Integrated Biocatalytic Process for Trehalose Production and

Article pubs.acs.org/IECR

Integrated Biocatalytic Process for Trehalose Production and Separation from Maltose Xiaogang Song,† Susu Tang,‡ Ling Jiang,*,# Liying Zhu,⊥ and He Huang*,† †

College of Biotechnology and Pharmaceutical Engineering, ‡Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), #College of Food Science and Light Industry, and ⊥College of Chemical and Molecular Engineering, Nanjing Tech University, Nanjing 210009, People’s Republic of China ABSTRACT: To develop cost-effective, biobased, industrial trehalose production from maltose, an integrated bioprocess for trehalose production and separation from maltose with recombinant trehalose synthase in permeabilized cells is proposed in this study. We have successfully established an efficient production system for recombinant trehalose synthase (TreS) (9234 U/mL) in a 50 L fermentor and efficient processes for separation and purification to achieve comprehensive utilization of the raw material and products. The trehalose conversion rate of 75% at 30 °C can be reached with 6% glucose generated as byproduct using maltose syrup (30 wt %) as substrate in the bioreactor system via whole-cell biocatalysis. After two-stage simulated moving bed chromatography (SMB), the trehalose could be successfully separated, crystallized, and identified with a 97.6% purity and 95.9% recovery yield, respectively. The yield of trehalose produced was 0.675 g per gram of maltose consumed in the whole process within 80.5 h.

1. INTRODUCTION Trehalose (α-D-glucopyranosyl α-D-glucopyranoside) is a unique sugar capable of protecting biomolecules against environmental stress. Trehalose is a stable, colorless, odorfree, and nonreducing disaccharide, which is widespread in nature.1,2 Trehalose plays a key role in the survival of some plants and insects, termed anhydrobionts, in harsh environments, even when most of their body water is removed.3 The properties of these types of organisms drove attention toward the study of trehalose. Trehalose proved to be an active stabilizer of enzymes, proteins, biomasses, pharmaceutical preparations, and even organs for transplantation. Recently, trehalose has been accepted as a safe food ingredient by the European regulation system following approval by the U.S. Food and Drug Administration. The wide range of applications of this sugar has increased the interest of many research groups in the development of novel and economically feasible production systems. Approximately 30,000 tons/year of trehalose is being produced utilizing this synthesis procedure, and trehalose can be found in over 8000 products in the food, cosmetic, and pharmaceutical fields.4 Currently, Hayashibara Biochemical Laboratories is the major producer of trehalose raw material, and Ferro Pfanstiehl (Waukegan, IL, USA) and Senn Chemicals AG (Dielsdorf, Switzerland) provide purified trehalose.3 However, information about their purification procedures is limited. Traditionally, there are many methods for the separation of trehalose, such as high-performance thinlayer chromatography (HPTLC),5 hydrophilic interaction chromatography (HILIC),6 and high-performance anionexchange chromatography (HPAEC),7 which are not suitable © XXXX American Chemical Society

for wholesale industrialization due to low separation efficiency and degree of automation. Among them, chromatographic techniques such as simulated moving bed (SMB) chromatography have been successfully applied in the food industry, particularly for sugar mixtures.8−10 Al-Eid11 used a chromatographic column filled with cation-exchange resin to separate fructose and glucose from date syrup. Lee12 used a two-section SMB to separate glucose and fructose from an aqueous mixture at a high concentration of 500 mg/mL. However, separation of trehalose on SMB has not been previously reported. Trehalose production has progressed from the gram scale to tons through the development of novel synthesis techniques. Trehalose can be isolated from yeast by alcohol extraction.13 Chemical synthesis involving the use of O-acetyl D-glucose has also been demonstrated.14 None of these methods were successfully implemented for large-scale production due to cost. Various enzymatic processes of trehalose synthesis have recently been reviewed.15 Three main pathways specifying the biosynthesis of trehalose have been identified in various organisms. Among them, the trehalose synthase (TreS) (EC 5.4.99.16) pathway (Figure 1) is more efficient, involving the direct conversion of maltose into trehalose by an intramolecular rearrangement of the α-1,4 linkage of maltose to the α-1,1 linkage of trehalose.16 This pathway allows one-step formation of trehalose, and an inexpensive substrate, maltose, is Received: June 12, 2016 Revised: August 7, 2016 Accepted: September 19, 2016

A

DOI: 10.1021/acs.iecr.6b02276 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

