Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii

Accepted Manuscript Title: Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii Author: Qian Liu Shuhuai Yu Tao Zhang Bo Jiang Wanmeng Mu PII: DOI: Reference:

S0144-8617(16)31332-7 http://dx.doi.org/doi:10.1016/j.carbpol.2016.11.057 CARP 11769

To appear in: Received date: Revised date: Accepted date:

3-10-2016 16-11-2016 19-11-2016

Please cite this article as: Liu, Qian., Yu, Shuhuai., Zhang, Tao., Jiang, Bo., & Mu, Wanmeng., Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.11.057 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Efficient biosynthesis of levan from sucrose by a novel levansucrase from Brenneria goodwinii

Qian Liua, Shuhuai Yua, Tao Zhanga, Bo Jianga, Wanmeng Mua,b,*

a

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, People’s Republic of China.

b

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, People’s Republic of China.

*

Corresponding author. Tel: +86 510 85919161. Fax: +86 510 85919161.

E-mail address: [email protected] (W. Mu). Research highlights

1. A novel levansucrase was characterized from B. goodwinii. 2. The polysaccharide biosynthesized from sucrose was determined to be levan. 3. The enzyme displayed maximal transfructosylation activity at pH 6.0 and 35 °C. 4. The enzyme produced 185 g/L levan from 50% (w/v) sucrose. 5. The molecular weight of the produced levan reached 1.3 × 108 Da.

1

ABSTRACT Levan, a unique homopolysaccharide consisting of fructose residues linked by β-(2, 6) bonds, possess promising physiochemical and physiological properties with numerous potential applications. In this study, a novel levan-producing levansucrase was characterized from Brenneria goodwinii. The polysaccharide produced by the recombinant enzyme from sucrose was structurally determined to be levan-type fructan connected by β-(2, 6) linkages. The optimum pH was measured to be pH 6.0 for both sucrose hydrolysis and transfructosylation. The optimum temperature was 35, 45, and 40 °C for transfructosylation, sucrose hydrolysis, and total activity, respectively. Higher sucrose concentration greatly favored to levan biosynthesis. The purified recombinant enzyme produced 185 g/L levan from 50% (w/v) sucrose at pH 6.0 and 35 °C for 12 h. The molecular weight of the produced levan polysaccharide reached 1.3 × 108 Da, which was much higher than the ones produced by many reported levansucrases.

Keywords Biosynthesis ∙ Brenneria goodwinii ∙ Fructan ∙ Levan ∙ Levansucrase

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Introduction Fructans are a group of naturally occurring polysaccharides composed of linear chains of fructose molecules, normally with a sucrose unit at the reducing end (Wang, Yu, Zhang, Jiang, & Mu, 2015). Inulin and levan are the two common types of fructans, in which the fructose chain is built by β-(2, 1) and β-(2, 6) fructosyl linkages, respectively. Fructans are commonly produced as a means of storing energy by organisms (Hendry, 1993). Generally, inulin is more extensive in plants (Zhu et al., 2016), however, microorganisms prefer to produce levan not only as an energy reserve (Daude, Remaud-Simeon, & Andre, 2012) but also as an important structural component for defense (Benigar et al., 2014). Levan-type fructan attracts increasing attention and has tremendous potential in food

and

pharmaceutical

industries

because

of

its

promising

physicochemical properties and physiological effects. As a kind of indigestible polysaccharide, levan has been considered as an ideal soluble dietary fiber (Peshev & van den Ende, 2014) with favorable prebiotic effects (Jang et al., 2003; Korakli, Pavlovic, Gänzle, & Vogel, 2003). It displays significant antioxidant effect and may effectively protect against oxidative stress linked atherosclerosis (Dahech et al., 2013). A recent study shows that levan exhibits great fibrinolytic activity and may be used as a strong fibrinolytic agent (Esawy et al., 2013). In addition, levan has many other beneficial properties, such as anti-obesity (Oh, Lee, Hwang, & Ji, 2014), hypocholesterolemic (Yamamoto et al., 1999), anti-inflammatory (Srikanth et al., 2015b), immunomodulatory (Xu et al., 2006), and anti-tumor effects (Yoo, Yoon, Cha, & Lee, 2004). Levan is also widely used in chemical industry due to its good physicochemical properties such as high molecular weight and low viscous nature (Srikanth, Reddy, Siddartha, Ramaiah, & Uppuluri, 2015a). It shows excellent tensile

