Enhancing the thermostability of recombinant cyclodextrin glucanotransferase via optimized stabilizer

Process Biochemistry xxx (xxxx) xxx–xxx

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Enhancing the thermostability of recombinant cyclodextrin glucanotransferase via optimized stabilizer ⁎

Jianguo Zhang , Mengla Li, Yan Zhang School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, 516 Jungong Road, Shanghai, 200093, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Chemical stabilizer Cyclodextrin Cyclodextrin glucosyltransferase Energy of inactivation Thermostability

Cyclodextrin is used widely in industry, and the thermostability of cyclodextrin glucanotransferase (CGTase) is important for high-yield cyclodextrin production. A chemical mixture (25% glycerol, 10% polyethylene glycol 400, and 0.5 mmol/L CaCl2) was used as stabilizer to enhance the thermostability of CGTase after optimizing its concentration, which resulted in a synergistic effect on CGTase stability. The β-CGTase and γ-CGTase activities increased by 7.6- and 9.4-fold, respectively, compared with controls, which resulted from an increase in the energy of inactivation, as determined by a thermokinetic analysis. The optimum temperature of CGTase activity increased from 50 to 60 °C after the stabilizer application. The stabilizer was applied to cyclodextrin production, and it resulted in enhanced CGTase activity, which showed higher increases of starch to CD conversion at 60 and 90 °C. These results demonstrate that using a stabilizer is an effective approach for CGTase storage and cyclodextrin production.

1. Introduction Cyclodextrins (CDs) attract much attention because they are used in the food, cosmetic, pharmaceutical, and chemical industries, as well as for drug delivery and in agriculture and environmental engineering [1]. CD production through a biocatalytic process using CD glycosyltransferase (CGTase), which is a 1,4-α-D-glucan 4-α-D-(1,4-α-D-glucano)-transferase, is beneficial because it is environmental friendly and easy to operate. For example, commercial CGTase Toruzyme 3.0 L and Amano CGTase catalyze starch to produce produces a mixture of CDs (α, β, γ) from the starch [2]. CGTase catalyzes mainly three kinds of reactions: disproportionation reaction that transfers a linear oligosaccharide chain to another linear oligosaccharide molecule, giving rise to a series of sugar molecules of different sizes, and hydrolytic reaction when water acts as an acceptor, and cyclization which is involved in the cleavage of α-(1,4) linked cyclomaltodextrins (six d-glucose units for αCD, seven d-glucose units for β-CD, eight d-glucose units for γ-CD) from starch, and intermolecular transglycosylation coupled the resulting maltooligosaccharide to various carbohydrate acceptors [3]. Many processes, such as isoamylase addition in 250-ml Erlenmeyer flasks [4], amylosucrase addition [5], ultrafiltration membrane reactor utilization, [6], and an integrated continuous stirred tank reactor – packed bed reactors process [7], have been explored by researchers for high-yield CD production. During CD production, a major issue is the short working time of CGTases, which is caused by CGTase inactivation [8].

⁎

Many CGTases have typical working temperatures in the range of 20–60 °C [3], with optimal temperatures of 50–60 °C, although some CGTases have an optimal temperature of 40 °C [9]. For example, the optimal temperature of Bacillus circulans CGTase is 55–60 °C [10], and Bacillus lehensis and Bacillus agaradhaerens CGTases showed the highest activity at 55 °C [11,12]. Therefore, high thermostability of CGTases is needed to achieve long working times [13]. High CGTase thermostability also has the advantages of low risk of contamination, lower viscosity of starch, and higher starch solubility [14]. And it has been obtained by microbial isolation [15], immobilization to a loofa sponge [16], sodium alginate [17], glutaraldehyde-activated chitosan spheres [18], and gene mutation [19,20]. Chemicals addition is an important approach for enhancing enzyme stability. And polyols, sugars, and salts are effective stabilizers [21]. Generally, hydroxy polyols and sugars engage in strong hydrophobic interactions with enzymes, which results in increased enzyme hydration. Salts have specific and non-specific effects on enzyme thermostability. However, the appropriate stabilizer depends on the nature of the enzyme because stabilizers are highly specific [21]. For example, the half-lives of trypsin and glucose oxidase are increased significantly by the addition of glycol chitosan [22]. Glucose oxidase is stabilized by salts addition [23]. The stability of chloroperoxidase is enhanced by the addition of polyhydroxy addition [24]. The stability of CGTase is enhanced by adding polyethylene glycol or polypropylene glycol for CD production [25]. In addition, enzymes tend to form aggregates with altered properties and that may alter the