Article

Industrial & Engineering Chemistry Research

Figure 1. Biosynthesis reaction of trehalose by trehalose synthase pathway and physicochemical properties of product (trehalose), byproduct (glucose), and substrate (maltose).

process for trehalose production and separation from maltose with recombinant trehalose synthase in permeabilized cells is proposed. In addition, we adopt the whole-cell catalysis with lower levels of impurities and endotoxin compared to crude enzyme after cell lysis, which can reduce the difficulty of the downstream trehalose purification. In this sense, large-scale fermentation of trehalose synthase, enzyme-catalyzed trehalose synthesis from maltose, and separation by SMB and crystallization of trehalose were here performed considering the above-mentioned strain and downstream process. The cell permeabilization and industrial process optimization analysis by intelligent visualization software were also investigated. Compared with other processes for trehalose production reported in the literature, we have significant innovation advantages via this strategy. Specifically, first, we use maltose syrup with high purity as raw material to eliminate impurities from the source and reduce the difficulty of the separation. At the same time, China is the world’s largest producing area of maltose, which has huge advantages of raw materials. Second, the trehalose synthase used here is a novel one with the highest conversion yield compared with others reported in the literature, and we use whole-cell catalyst technology to reduce the difficulty of the subsequent separation and improve the production efficiency. Third, we choose high automatic SMB combined with crystallization technology for the purification of trehalose instead of the exchange resin separation. It is characterized by low pollution, high efficiency, simple technique, and low-cost advantages, which are suitable for industrial production.

employed, which provides a simple, fast, and low-cost method for the future industrial production of trehalose. Various TSases from different species have been isolated and characterized.17−28 However, these TSases were still not satisfactory in practical application with regard to either their activities or conversion efficiency. Recently, we successfully cloned a novel recombinant trehalose synthase (TreS) from uncultivable microorganisms with a high catalytic efficiency of 78% trehalose bioconversion yield at high maltose concentration (30 wt %).29 The purified recombinant enzyme (TreS) could catalyze the reversible interconversion of maltose and trehalose without other disaccharides, including isomaltulose. The recombinant TreS exhibited a 4.1-fold higher catalytic efficiency (Kcat/Km) for maltose than for trehalose and could be used effectively as a potential biocatalyst for the production of trehalose from maltose in a one-step reaction. Usually, the complete process from fermentation to purification is short; most studies on trehalose production focus on the upstream processes, such as enzyme modification by genetic engineering or fermentation for enzyme production and enzyme immobilization,30,31 but these works aim to improve the enzyme activity and conversion efficiency. As for the difficulties encountered in the separation of trehalose from other disaccharides and monosaccharides, the enzymatic processes, such as separation, cannot be easily achieved with conventional separation operations, such as extraction, adsorption, or precipitation, due to the similarity in their structural and physicochemical properties (Figure 1). Although some methods involving analytical liquid column chromatography32 for the separation of maltose and glucose or converting glucose to other products, such as bioethanol,33 glucose acid,34 and sorbitol,35 have been reported, they may not be costeffective for the separation of trehalose from maltose and glucose on an industrial scale. Therefore, to make the trehalose product more economically attractive, it is urgently necessary to develop complete technology and to search for suitable separation and purification processes to achieve comprehensive utilization of the raw material and products. In this study, an integrated

2. MATERIALS AND METHODS 2.1. Materials. All saccharides, including maltose and trehalose, were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The protein assay reagents and dyes were from Bio-Rad Laboratories. Columns for protein separations were obtained from Pharmacia Biotech. Other chemicals and reagents were of analytical grade. The cultivation medium of the large-scale fermentor system contained (per L, pH 7.0) 4 g of (NH4)2·HPO4, 13.5 g of KH2PO4, 1.39 g of MgSO4·7H2O, 2.7 g of NH4Cl, 3 g of yeast B


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to our previous preliminary experiment (data not shown), and the cell pellets were collected by centrifugation, washed twice with 50 mM (pH 7.0) phosphate buffer, and resuspended in a half volume of 50 mM (pH 7.0) phosphate buffer. The bacterial suspension containing permeabilized cells was kept at 4 °C for further trehalose production using a bioreactor system in recycle use. For further trehalose production, all experiments used highmaltose syrup as a substrate and were performed in a 50 L fermentor as the bioreactor system. The bioconversion reaction was started by adding pretreated permeabilized cells containing trehalose synthase into 40 L of high-maltose syrup (30 wt %) with a 100 rpm agitation rate at 30 °C for 0−24 h to optimize the weight conversion of trehalose. After the reaction solution was collected by centrifugation for further separation and purification of trehalose, the permeabilized cells were repeatedly used 10 times, and then the permeabilized cells were utilized for protein feed. The trehalose conversion rate was calculated by high-performance liquid chromatography (HPLC) analysis.