3

and shear strength properties and can be used as a natural adhesive and surfactant (Barone & Medynets, 2007). It displays anti-inflammatory and cell-proliferative effects in skin and thus has a great potential as a cosmeceutical agent (Kim et al., 2005). Levan does not exist in plants abundantly, however, it is convenient to biologically produce levan from sucrose by microbial levansucrase (Srikanth et al., 2015a). Levansucrase (EC 2.4.1.10, sucrose:2,6-β-D-fructan 6-β-D-fructosyltransferase) is distributed in a wide range of microorganisms (Li, Yu, Zhang, Jiang, & Mu, 2015). Levan can be easily produced by microbial fermentation by many microorganisms harboring levansucrase activity, and can also be easily synthesized from sucrose by various levansucrases in crude, purified, recombinant, or immobilized forms (Li et al., 2015). So far, levan biosynthesis has been reported using levansucrases from many microorganisms, especially the Bacillus, Geobacillus, Lactobacillus, and Zymomonas species strains (Li et al., 2015; Srikanth et al., 2015a), such as Bacillus methylotrophicus SK 21.002 (Zhang et al., 2014a), Bacillus licheniformis RN-01 (Nakapong, Pichyangkura, Ito, Iizuka, & Pongsawasdi, 2013), Geobacillus stearothermophilus (Inthanavong, Tian, Khodadadi, & Karboune, 2013), Bacillus amyloliquefaciens (Tian, Inthanavong, & Karboune, 2011), Lactobacillus reuteri (van Hijum, Szalowska, van der Maarel, & Dijkhuizen, 2004), Lactobacillus panis (Waldherr, Meissner, & Vgel, 2008), Lactobacillus sanfranciscensis (Tieking et al., 2005), and Zymomonas mobilis (Sangiliyandi, Raj, & Gunasekaran, 1999). In this study, a levansucrase was identified from a new strain, Brenneria goodwinii, with a high levan-producing activity. The enzyme was heterologously expressed in Escherichia coli and purified by metal-affinity chromatography and the enzymatic properties were studied and compared with other reported ones. The biosynthesized

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polysaccharide product was structurally determined to be levan. In addition, the optimization of enzymatic levan biosynthesis by B. goodwinii levansucrase was preliminarily studied. To our best knowledge, it is the first report on the levan biosynthesis by levansucrase from a Brenneria species strain.

2. Materials and methods 2.1. Cloning and expression of B. goodwinii levansucrase B. goodwinii genomic DNA has recently been completed and deposited in GenBank database

(accession

No.

CGIG01000001.1),

harboring

a

putative

levansucrase-encoding gene (locus_tag: BN1221_00994c, protein ID: CPR14579.1). The full-length nucleotide sequence of the levansucrase-encoding gene, linked with a 6×histidine-tag sequence at 3’-terminal, was commercially synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China) without codon modification and inserted into pET-22b(+) expression vector with NdeI and XhoI restriction sites in 5’- and 3’-terminal of the fusion gene, providing a recombinant plasmid, termed pET-Brgo-Lev. The recombinant plasmid was then transformed into host E. coli BL21(DE3). For levansucrase expression, the E. coli BL21(DE3) harboring pET-Brgo-Lev was grown in 400 mL LB broth containing 100 μg/mL ampicillin at 37 °C. When the optical density at 600 nm reached 0.6, the culture was induced with 0.2 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and then grown at 28 °C for another 6 h.

2.2. Purification of the recombinant B. goodwinii levansucrase Cells were harvested by centrifugation at 4 °C and 15,000 g for 20 min, washed twice with lysis buffer (50 mM sodium phosphate buffer, 100 mM NaCl, pH 7.4),

5

resuspended in the same buffer, and then disrupted by sonication on ice for 10 min (pulse for 1 s with interval for 2 s) using a Vibra-Cell 72405 Sonicator (Bioblock, Illkirch, France). Cell debris was removed by centrifugation at 20,000 g for 30 min. The recombinant enzyme was purified using Fast Protein Liquid Chromatography (FPLC, ÄKTA Purifier System, GE Healthcare). The crude extract was loaded onto chelating Sepharose Fast Flow resin column (GE Healthcare), preloaded Ni2+ and pre-equilibrated with binding buffer (50 mM sodium phosphate buffer, 500 mM NaCl, pH 6.0). Unbound proteins were washed with washing buffer containing 50 mM imidazole. The recombinant levansucrase fused with a 6×histidine-tag was eluted from the column by elution buffer (50 mM sodium phosphate buffer, 500 mM NaCl, 500 mM imidazole, pH 6.0) and the purified enzyme was dialyzed against 50 mM sodium phosphate buffer (pH 6.0) using a 10 kDa cut-off dialysis bag for 24 h to remove imidazole. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the purity and molecular mass of the purified enzyme, using a 5% stacking gel and 12% separating gel. Protein bands were stained by Coomassie brilliant blue R250.

