversion continued) in the case of adding intermediates Rd, F2 (e.g., CK reached maximums of 7.1, and 6.2 mM at 3 mM F2, and 2 mM Rd, respectively). CK yield was most hindered by CK concentrations above 3 mM because they inhibited the forward reaction of the reversible reaction (Palmerín-Carreño et al., 2015). Therefore, accumulation of the final CK product was another important factor in inhibiting catalytic conversion. To eliminate the inhibition by substrate or product, a substrate fed-batch strategy, which also prevents high viscosity at the beginning of the hydrolysis process (Sotaniemi et al., 2016), was adopted. Compared to DES-buffer and traditional buffer conversions without fed-batch, the optimized conversion method (DES/fed-batch) improved the final CK conversion by 29.2 and 44.0%, respectively (Fig. 5c). We demonstrated that 2.4 mM Rb1 was converted to 2.2 mM CK within 48 hours in the fed-batch process, reaching a

19

productivity of 142 mg L−1 h−1. The fed-batch process led to a smaller decline in activity, with 38.9% higher final residual activity than that of traditional batch method (large amounts of substrate added at once at the beginning of the reaction) (Fig. 5d). The significant enhancement of enzyme stability during CK production may be thanks to the relatively constant reaction environment (Guajardo et al., 2019), with low substrate concentration and low viscosity maintained in the DES-buffer of the substrate fed-batch strategy. 4. Conclusion This study was the first demonstration of a fed-batch strategy with DES (ChCl: EG 2: 1) buffer for enzymatic hydrolysis of ginsenosides. Because the solvent ameliorated the reaction limitation by Rd, with minimum disruption to enzyme activity, continuous hydrolysis of Rb1 was promoted and the CK conversion increased by 29.1%. The substrate fed-batch process, which mitigated the inhibition caused by the high-viscosity of the initial substrate and accumulation of end-product, achieved a CK productivity of 142 mg L−1h−1. The fed-batch strategy with DES represents a promising sustainable process that may be applied to large-scale ginsenoside production. Acknowledgements This study was financially supported by the National Natural Science Foundation of China (21706211, 21576160, 21776227, 21838009); Shaanxi Key Laboratory of Degradable Biomedical Materials Program (17JS124); the Shaanxi Provincial Scientific Technology Research and Development Program (2019JQ-720, 2017KJXX-02).

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Appendix A. Supplementary data E-supplementary data of this work can be found in online version of the paper. References 1.

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Figure captions Fig. 1. Production and purification of β-glucosidase from Aspergillus terreus. (a) The relationship between β-glucosidase activity and dry cell weight during the fermentation process; (b) SDS-PAGE purification analysis of β-glucosidase. Fig. 2. Dynamic changes in ginsenoside content during hydrolysis by β-glucosidase. 3 mM substrate Rb1, pH 5.0, 55℃. Fig. 3. Effect of choline chloride-based DES on β-glucosidase catalytic performance. (a) Hydrolytic activity and thermostability of β-glucosidase in different types of DES (30 vol%)-buffer. (b) Equilibrium solubility of ginsenoside Rd with temperature in 30 vol% DES. DES refers to ChCl:EG 2:1, ChCl:G 1:2, ChCl:U 1:1. (c) Hydrolytic activity of βglucosidase in different ratios of DES (10–60 vol%)-buffer. (d) Effect of ChCl: EG (2:1) concentration (pH 5.0) on CK conversion at 48 h, 8 mM Rb1, 55 °C. The relative activity (%) refers to the percentage of the initial enzyme activity in the buffer solution containing DES relative to the pure buffer. The enzyme half-life (hours) was obtained at 55 °C. Fig. 4. Effects of reaction pH (a) and temperature (b) on β-glucosidase activity and CK conversion in both buffer and DES-buffer. The CK conversion reaction was carried out in 20 mM acetate buffer containing 6 mM Rb1 for 48 h. Fig. 5. Process optimization for CK production using a substrate fed-batch strategy. (a) Substrate and (b) product inhibition on CK yield with different concentrations of Rb1, Rd, F2 and CK in DES-buffer. The reaction was performed in the system containing 6 mM Rb1 for 48 h, and the detection index was the actual molar yield of CK. (c) CK

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conversion and (d) residual activity under the fed-batch program, compared with traditional batch processing from 12 mM Rb1. The reaction was performed under the conditions of pH 5.0 and 55 °C, and the control was the traditional conversion mode without DES or fed-batch technique.

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Table 1. Purification of β-glucosidase from Aspergillus terreus. Purification steps

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Purification fold

Yield (%)

Fermented supernatant

4704.20±131.24

1938.38±38.05

2.43±0.11

1

100

(NH4)2SO4 fractionation

3142.93±38.98

826.07±25.89

3.81±0.16

1.57

66.81

DEAE-Cellulose-52

1084.80±26.15

74.75±7.63

14.62±1.60

6.02

23.06

Table 2. Kinetic parameters of β-glucosidase for various substrates in acetate buffer and DES-buffer Substrate

Rb1

Rd

F2

Pathway

Hydrolysis of 20-O-Glc

Hydrolysis of 3-O-

Hydrolysis of 3-O-Glc

of Rb1 to Rd

Glc of Rd to F2

of F2 to CK

Km

Buffer

9.61 ± 0.80

14.74 ± 2.58

6.71 ± 1.69

(mM)

DES-buffer

9.45 ± 0.59

9.86 ± 1.50

5.24 ± 1.14

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k cat

Buffer

33.40 ± 3.93

10.74 ± 2.16

14.81 ± 1.08

(s-1)

DES-buffer

32.04 ± 1.08

12.06 ± 1.16

16.18 ± 0.50

k cat / Km

Buffer

3.50 ± 0.55

0.76 ± 0.28

2.37 ± 0.57

(s mM)-1

DES-buffer

3.39 ± 0.21

1.25 ± 0.28

3.16 ± 0.53

Km, the Michaelis constant; k cat, catalytic number; k cat / Km, the catalytic efficiency of different substrates catalyzed by β-glucosidase. The data were measured during the initial reaction time of 5-10 min.

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32

33

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Highlights 

DES-buffer can be used in continuous catalytic process of ginsenoside hydrolysis.



DES improved the catalytic efficiency by 64% and CK conversion by 29.1%.



The conformation of β-glucosidase was quite well kept in the presence of DES.



The substrate fed-batch strategy reached a final CK productivity of 142 mgL-1h-1.

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CRediT authorship contribution statement Xin Han: Visualization, Writing-original draft, Data curation, Writing-review & editing; Xiaoxuan Ma: Validation, Investigation, Project administration; Zhiguang Duan: Validation, Formal analysis, Visualization; Weina Li: Writing-review & editing, Formal analysis, Funding acquisition, Constructive discussions, Supervision; Daidi Fan: Conceptualization, Resources, Project administration, Funding acquisition, Supervision.

Declaration of Competing Interest

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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