extract, 1.89 g of citric acid, and 8.8 g of glucose. The supplementary carbon source contained (per L) 660 g of glucose and 18 g of MgSO4·7H2O. The supplementary nitrogen source contained (per L) 300 g of yeast extract. All media were sterilized by autoclaving at 121 °C and 15 psig for 20 min. 2.2. Bacterial Strains and Culture Conditions. The transformant of the engineered Escherichia coli strain BL21 (DE3) (Novagen), containing a new trehalose synthase (TreS) gene, was constructed previously by our laboratory.29 All recombinant E. coli strains were grown on Luria−Bertani (LB) medium, and cell densities were measured with a spectrophotometer at 600 nm (OD600). For the production of trehalose synthase, recombinant strains were grown in LB medium containing 100 g/mL ampicillin (Bio basic, USA) at 37 °C for 12 h and were then inoculated into shake flasks containing fresh LB medium. The shake flask cultures were placed in an orbital shaker set at 200 rpm. When the OD600 reached 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1.0 mM. The incubation was continued for another 12 h at 30 °C, and the cells were harvested from the culture broth by centrifugation at 12000g for 10 min. The collected cell pellets were washed with 50 mM (pH 7.0) phosphate buffer and were subsequently resuspended and stored at 18 °C until use. 2.3. Fermentation for Trehalose Synthase Production. The fermentation of trehalose synthase included three levels of culture: (i) shake flask, (ii) level 1 fermentor, and (iii) level 2 fermentor. E. coli cells were cultivated overnight and were then inoculated into shake flasks containing fresh LB medium with 100 g/mL ampicillin at 37 °C overnight in an orbital shaker set at 200 rpm. Then, the production level was increased to a 15 L fermentor system (working volume = 10 L) with 2 vvm (air volume/culture volume/min) aeration and an agitation speed of 300 rpm at 37 °C. The pH was controlled at 7.0 by adding NH4·OH with an online sensing and dosing system. After the cell density reached 10, according to a 5% inoculum dose, the cell broth was inoculated into the 50 L fermentor system (FUS50L, GuoQiang, China) with a working volume of 30 L, which was the same operation as in the 15 L fermentor system. When the OD600 value of the cell broth reached 10, IPTG was added to induce at 30 °C for 20 h. During induction, the addition of carbon source and organic nitrogen source was adjusted according to the glucose consumption and dissolved oxygen level by the dosing system. The cells were then harvested for the catalytic process. During the large-scale fermentation process, the OD600 value, dissolved oxygen, glucose consumption, and enzyme activity of the sampling culture broth were determined and recorded. The cells were harvested from the sampling culture broth by centrifugation at 12000g for 10 min. The collected cell pellets were resuspended in 50 mM (pH 7.0) phosphate buffer and were subsequently lysed by ultrasonication. The crude cell lysate containing the enzyme was collected by centrifugation at 12000g for 10 min for further analysis of the trehalose synthetic activity. 2.4. Enzymatic Preparation of Trehalose from Maltose in Permeabilized Cells Using a Bioreactor System. For the preparation of trehalose synthase in permeabilized cells, after being harvested from the fermentation broth by centrifugation and washed twice in 50 mM (pH 7.0) phosphate buffer, the cells were permeabilized by shaking a mixture of 1.5 g/L colistin sulfate for 72 min at 35 °C at 200 rpm, according

conversion of trehalose (%) wt (g) of trehalose production = × 100% wt (g) of total maltose in initial substrate