Corresponding author. E-mail address: [email protected] (J. Zhang).

https://doi.org/10.1016/j.procbio.2018.02.006 Received 11 December 2017; Received in revised form 27 January 2018; Accepted 6 February 2018 1359-5113/ © 2018 Elsevier Ltd. All rights reserved.

Please cite this article as: Zhang, J., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.02.006

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Fig. 1. Effect of chemicals on CGTase activities (a: glycerol, PEG400, Ethylene glycol on β- CGTase activities; b: glycerol, PEG400, Ethylene glycol on γ-CGTase activities; c: gelatin, chitosan, mycose on β-CGTase activities; d: gelatin, chitosan, mycose on γ-CGTase activities; e: CaCl2, MgSO4, NaAC on β- CGTase activities; f: CaCl2, MgSO4, NaAC on γ-CGTase activities).

2. Materials and methods

results of the activity and stability studies [26]. Therefore, the effect of combined stabilizers should be investigated for enzyme stability enhancement. In the present study, chemicals mixture was established to enhance the stability of a recombinant CGTase from Komagataella phaffii was enhanced by the addition of a stabilizer. The thermokinetic parameters were analyzed to clarify the changes of stabilizer addition. This recombinant K. phaffii harbored a codon-optimized cgt gene encoding a CGTase from Bacillus pseudalcaliphilus 8SB. This CGTase catalyzed starch to produce β-CD and γ-CD only, without α-CD [27,28]. These results built a base for enzyme stabilizer enhancement by chemicals mixture.

2.1. Materials Recombinant K. phaffii harboring a codon-optimized cgt gene encoding a CGTase from Bacillus pseudalcaliphilus 8SB [15], according to the Codon Usage Database (http://www.kazusa.or.jp/codon/), was constructed in a previous study for high-level CGTase expression [28]. Only β-CGTase activity and γ-CGTase activities were detected with this CGTase. CGTase (27.6 ± 0.7 × 103 U/mL β-CGT activity, 111 ± 3 × 10−2 g protein/L) was used in this research. Glycerol, CaCl2, MgSO4, and sodium acetate (NaAC) were purchased from the Sino Chemical Company (Shanghai, China). Polyethylene glycol 400 (PEG400), ethylene glycol, gelatin, chitosan, and mycose were purchased from Sangon Biotech (Shanghai, China). Liquid chemicals (glycerol, PEG400, ethylene glycol) solution were prepared using 2

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Fig. 2. Effect of temperature on recombinant CGTase activities with stabilizer (a: β-CGTase activities; b: γ-CGTase activities).

2.4. Effect of temperature on activities of CGTase

volume percentage. And solid chemicals (CaCl2, MgSO4, NaAC, gelatin, chitosan, and mycose) were prepared using gram percentage. All chemicals were biochemical grade.

The recombinant CGTase (66.67 mmol/L phosphate salt buffer, pH 6.0) activities were measured with and without stabilizer at 40, 50, 55, 60, 65, 70, and 80 °C at pH 6.0, respectively. The CGTase activities without stabilizer at 50 °C were used as 100% relative activity.