2.3. Enzyme assay Levansucrase activity was assayed using sucrose as a sole substrate by total, hydrolysis, and transfructosylation activity. Total activity was determined by calculating the release of glucose from sucrose, hydrolysis activity was measured based on the release of fructose from sucrose, and transfructosylation activity was calculated as total activity minus hydrolysis activity and determined by measuring the amount of glucose minus the amount of fructose from sucrose (Tieking, Ehrmann,

6

Vogel, & Ganzle, 2005). A reaction mixture of 1 mL included 20% (w/v) sucrose, 50 mM sodium phosphate buffer (pH 6.0), and purified enzyme with the final concentration ranged from 5 to 10 μg/mL. The reactions were performed at 40 °C for 20 min and stopped by heating in boiling water for 5 min. One unit of total activity and hydrolysis activity were defined as the amount of enzyme catalyzing the release of 1 μmol glucose and fructose per min, respectively. Transfructosylation activity was defined as total activity minus hydrolysis activity. Unless stated otherwise, the enzyme activity was described as the total activity in this article.

2.4. Structural determination of polysaccharide produced by the recombinant B. goodwinii levansucrase

2.4.1 Isolation and purification of the produced polysaccharide The polysaccharide was produced from 50% (w/v) sucrose by 6 U/g sucrose of recombinant B. goodwinii levansucrase at pH 6.0 and 35 °C for 12 h. The reaction mixture was treated with Sevag reagent (n-butanol : chloroform = 1 : 4, v/v) for 4 times to remove proteins (Sevag, 1938). Three volumes of anhydrous ethanol was added to the reaction mixture, mixed thoroughly, and stored at 4 °C for overnight to allow the polysaccharide precipitated. The mixture was then centrifuged at 10,000 g and 4 °C for 30 min. The precipitated polysaccharide was collected and freeze-dried to constant weight using a 4.5 L FreeZone freeze-dry system (Labconco Corp, MO, U.S.A.).

2.4.2 Fourier-transform infrared (FTIR) spectroscopy FTIR spectroscopy analysis was performed to determine the functional groups in

7

the polysaccharide enzymatically produced from sucrose. The FTIR spectra of the dried polysaccharide film were recorded over a wavenumber range of 400 - 4000 cm-1, with an infrared spectrometer (Perkin-Elmer 16 PC spectrometer, Boston, USA) in the internal reflectance (Attenuated Total Reflectance, ATR) mode. The resolution was set as 4 cm-1 for the spectrum scanning.

2.4.3. Nuclear magnetic resonance (NMR) measurement NMR analysis was performed to determine the structure and the linkages of the polysaccharide. The dried polysaccharide sample (25 mg) was dissolved in deuterium oxide (D2O) and lyophilized. The spectra of 1H and 13C were recorded at 60 °C on an AVANCE III 400MHz Digital NMR spectrometer (Brucker Co., Billerica, MA, USA). The chemical shifts (δ) were expressed in ppm and determined using acetone (δH = 2.225 ppm) and 1,4-dioxan (δC = 66.50 ppm) as internal reference standards.

2.5. Effect of pH on enzyme activity Effect of pH on levansucrase activity was studied using three different buffer systems, acetate buffer (50 mM, pH 4.0 – 6.0), sodium phosphate buffer (50 mM, pH 6.0 – 7.5), and Tris-HCl (50 mM, pH 7.5 – 9.0) at 40 °C. The activity of the enzyme which presented the maximum activity was considered as 100%.

2.6. Effect of temperature on enzyme activity and thermostability The optimum temperature of the recombinant levansucrase was determined under the standard assay conditions at temperatures ranged from 30 to 60 °C. Thermostability was examined by measuring the residual activity of levansucrase after incubating the enzyme at various temperatures (35, 40, 45, and 50 °C) for

8

different amounts of time. The activity of the enzyme without incubation was considered as 100%.