Furthermore, to optimize the enzymatic procedure, a threelevel, four-factor, orthogonal design was used, and the yield of trehalose was chosen as the index (Table 1). For each treatment above, two replications were performed for statistics analysis. Table 1. Factors and Levels of Orthogonal Experimental Design L9 (34) temperature (°C)

pH

time (h)

concentration of substrate (%)

level

A

B

C

D

1 2 3

30 40 45

5.5 6.0 7.0

16 20 24

25 30 35

2.5. Separation and Purification of Trehalose. The enzymatic conversion solution was further decolored by a twolayer filter containing diatomite and activated carbon, and the ion-exchange treatment was utilized to remove the salts and proteins of each filtrate in a series connection with both cationic (DIAION PK216, Taiwan) and anionic (DIAION WA30LL, Taiwan) exchange resin, as described by other groups.33,36 After the residual minerals and color were removed, the separation of trehalose from glucose and maltose in the sugar mixture solution was conducted with a two-stage separation system of SMB; the total column volume of the SMB device with eight columns is 10 L, and the size of each column is 50 cm × 5 cm (height × diameter). Purolite CGC 100 × 8 Ca ion-exchange gel-type resin was used to pack the column as the stationary phase, and deionized water was used as the eluent and solvent in all of the experiments. Finally, the trehalose-rich solution with high purity was further concentrated to 66−82% supersaturated trehalose liquid at 70 °C in a reliever and was crystallized by fed-batch by adding 99% ethanol during cooling crystallization in a crystallizer. The total amount of ethanol was 4-fold that of water in the trehalose supersaturated liquid. Crystalline trehalose was then obtained by filtration and was washed with a small amount of warm C


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Figure 2. Simplified flow sheet for integrated process of trehalose production and separation from maltose syrup. The whole technological scheme integrates fermentation, enzyme catalysis, separation, purification, crystallization, etc. SMB, simulated moving bed chromatography.

as the amount of enzyme that catalyzed the formation of 1 nmol of trehalose per minute under the assay conditions.

ethanol. Because residual ethanol might remain on the surface of the crystalline trehalose, the collected samples were dehydrated at 40 °C by vacuum-drying for 12 h for further yield calculations. For the product yield calculation, the purification step of the trehalose solution, including filtration, decolorization, ion exchange, SMB, and crystallization, was evaluated, and the yield of the product was defined as

trehalose synthase activity (U/mL) = × dilution fold ×

wt of crystal trehalose (g) × 100% total trehalose amount in enzymatic mixture

For the evaluation of the purity of the crystalline product, 0.5 M trehalose standard was prepared to compare with the same concentration of our crystalline sample and was analyzed by HPLC. The purity of crystalline trehalose was defined as purity of crystalline trehalose (%) =

1mL diluted enzyme amout (mL)

Quantitative analysis of the sugar was conducted with an HPLC system equipped with a refractive index detector (RID). Samples were injected into an HPLC system (Dionex, USA) equipped with an NH2 column (250 mm × 4.6 mm, Sepax, USA). The column was maintained at 35 °C and was eluted isocratically with a mobile phase consisting of acetonitrile and Milli-Q water at a volume ratio of 75:25 and a flow rate of 1.0 mL/min. The permeabilized cells were freeze-dried for transmission electron microscopy (TEM) observation. For the TEM studies, the permeabilized cells were double-stained with phosphotungstic acid (PTA) and were observed on a transmission electron microscope (JEM-2100, JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.

yield of crystalline trehalose (%) =

released trehalose (U/mL) reaction time (30 min)

concentration of crystalline sample (mM) × 100% concentration of trehalose standard (mM) × 0.99

3. RESULTS AND DISCUSSION For a large-scale system, the overall cost is greatly affected by the enzyme production and trehalose purification. A simplified and efficient purification procedure to separate trehalose from residual sugar mixtures with similar characteristics is another obstacle to overcome for better market competitiveness. To make the biocatalyst production of trehalose economically attractive, the development and analysis of new fermentation and downstream processes are required. In this study, an integrated process for the fermentation of recombinant trehalose synthase and concomitant trehalose production and separation from maltose is proposed, as shown in Figure 2. The performance of the biocatalyst obtained for the transformation of maltose into trehalose was also investigated. 3.1. Fermentation of the Recombinant Trehalose Synthase. The expression of our recombinant TreS production was estimated on the basis of a small-scale flask