2.2. Recombinant CGTase preparation Recombinant K. phaffii was cultivated in 50 mL of YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) at 30 °C with shaking 200 rpm for 18–24 h until the optical density at 600 nm reached 2.0–8.0. Subsequently, the recombinant cells were transferred into 25 mL of BMGY medium (10 g/L yeast extract, 20 g/L peptone, 100 mmol/L phosphate buffer (pH 7.0), 20 g/L glycerol, 13.4 g/L yeast nitrogen base, and 4 × 10−4 g/L biotin) at a 6% inoculation ratio, with shaking at 200 rpm, for 24 h. Then, after centrifugation (4200g, 5 min), the BMGY medium was replaced with 50 mL of BMMY medium (10 g/L yeast extract, 20 g/L peptone, 100 mmol/L phosphate buffer (pH 7.0), 5 mL/L methanol, and 4 × 10−4 g/L biotin); 1.5% methanol was added every 24 h to induce cgt gene expression. The culture supernatant was obtained by centrifugation (4200g, 2 min), and ammonium sulfate was added slowly to a proportion of 40%. Subsequently, the mixture was centrifuged (4200g, 45 min) to separate the supernatant and precipitate, and ammonium sulfate was added sequentially to the supernatant to proportions of 60% and 80%. After centrifugation (4200g, 45 min), the precipitate was dissolved in 66.7 mmol/L phosphate buffer (pH 6.0) and dialyzed using a 7-kDa dialysis bag at room temperature overnight.

2.5. Stability of the CGTase and thermokinetic parameter calculations A 1.0-ml CGTase solution with stabilizer was stored at 4, 25, 37, 40, 50, 60, 70, 80, and 90 °C for sampling at intervals. Samples (100 μL) were collected every 7 d at conditions of 4, 25, and 37 °C, and every 15 min at conditions of 40, 50, 60, 70, 80, and 90 °C for the determination of CGTase activities. And CGTase samples were taken at intervals to determine CGTase activities according to method of “Measurement of CGTase activities”. The CGTase activities without stabilizer at 50 °C were used as 100% relative activity. Tm was the temperature of denaturation midpoint. Tm was calculated through following process. The CGTase activities from 60 °C to 90 °C were fitted to obtain quadratic equations. And the Tm was determined through these quadratic equations. A first-order kinetic model by heat inactivation was obtained using Eq. (1). Eqs. (2) and (3) were used for the KD calculation. KD

2.3. Optimization of chemicals addition on activities of CGTase

N ⎯→ ⎯ D

(1)

d [N ] = −KD [N ] dt

(2)

[N0]=[Nt ] + [D]

(3)

where N represents the active CGTase; D represents the inactive CGTase; and KD (d−1) represents the first-order inactivation constant. Half-life (t 1 ) was calculated using Eq. (4).

Optimization of chemicals was conducted in two steps. The first step was chemical addition to a CGTase solution with 1.0 mL final volume. Glycerol, PEG400, and ethylene glycol were added respectively to 0.5 mL of a CGTase solution at final concentrations of 1%, 5%, 10%, 20%, and 30% (w/v). Gelatin, chitosan, and mycose were added respectively to 0.5 mL of a CGTase solution at final concentrations of 0.2, 0.5, 1.0, 2.0, and 3.0 g/mL. CaCl2, MgSO4, and NaAC were added respectively to 0.5 mL of a CGTase solution at final concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 mmol/L. CGTase activities were determined after a 2-h cultivation in a water bath at 60 °C. The second step was a three-factor and three-level orthogonal experimental design to optimize the proportions of glycerol (15%, 20%, and 25%; w/v), PEG400 (5.0%, 10.0%, and 15.0%; w/v), and CaCl2 (0.5, 1.0, and 1.5 mmol/L) in a CGTase solution with 1.0 mL final volume. Nine trials were conducted, with β- and γ-CGTase activities as responses. The CGTase activities were determined after a 2-h cultivation at 60 °C in a water bath.

2

0.693 t1 = 2 KD

(4)

The decimal reduction time (t) was calculated using Eq. (5).

log10

[Nt ] t =− [N0] D

(5)

The energy of inactivation was calculated using Eqs. (6) and (7).

nKD = lnA −

Ea RT

KD = Ae−Ea/ RT

(6) (7)

The frequency factor A (/d) is a parameter related to the total number of collisions that took place during the reaction. Ea (kJ/mol) represents the energy of inactivation. R (kJ/(mol K)) represents the universal gas 3

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Fig. 3. Effect of chemical stabilizers on stability of CGTase (a: β-CGTase activities at 4, 25, 37 °C without and with stabilizer; b: γ-CGTase activities at 4, 25, 37 °C without and with stabilizer; c: β-CGTase activities at 40, 50, 60. 70, 80, 90 °C without stabilizer; d: β-CGTase activities at 40, 50, 60. 70, 80, 90 °C with stabilizer; e: γ-CGTase activities at 40, 50, 60. 70, 80, and 90 °C without stabilizer; f: γ-CGTase activities at 40, 50, 60. 70, 80, and 90 °C with stabilizer).

absorbance was measured at 630 nm after the addition of 2.0 mL of 100 mmol/L citrate buffer (pH 4.2). β-CD and γ-CD contents were calculated according to standard curves of β-CD and γ-CD standards. Conversion ratio was calculated as the percentage of yield of both β-CD and γ-CD from initial starch.

constant, and T (K) represents the absolute temperature.