2.7. Effect of metal ions Enzyme activity was measured under standard assay conditions by using different divalent metal salts in sodium phosphate buffer (50 mM, pH 6.0). The metal ions in the form of CaCl2, CuSO4, FeSO4, ZnSO4, MgSO4, MnSO4, and NiSO4 were used at a final concentration of 1 mM. Enzyme activity in the absence of the divalent metal salts was considered as 100%.

2.8. Effect of enzyme dosage on levan biosynthesis The reaction mixtures for levan biosynthesis were prepared with 20% (w/v) sucrose by varying the enzyme amounts from 1 to 16 U/g sucrose, and the reactions were carried out at pH 6.0 and 35 °C for 12 h.

2.9. Effect of sucrose concentration on levan biosynthesis The effect of sucrose concentration was determined by measuring the levan biosynthesis from 10% – 60% (w/v) sucrose by 6 U/g sucrose of levansucrase at pH 6.0 and 35 °C for 12 h.

2.10. Levan biosynthesis The purified recombinant B. goodwinii levansucrase was used to enzymatic synthesis of levan from sucrose under the optimized conditions. Samples were taken at 2-h intervals and analyzed for sugar content measurement. The bioconversion kinetics was investigated over 12 h.

9

2.11. HPLC analysis The concentrations of different sugars were analyzed by HPLC measurements. HPLC was performed by a Waters e2695 system (Waters Corporation, MA, USA) equipped with a Sugar-Pak I column (6.5 mm × 300 mm, Waters, USA) and a Waters 2414 RI detector. Samples were eluted with deionized water at a flow rate of 0.4 mL/min, with the column temperature maintained at 85 °C.

2.12. Molecular weight measurement The molecular weight of levan polysaccharide was measured by using a High Pressure Size Exclusion Chromatography (HPSEC) system. Detection consisted of an Optilab® T-rEX refractive-index (RI) detector (Wyatt Technology, Santa Barbara, CA, USA) and a DAWN HELEOS-II multi-angle laser light scattering (MALLS) detector (Wyatt Technology, Santa Barbara, CA, USA) with the He-Ne light wave length at 658.0 nm. A Shodex OH-pak SB-806 HQ column (8 mm × 300 mm, Showa Denko K.K., Tokyo, Japan) with an OH-pak SB-G guard column was used for chromatography. The column temperature was set as 25 °C. The column was eluted by 0.1 M NaNO3 at a flow rate of 0.5 mL/min. The molecular weight was calculated based on the data processing by Wyatt Astra software (Version 5.3.4.14, Wyatt Technology, USA).

3. Results and discussion 3.1. Cloning, expression, and purification of B. goodwinii levansucrase The whole genomic sequence of B. goodwinii has recently been determined and released in GenBank database with accession No. CGIG01000001.1. Based on the

10

genome sequence, there is a putative gene (locus_tag: BN1221_00994c) encoding the hypothetical protein of levansucrase (protein ID: CPR14579.1) in the genome. The full-length levansucrase-encoding gene was commercially synthesized and cloned into pET-22b(+) expression vector. An in-frame 6×histidine-tag was fused at the C-terminal of the protein. The generated recombinant plasmid, pET-Brgo-Lev, was then transformed into E. coli BL21(DE3) as host cell for heterogeneous expression of B. goodwinii levansucrase. The recombinant E. coli harboring pET-Brgo-Lev was cultured by IPTG induction. The whole cells were collected by centrifugation and disrupted by sonication. Compared to the control of E. coli without transformation, the crude enzyme from recombinant E. coli showed a significant sucrose-splitting activity (Fig. S1) and a large amount of proteins around 49 kDa (Fig. 1), which was consistent with the predicted molecular mass of the B. goodwinii levansucrase. These results indicated that the recombinant enzyme was expressed. Because of fusion with a 6×histidine-tag, the recombinant levansucrase was conveniently purified to electrophoretic homogeneity using a single-step purification by nickel-affinity chromatography. The purified enzyme showed a single protein band at 49 kDa on SDS-PAGE (Fig. 1).