The purity of the trehalose standard was 0.99. 2.6. Analytical Methods. The protein concentrations of all samples were analyzed by using the Bradford method with protein dye (Bio-Rad, USA) at 595 nm. SDS-PAGE with 12% polyacrylamide gel stained with Coomassie blue was used for the analysis of the protein purity, and the cell densities were measured with a spectrophotometer at a wavelength of 600 nm (OD600). The enzyme activity of trehalose synthase was assayed by measuring the trehalose produced from maltose. The reaction mixture consisting of 800 μL of 150 mM maltose solution in 50 mM sodium phosphate buffer (pH 7.0) and 200 μL of crude enzyme solution in a final volume of 1 mL was incubated at 30 °C for 30 min. Then, this reaction mixture was heated at 100 °C for 10 min to stop the reaction. The trehalose produced was analyzed by HPLC as described below and was measured using a standard curve. One unit (U) of enzyme activity was defined D


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Figure 3. Time course of cell growth and the trehalose synthase activity in a 50 L fermentor system at 30 °C for 24 h. The enzyme activity was analyzed by using 150 mM maltose as substrate at 30 °C for 30 min. Error bars show SD for n = 3.

experiment in our previous study,29 but it is necessary and practical to develop a functional system for the large-quantity production of our active recombinant enzyme for further industrial applications. Figure 3 shows the time course of the cell growth and the trehalose synthase activity in a 50 L fermentor system at 30 °C for 24 h. The level of glucose consumed during fermentation was measured by a commercialized SBA-40C biosensor (SBA-40C, Shandong, China). The glucose concentration declined gradually from 60 g/L, accompanied by a steady increase in cell density from the initial cell concentration. When cell growth reached the logarithmic phase at 8 h, trehalose synthase was induced, and the enzyme activity was measured throughout the course of the fermentation. The maximum OD600 value was 42 in the stationary phase of cell growth after 20 h. After this fermentation time, the increase of enzyme activity was very small, and the cells began to die. The highest activity (9234 U/ mL) of trehalose synthase was obtained at 30 °C for 24 h, which indicated that the expression of our recombinant TreS was stable and workable in a 50 L fermentor system. 3.2. Characterization of Permeabilized Cells. Because colistin sulfate can induce alterations in the permeability of the bacterial cytoplasmic membrane,37,38 after being permeabilized, the plasma membrane is damaged, whereas the morphology of the cells remains intact. However, low molecular weight molecules can freely enter and leave the cells.39 In the experiment, the expression of intracellular trehalose synthase activity in permeabilized cells increased 2.09-fold with respect to the untreated cells. To study the effect of permeabilization on cells, E. coli cells before and after permeabilization were compared by TEM. Figure 4 compares the micrographs of the treated and untreated cells, indicating that the shape of the cells did not change after permeabilization. Therefore, the trehalose synthase is embedded in the cells, and the substrate (maltose) and the product (trehalose) can freely enter and leave the cells. The trehalose synthase in freeze-dried permeabilized cells is then obtained. 3.3. Optimization of the Enzymatic Reaction Conditions for the Conversion of Trehalose from Maltose.

Figure 4. Transmission electron micrographs of Escherichia coli cells after permeabilization (a) as compared with those without treatment (b).

The effects of the reaction factors, that is, temperature, pH, and substances, on the enzyme reaction activity were investigated in our previous study.29 On the basis of the results of the previous study, an L9 (34) orthogonal experimental design was performed. Four independent variables were selected: A, temperature; B, pH; C, time; and D, concentration of substrate. Table 1 presents the details of the orthogonal experimental design, and the results are given in Table 2. Table 2 shows that the larger the range of the factor, the greater the effect of the factor on the yield. According to the range analysis of the orthogonal experimental design, the strength of the effect of the factors on trehalose yield was in the order pH > temperature > concentration of substrate > time, and the following results were observed: (a) 30 °C produced a higher yield than 40 and 45 °C; (b) the trehalose yield at pH 7.0 was higher than that at pH 6.0 and 5.5; (c) the yield of trehalose did not significantly change as the reaction time increased; and (d) a high concentration of substrate required a longer time to achieve the highest yield. In industry, high productivity requires a high substrate concentration. The optimum reaction conditions were identified as a temperature of 30 °C, pH 7.0, reaction time of 24 h, and concentration of substrate of 35%, under which a 75% yield of trehalose was obtained. To further predict the optimal process conditions, a novel visual method for industrial process optimization40,41 was used to analyze the trehalose preparation process conditions. Using E


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Industrial & Engineering Chemistry Research Table 2. Design of the Orthogonal Experiment and Results factor no. of

temperature (°C)

pH

time (h)

concentration of substrate (%)

conversion of

experiments

A

B

C

D

trehalose (%)