2.6. CD production from potato starch by the recombinant CGTase The reaction system consisted of 1.0% potato starch and 10.0 μL of a CGTase solution with stabilizer in 66.7 mmol/L phosphate buffer, pH 6.0. The reaction was conducted at 40, 50, 60, 70, 75, 80, and 90 °C using a 1.0-mL reaction system. β-CD yield of sample was analyzed every 30 min after the reaction was stopped by heating at 100 °C for 10.0 min. Subsequently, 0.5 mL of 0.02% (w/v) phenolphthalein was added to the sample and incubated at room temperature for 15 min, and the absorbance was measured at 550 nm. For γ-CD yield determination, 100 μL of a 5 mmol/L bromocresol green solution was added to the sample and incubated for 20 min at room temperature and the

2.7. Measurement of CGTase activities To measure the β-CGTase activity, the recombinant CGTase solution (100 μL) was mixed with 1 mL of a 4% starch buffer solution (pH 6.0) and incubated in a water bath at 50 °C for 20 min. The reaction was stopped by adding 3.5 mL of 30 mmol/L NaOH. Subsequently, 0.5 mL of 0.02% (w/v) phenolphthalein was added to the mixture and incubated at room temperature for 15 min, and the absorbance was measured at 4

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Table 1 Thermokinetic parameter analysis of β-CGTase and γ-CGTase. Temp (°C) β-CGTase activity

β-CGTase activity with stabilizer

γ-CGTase activity

γ-CGTase activity with stabilizer

KD (/min) t1/2 (min) Ea (kJ/mol) KD (/min) t1/2 (min) Ea (kJ/mol) KD (/min) t1/2 (min) Ea (kJ/mol) KD (/min) t1/2 (min) Ea (kJ/mol)

4

25

37

40

50

60

70

80

90

4.0 × 10−6 1.7 × 105 117.4 4.0 × 10−7 2.5 × 106 132.1 5.0 × 10−6 2.0 × 105 115.71 6.0 × 10−7 1.7 × 106 125.3

8.0 × 10−6 8.7 × 104

2.0 × 10−5 3.5 × 104

7.0 × 10−4 8.7 × 102

3.9 × 10−3 1.4 × 102

2.6 × 10−2 26.7

3.4 × 10−2 20.4

7.2 × 10−2 9.7

8.9 × 10−2 7.8

1.0 × 10−6 1.0 × 106

1.0 × 10−6 1.0 × 106

1.9 × 10−4 5.31.0 × 103

1.1 × 10−3 909.1

3.1 × 10−3 322.6

1.3 × 10−2 78.1

2.2 × 10−2 44.6

4.0 × 10−2 24.6

1.0 × 10−5 1.0 × 105

2.0 × 10−5 5.0 × 104

1.4 × 10−3 1.0 × 103

3.9 × 10−3 666.7

3.1 × 10−2 32.1

7.5 × 10−2 13.3

9.1 × 10−2 11.1

9.4 × 10−2 10.7

9.0 × 10−6 1.1 × 105

3.0 × 10−4 3.3 × 103

1.1 × 10−3 555.6

2.1 × 10−3 243.9

3.8 × 10−3 322.6

5.2 × 10−2 19.1

5.7 × 10−2 17.5

0.1 7.7

Fig. 4. CD production by CGTase with stabilizer at different temperatures (a: without stabilizer; b: with stabilizer).

Fig. 5. Conversion ratio increased by CGTase at the condition of stabilizer addition (a: conversion ratios at different temperatures; b: β-CD percentages at different temperatures).