1 2 3 4 5 6 7 8 9 K1 K2 K3 k1 k2 k3 R

30 30 30 40 40 40 45 45 45 184 177 148 61.333 59.000 49.333 12.000

5.5 6.0 7.0 5.5 6.0 7.0 5.5 6.0 7.0 131 175 203 43.667 58.333 67.667 24.000

16 20 24 24 16 20 20 24 16

25 30 35 30 35 25 35 25 30

168 169 172 56 56.333 57.333 1.333

48 61 75 45 62 70 38 52 58

170 164 175 56.667 54.667 58.333 3.666

a

K, sum of yields at each level; k, average of K at each level; R, range, the difference between the maximal k and minimal k. The value is expressed as the mean (n = 3), and the SD is 65% conversion. 3.4. Separation of Trehalose from Sugar Mixtures by SMB. Because the bioconversion of high-maltose syrup was not complete for the enzymatic process, all final conversion solutions were complex mixtures containing multiple sugars and substances, such as trehalose, maltose, glucose, protein, and mineral. Therefore, it is difficult to obtain high-purity trehalose for further industrial applications. For this reason, we attempted to develop biotreatment methods to separate and purify trehalose into useful products. To obtain high-purity trehalose, the sugar mixture solution of 22.5% trehalose and residual sugars (i.e., glucose and maltose) has to be separated by SMB due to the similarity in their structural and physicochemical properties (Figure 1). Primary separation is used for monosaccharide and disaccharide purification, whereas secondary separation is used for maltose and trehalose purification. As the resolution of the two sugars was 0.59 in a typical chromatogram, which is not good enough to completely separate them, we therefore selected a highperformance resin as the stationary phase to separate trehalose and maltose in SMB. The potential of a cation-exchange resin was investigated as the stationary phase for separating trehalose and maltose, using deionized water as the mobile phase. On the basis of a previous paper43 about a single-column experiment and the equilibrium dispersive model, some parameters, including the column void, retention time, and adsorption isotherms, were applied to simulate the elution curves of trehalose and maltose. Under the linear conditions, the theoretical parameters in the triangular region of the complete separation of glucose, maltose, and trehalose for an SMB were predicted according to equilibrium theory. The theoretical parameters were constantly optimized in the actual operating stage. Optimized parameters are critical to the chromatographic separation of trehalose and maltose on an industrial scale. The effects of some operating conditions (switching time, extract and feed flow rates) were investigated in view of process optimization, and the process performance was analyzed through variables such as product purity, eluent consumption, and productivity. It was verified that there are combinations of operating variables under which high purity in the extract stream, good productivity, and low solvent consumption can be obtained.44 The optimal operating conditions of a two-stage separation system for the three components by SMB are shown in Table 3. On the basis of these typical operating conditions, the glucose and disaccharides (maltose and trehalose) were separated by primary separation of SMB; 98.8% purity glucose was obtained in raffinate, whereas the purity and yield of the disaccharides

Figure 6. Time course of maltose hydrolysis with the trehalose synthase in a batch reactor. Maltose hydrolysis was carried out at 30 °C and pH 7.0 with immobilized trehalose synthase in permeabilized cells. Error bars show SD for n = 3.

catalytic pockets of the enzyme, which leads to the transformation of a maltose molecule into two glucose molecules.19 The formation of glucose during the process has been shown to be irreversible and thus could lead to a decline in trehalose yield.19 The presence of glucose in the reaction mixture could further hamper trehalose synthesis by acting as a competitive inhibitor.23 The operational stability31,42 of trehalose synthase in permeabilized cells was evaluated by measuring the residual conversion of trehalose synthase in a batch reactor. On the basis of the time course shown in Figure 7, each cycle of

Table 3. Optimal Operating Conditions of SMB

Figure 7. Reusability of permeabilized cells in a batch reactor. Maltose hydrolysis was carried out at 38 °C and pH 7.0 with immobilized trehalose synthase in permeabilized cells. Error bars show SD for n = 3.

SMB

maltose hydrolysis was conducted at 30 °C for 24 h to ensure complete conversion of maltose. As shown in Figure 7, no observable decline in conversion was observed for the first 5 cycles, and the conversion of maltose to trehalose was maintained at 0.65 after 10 cycles. The decline in conversion is attributed to enzyme inactivation and/or desorption in permeabilized cells. On the basis of the trend of a steady, slight G

operating conditions

primary separation

secondary separation

operating temperature (°C) switching time (min) feed flow rate (mL/min) eluent flow rate (mL/min) extract flow rate (mL/min) raffinate flow rate (mL/min) recycle flow rate (mL/min)

58 11 13.8 29.7 20 23.5 17.6

50 3.9 1.0 5.6 3.6 3.0 12.4


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processing of maltose, and (iii) separation and purification of trehalose. For the upstream process of fermentation, trehalose synthase was produced through three levels of fermentation, and cell pellets containing trehalose synthase were obtained by centrifugation and washed twice with phosphate buffer. For the whole-cell biocatalysis with permeabilized cells, the catalytic process was performed in a 50 L fermentor system packed with immobilized permeabilized cells at pH 7.0 and 38 °C and fed with 40 L of high-maltose syrup (30 wt %) as the substrate for maltose hydrolysis. The operational stability of the immobilized enzyme in the permeabilized cells during the batch operations was investigated. After each cycle, the immobilized permeabilized cells were recovered by filtration for subsequent operations. The product mixture (40 L) containing 22.5% trehalose, 5.7% maltose, and 1.8% glucose was obtained in catalytic solution after the catalytic reactor and filter after the catalysis of immobilized permeabilized cells containing recombinant trehalose synthase. However, due to the similarity of their structural and physicochemical properties, this separation is not easily achieved by conventional separation operations, such as extraction, adsorption, and precipitation. Therefore, to further increase the purity of trehalose for food and therapeutic applications, it is necessary to develop an efficient process for the separation and purification of trehalose from residual maltose and glucose in the product streams. After sequential pretreatment of the sugar liquid, including decolorization with activated carbon, filtration of insoluble substances and carbon, ion-exchange treatment to remove salts and proteins, and concentration of the suspension by evaporation, to facilitate the separation of trehalose from glucose and unreacted maltose, SMB was studied as a novel separation method. Extraction solution 1 containing 98.8% glucose was used as the single-cell protein feed with the 11th batch cells after the primary separation of SMB, and the 99.5% disaccharide obtained in raffinate 1 (54.6 g/L maltose and 215.7 g/L trehalose) was further separated by secondary separation of SMB. The maltose in extraction solution 2 can be recycled, and the 97.6% trehalose-rich fraction, 205.4 g/L, in raffinate 2 was obtained as high-purity trehalose for further refinement by crystallization. After the high-purity trehalose was recrystallized with a 97.6% purity and a 99.2% yield in a crystallizer and dried in a dryer, approximately 8.1 kg of highpurity trehalose crystals was obtained. To sum up, 40 L of maltose syrup (30 wt %) can produce approximately 8.1 kg of trehalose for each batch, the trehalose productivity reached being 9.37 g/L/h during enzymatic catalysis process and the yield of trehalose produced being 0.675 g/g maltose consumed

(maltose and trehalose) in the extract solution were 99.5 and 95.9%, respectively. After the disaccharides obtained above were separated through secondary separation by SMB, the purity and yield of trehalose in raffinate were 97.6 and 95.2%, respectively. 3.5. Refinements for Crystalline Trehalose Production. Cooling and dilution crystallization coupling technology was used in this study. The trehalose-rich solution was easily crystallized by ethanol precipitation with 97.6% purity and 99.2% recovery of trehalose. White, single rhombus, trehalose crystals were obtained, as shown in Figure 8. The crystals had a

Figure 8. Optical photomicrographs of anhydrous trehalose crystals (×100 for Leica microscope).

smooth surface, clearly visible sides and edges, as found in a previous study,45 which indicates that cooling and dilution crystallization coupling technology does not change the crystal structure of trehalose. Compared with traditional cooling crystallization, cooling and dilution crystallization coupling technology can produce higher purity crystals with uniform and stable particle size distributions, avoiding fine crystals, and can increase the trehalose crystal growth rate, shorten the crystallization period, and improve the efficiency of crystal production. Therefore, this crystallization process is feasible for industrial production. 3.6. Integration Process Description. The whole technological scheme for trehalose production was divided into three main stages, as shown in Figure 2. These stages are (i) fermentation for trehalose synthase production, (ii) catalytic