550 nm. One unit of β-CGTase was defined as the amount of β-CGTase required to produce 1 μmol β-CD per min at 50 °C. To evaluate the γCGTase activity, 50 μL of the recombinant CGTase solution was mixed with 450 μL of a 4% starch solution (pH 8.0) and incubated at 50 °C for 20 min. The reaction was stopped by adding 500 μL of 100 mmol/L HCl. Subsequently, 100 μL of a 5 mmol/L bromocresol green solution was added to the mixture and incubated for 20 min at room temperature and the absorbance was measured at 630 nm after the addition of 2.0 mL of 100 mmol/L citrate buffer (pH 4.2). One unit of γ-CGTase was defined as the amount of γ-CGTase required to produce 1 μmol γ-CD per min at 50 °C.

3. Results and discussion 3.1. Effects of chemicals on CGTase activity The effects of polyols (glycerol, PEG400, and ethylene glycol) on βCGTase and γ-CGTase activities are shown in Fig. 1a and b, respectively. The results showed that with increasing concentrations of glycerol and ethylene glycol (up to 20% v/v), the β-CGTase activity increased by 9.8%, 65.4%, and 35.1%, respectively, and the γ-CGTase activity increased by 7.8%, 66.7%, and 39.0%, respectively, compared with that of control. The β-CGTase activities remained stable as glycerol and ethylene glycol concentrations increased from 20% to 30%. While the γ-CGTase activities decreased slightly as glycerol and ethylene glycol increasing from 20% to 30%. In the case of PEG400, the βCGTase and γ-CGTase activities increased by 50.4% and 55.3% compared with that of control at the condition of 10% PEG400. And the residual activities of β-CGTase remained stable, while the residual

2.8. Data analysis All experiments were conducted in triplicate. The relative CGTase activity was defined as the percentage of its initial value. The data was processed by Excel software (Microsoft, Redmond, WA, USA). 5

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activity of γ-CGTase decreased slightly when the PEG400 concentration was higher than 10%. In summary, with 20% glycerol, 10% PEG400, and 20% ethylene glycol, the β-CGTase and γ-CGTase activities increased by 6.5-, 5.2-, and 3.5-fold and 6.7-, 5.7-, and 3.9-fold, respectively, compared with that of control. The effects of gelatin, chitosan, and mycose on the β-CGTase and γCGTase activities are shown in Fig. 1c and d, respectively. With increasing gelatin concentrations, both the β-CGTase and γ-CGTase activities showed bell-shaped curves, with the highest increases of activities of 48.9% and 38.6%, respectively, at 1.0% gelatin concentration. The β-CGTase and γ-CGTase activities increased as the chitosan and mycose concentrations increased to 2.0%. Additionally, the β-CGTase activity fluctuated, and the γ-CGTase activity decreased slightly at chitosan and mycose concentrations of higher than 2%. The highest βCGTase activities (37.0% and 37.7% higher than that of control) were obtained at 3.0% chitosan and 2.0% mycose, respectively, which were 5.0- and 3.9-fold higher, respectively, than that of control. The highest γ-CGTase activities (45.5% and 43.1% higher than that of control) were obtained at 2.0% chitosan and 2.0% mycose concentrations, and they were 5.9- and 5.5-fold higher, respectively, than that of control. The effects of CaCl2, MgSO4, and NaAC on the β-CGTase and γCGTase activities are shown in Fig. 1e and f, respectively. All the βCGTase activities and γ-CGTase activities demonstrated bell-shaped curves in the ranges of 0–4.0 mmol/L CaCl2, MgSO4, and NaAC. The highest increasement in β-CGTase activities were 54.3%, 46.4%, and 46.9% at 1.0 mmol/L CaCl2, 2.0 mmol/L MgSO4, and 1.0 mmol/L NaAC, respectively, which were 5.6-, 4.8-, and 5.8-fold higher, respectively, than that of control. The highest increasement in γ-CGTase activities were 55.5%, 45.9%, and 39.1% at 1.0 mmol/L CaCl2, 3.0 mmol/L MgSO4, and 2.0 mmol/L NaAC, respectively. And they were 7.2-, 5.9-, and 5.0-fold higher, respectively, than that of control.