Table 4. Summary of the Main Material Stream Results in Experimental Work for the Trehalose Production and Separation Process enzymatic catalysis process temperature (°C) mass flow (kg/h) water maltose trehalose glucose mineral

maltose syrup

catalytic solution

primary separation sugar liquid

extraction solution 1

secondary separation

raffinate 1

extraction solution 2

raffinate 2

30

30

30

58

58

50

50

693 300 0 0 7

693 57.0 225 18.0 7

693 57.0 225 18.0 0

28.7 16.6 0.763 0.193 0

671 54.7 216 1.41 0

32.2 50.2 2.90 1.29 0

639 4.51 205 0.124 0

H


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maltose syrup. To make the biocatalyst production of trehalose economically attractive, the development and analysis of new fermentation and downstream processes are required. Without any chemical addition, we expect that this efficient green bioprocess can be applied to maltose low-value agricultural produce for wide industrial application.

in the whole process. The total duration of the whole process is approximately 80.5 h in the first batch of immobilized cells, including 36 h of two-stage fermentation of microbial cells, 2 h of cell permeabilization, 24 h of enzymatic catalysis, 3.5 h of two-stage separation of trehalose on SMB, 10 h of crystallization of trehalose, 5 h of filtered wash, and so on. The subsequent continuous process can start with cyclic catalysis of immobilized cells instead of the fermentation of microbial cells, which can greatly save time and improve production efficiency. In brief, compared with the existing technology, it is characterized by a short process, mild reaction conditions, high yield, high purity of the products, and less waste. According to experimental results in each procedure combined with the material balance calculation and simulation analysis by Aspen Plus (Aspen Technologies Inc., USA) on the basis of the technological conditions and parameters adopted in the former pilot test, the main material stream results of the scale-up test were evaluated for enzymatic catalysis and twostage separation of SMB, as shown in Table 4. If the loss of twice the concentration by evaporation was ignored, for the whole trehalose production process, the production of trehalose from high-maltose syrup per ton was 203.7 kg with approximately 80.5 h in the first batch of immobilized cells through this integrated technique. Furthermore, the single-cell protein feed mixed from the waste cells and the glucose-rich solution can be utilized as high-value-added products in agriculture.

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AUTHOR INFORMATION

Corresponding Authors

*(L.J.) E-mail: [email protected]. Phone: +86-2558139942. Mail: College of Food Science and Light Industry, Nanjing Tech University, Nanjing 210009, PR China. *(H.H.) E-mail: [email protected]. Phone: +86-2558139942. Mail: College of Pharmaceutical Science, Nanjing Tech University, Nanjing 210009, PR China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation for Young Scholars of China (21225626, 21506101), the State Key Laboratory of Bio-organic and Natural Products Chemistry, CAS (SKLBNPC15429), the Six Talent Peaks Project in Jiangsu Province (2015-JY-009), and the Environmental Research Foundation of Jiangsu Province (2016053).

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REFERENCES

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4. CONCLUSIONS A preliminary integrated technique design and analysis of trehalose, including fermentation, enzyme catalysis, separation, purification, and crystallization, was performed for trehalose production from maltose syrup. Although an economic assessment was not conducted for this process, the largequantity production of the recombinant trehalose synthase in permeabilized cells to convert maltose into trehalose product in a 50 L fermentor system was achieved. The highest activity of the recombinant trehalose synthase reached 9234 U/mL, which resulted in a conversion of trehalose up to 75% with 6% glucose generated as a byproduct. By using the integrated biocatalytic process, the trehalose products could be successfully separated, crystallized, and identified with a 97.6% purity and 95.9% recovery yield, respectively. The yield of trehalose produced was 0.675 g per gram of maltose consumed in the whole process within 80.5 h. This work demonstrated the feasibility of recombinant enzyme-catalyzed trehalose synthesis by wholecell catalysis from maltose syrup in one step and the purification of trehalose using SMB. In conclusion, when the scale-up test of this integrated biocatalytic process is to be accomplished on the basis of the technological conditions and parameters adopted in the former pilot test, each ton of maltose syrup (30 wt % maltose) can produce approximately 203.7 kg of high-purity trehalose in addition to a large quantity of single-cell protein feed as a highvalue-added product simultaneously through this integrated technique within 80.5 h. Colistin sulfate, as a biosurfactant used for the permeabilized treatment of E. coli, is more environmentally friendly and effective compared with chemical reagents. These results suggest that the currently available strains and technologies could be significantly improved by bioengineering and process analysis tools (e.g., intelligent visualization software) for the production of trehalose from I


Article

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Integrated Biocatalytic Process for Trehalose Production and

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