92.2%, 64.2%, and 9.8%, and 89.0%, 65.9%, and 7.8%, respectively, of initial value after 120 min. At temperatures higher than 60 °C, the βCGTase and γ-CGTase activities decreased sharply within 15 min, and decreased continually to 0–5.0% of the initial activities after 120 min without stabilizer (Fig. 3c and e). With stabilizer, the CGTase activities were higher than that of without stabilizer at every temperature group (Fig. 3a, b, d, and f). Notably, the β-CGTase and γ-CGTase activities remained stable for 37 d at 4, 25, and 37 °C, while their activities decreased continually without stabilizer. The highest increasement of CGTase activity was obtained at 60 °C, as the β-CGTase and γ-CGTase activities increased from 9.7% and 7.8% to 73.3% and 65.3%, respectively, of initial value. The thermokinetics parameters of CGTase were analyzed to investigate the effect of stabilizer on CGTase stability. For the β-CGTase activity, the KD decreased significantly from 4.0 × 10−6, 8.0 × 10−6, 2.0 × 10−5, 7.0 × 10−4, 3.9 × 10−3, 2.6 × 10−2, 3.4 × 10−2, 7.2 × 10−2, and 8.9 × 10−2 min−1 without the stabilizer to 4.0 × 10−7, 1.0 × 10−6, 1.0 × 10−6, 1.90 × 10−3, 1.1 × 10−3, 3.1 × 10−3, 1.3 × 10−2, 2.2 × 10−2, and 4.0 × 10−2 min−1 at 4, 25, 37, 40, 50, 60, 70, 80, and 90 °C, respectively, with the stabilizer (Table 1). For the γ-CGTase activity, the KD decreased significantly from 5.0 × 10−6, 1.0 × 10−5, 2.0 × 10−5, 1.4 × 10−3, 3.9 × 10−3, 3.1 × 10−2, 7.5 × 10−2, and 9.1 × 10−2, 9.4 × 10−2 min−1 without the stabilizer to 6.0 × 10−7, 9.0 × 10−6, 3.0 × 10−4, 1.1 × 10−3, 2.1 × 10−3, 3.8 × 10−3, 5.2 × 10−2, 5.7 × 10−2, and 0.1 min−1 at 4, 25, 37, 40, 50, 60, 70, 80, 90 °C, respectively, with the stabilizer (Table 1). The t1/2 of β-CGTase and γ-CGTase increased significantly with the addition of the stabilizer. The Ea of the β-CGTase and γCGTase activities increased from 117.4 kJ/mol and 115.7 kJ/mol to 132.1 kJ/mol and 125.3 kJ/mol, respectively, with the addition of the stabilizer (Table 1). The Ea of the β-CGTase activities increased by 14.7 kJ/mol which was higher than that of trypsin (9.9 kJ/mol) by glycol chitosan addition [22]. The increase of Ea was attributed to the compact structure of the CGTase with stabilizer, because a salt bridge of the CGTase is more important for stability than other interactions according to a molecular dynamics simulation [29]. Glycerol and PEG400 increased the hydrophobic interactions of a CGTase, which increased its stability [21]. For example, the stability of B. agaradhaerens KSU-A11 CGTase was enhanced significantly in the presence of 10 mmol/L CaCl2 [12]. It had been shown that sugars and polyols strengthen hydrophobic interactions among non-polar amino acid residues, leading to protein rigidification and resistance to thermodeactivation [21]. Costa et al. showed that glycerol and polyethylene glycol increased the stability of catalase [30]. Metal ions also stabilized enzymes, although different enzymes need different metal ions [31]. For example, calcium and zinc changed a protease structure drastically by increasing the proportion of random coil conformation, as well as the enzyme stability and activity [32]. Several calcium binding sites in a CGTase [33] lead to a compact CGTase structure under hydrophilic conditions [34]. As shown in Fig. 3e and f, the stabilizer increased the β-CGTase and γ-CGTase activities by 73.3% and 65.3%, respectively, which was higher than the activities resulting from the addition of any single chemical. This indicates that the stabilizer had a synergistic effect on CGTase stability. The synergistic effect of stabilizer was attributed to the better hydrogen bonds formation because of different sizes of glycerol, PEG400 and Ca2+, which was termed preferential hydration [35].

3.2. Orthogonal experimental design for chemicals optimization A three-factor and three-level orthogonal experimental design was conducted based on the results shown in Fig. 1. The results of the orthogonal experiment are shown in Table S1 and Table S2, with the highest increasement of β-CGTase activity of 73.8% and the highest increase of γ-CGTase of 73.1% with 25% glycerol, 10% PEG400, and 0.5 mmol/L CaCl2. The β-CGTase and γ-CGTase activities were 7.6- and 9.4-fold higher, respectively, than that of control. The mixture of 25% glycerol, 10% PEG400, and 0.5 mmol/L CaCl2 was subsequently used as the stabilizer. 3.3. Effect of temperature on activities of CGTase The β-CGTase and γ-CGTase activities showed bell-shaped curves in the range of 40–90 °C with and without stabilizer (Fig. 2a and b). Without stabilizer, the highest β-CGTase and γ-CGTase activities were obtained at 50 °C. With stabilizer, the optimum temperature of the βCGTase and γ-CGTase activities increased to 60 °C, with the highest increases of β-CGTase and γ-CGTase activities of 110.3% and 107.9%, respectively. The Tm values of the β-CGTase and γ-CGTase activities increased from approximately 70.2 °C to 88.1 °C and from 64.5 °C to 76.0 °C, respectively. 3.4. Effect of stabilizer on activities of CGTase The CGTase activities at different temperatures are shown in Fig. 3. The β-CGTase and γ-CGTase activities decreased continually without stabilizer (Fig. 3a–c, and e). Higher temperatures led to greater decreasement of CGTase activities, as the β-CGTase activity decreased to 79.3%, 65.6%, and 36.9% of the initial activities at 4, 25, and 37 °C, respectively, after 37 d (Fig. 3a). The γ-CGTase activities decreased to 75.5%, 53.1%, and 26.7%, respectively, of initial value (Fig. 3b). At 40, 50, and 60 °C, the β-CGTase and γ-CGTase activities decreased to

3.5. CD production by the CGTase at different temperatures The CD production process was conducted in the range of 40–90 °C. With and without the stabilizer, the starch to CD conversion ratio increased over time (Fig. 4). The conversion ratio decreased as the temperature increased from 40 to 90 °C. The highest conversion ratio was 28.24% which was obtained at 40 °C, although the CGTase activity was higher at 60 °C because the CGTase was more stable at 40 °C than 60 °C. 6

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With the stabilizer, the conversion ratio was higher than that without the stabilizer (Fig. 4b). In the presence of stabilizer, the highest CD production yield, a 21.9% increase, was obtained at 60 °C. CD production increased by 25% at 90 °C, while it increased by approximately 10% at 40, 50, and 70 °C (Fig. 5a). The percentages of β-CD and γ-CD did not change after stabilizer addition, although the conversion ratio increased with increasing temperatures (Fig. 5b). It is also of interest to obtain high-purity β-CD during CD production [36]. At high temperatures with the stabilizer, the increasing β-CD percentage indicates the potential for high-purity β-CD production. In this research, the β-CD purification process was still needed because of no 100% β-CD produced [37]. In conclusion, a chemical mixture for CGTase stability enhancement was established and used as a stabilizer. The components of this stabilizer were 25% glycerol, 10% PEG400, and 0.5 mmol/L CaCl2. The βCGTase and γ-CGTase activities remained stable for many days under the storage conditions. The higher stability of the CGTase was attributed to the increased Ea following stabilizer addition. For CD production, the CGTase with stabilizer showed a higher CD production yield, which increased by 25% at 90 °C, and the highest production yield, a 21.9% increase, was obtained at 60 °C.

[12]

[13]

[14] [15]

[16]

[17]

[18]

[19]

[20]

Acknowledgements

[21]

Project supported by the National Natural Science Foundation of China (No. 21306112), Shanghai Municipal Natural Science Foundation (No.13ZR1429100), The Innovation Fund Project for Graduate Student of Shanghai. All authors declare they have no other competing interests.

[22]

Appendix A. Supplementary data

[25]

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.procbio.2018.02.006.

[26]

[23]

[24]

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