Many levan-producing levansucrases have been cloned and expressed from various microorganisms, including Acetobacter xylinum NCI 1005 (GenBank accession No. BAA93720.1)

(Tajima

et

al.,

2000),

B.

amyloliquefaciens

(ACD39394.1)

(Rairakhwada et al., 2010), B. licheniformis 8-37-0-1 (AAU42526.1) (Lu et al., 2014), Bacillus megaterium DSM319 (ADF38395.1) (Homann, Biedendieck, Gotze, Jahn, & Seibel, 2007), L. reuteri 121 (AAO14618.1) (van Hijum, Bonting, van der Maarel, & Dijkhuizen, 2004), Leuconostoc mesenteroides B-512 FMC (AAT81165.1) (Kang et

11

al., 2005), L. sanfranciscensis TMW 1.392 (CAD48195.1) (Tieking et al., 2005), Pseudomonas aurantiaca S-4380 (AAL09386.1) (Jang et al., 2002), Pseudomonas syringae pv. tomato (CCM43846.1) (Visnapuu et al., 2011), and Z. mobilis (BAA04475.1) (Kyono, Yanase, Tonomura, Kawasaki, & Sakai, 1995). Herein, a novel levansucrase was expressed from B. goodwinii, which was from Brenneria species strains for the first time. The levansucrase from B. goodwinii exhibited 81%, 80%, 72%, and 51% amino acid sequence identities with the ones from P. aurantiaca S-4380, L. mesenteroides B-512 FMC, P. syringae pv. tomato, and Z. mobilis, respectively, but less than 50% identities with other ones.

3.2. Structure determination of biosynthesized polysaccharide The structure analysis of the polysaccharide produced by the recombinant B. goodwinii levansucrase was performed by both FTIR and NMR measurement (Fig. 2). Shown in the FTIR spectrum (Fig. 2A), two typical bands at approximately 3,280 and 2,930 cm-1 represented O-H and C-H stretching, respectively (Zhu, Sheng, & Tong, 2014). The O-H stretching vibration was observed within the wavenumber range of 3,600 – 3,200 cm-1, and the broad and pure peak at 3,280 cm-1 indicated the intermolecular hydrogen bonding (Zhang, Tian, Jiang, Miao, & Mu, 2014b). A shoulder peak at 2,930 cm−1 was attributed to C-H stretch of alkane. Further, the spectrum results showed a peak at 1,644 cm−1 which was due to C-O stretching (Ahuja, Singh, & Kumar. 2013). And several sharp peaks between 1,000 and 800 cm−1 were the typical peaks of carbohydrates.

The

13

C and 1H NMR spectrum were shown in Fig. 2B and 2C, respectively. The

13

C NMR spectrum exhibited six broad resonance signals at 104.568 (C2), 80.675

12

(C5), 77.273 (C3), 75.914 (C4), 63.830 (C6), 60.953 (C1) ppm corresponding to the peak position for β-(2, 6)-levan (Fig. 2B). These carbon chemical shifts were ascribed to β-configurated fructofuranose units by comparison with those of the standard methyl glycoside (Bock & Pedersen, 1983), and were almost similar to those for the reported levans (Table 1) produced by levansucrases from B. methylotrophicus SK 21.002 (Zhang et al., 2014a), B.

amyloliquefaciens (Tian et al., 2011),

Bacillussubtilis (Natto) Takahashi (Shih, Yu, Shieh, & Hsieh, 2005), L. reuteri 121 (van Hijum et al., 2001), and B. licheniformis RN-01 (Nakapong et al., 2013). The 1H NMR spectrum (Fig. 2C) was almost identical to that of levan produced by B. methylotrophicus SK 21.002 levansucrase (Zhang et al., 2014a). All these data indicated the structure of β-(2, 6)-levan polysaccharide biosynthesized from sucrose by the recombinant levansucrase from B. goodwinii.

3.3. Effect of pH and temperature on levansucrase activity The total, hydrolysis, and transfructosylation activities of the recombinant B. goodwinii levansucrase were measured at 40 °C and various pHs ranged from pH 4.0 to 9.0. Shown in Fig. 3A, all the highest activities were shown at pH 6.0. The transfructosylation activity was rather sensitive to pH, which was only relatively high at pH 5.5 and 6.0 but dropped remarkably below pH 5.5 or above pH 6.0 and was almost completely inhibited at pH 4.0 and 9.0. By comparison, the enzyme showed relatively high hydrolysis activity over a wide range of pHs from 5.5 to 8.0. The transfructosylation activity was higher than the hydrolysis activity at pH 5.5 and 6.0 but lower than the hydrolysis activity when the pH was less than 5.5 or more than 6.0. By comparison, levansucrase from P. syringae pv. tomato showed the highest transfructosylation activity the pH ranges from 5.0 to 7.0 with no pH-dependence 13

(Visnapuu et al., 2011). Most of the reported levan-producing levansucrase also exhibited the highest activities at slightly acidic pHs, including the ones from Acetobacter diazotrophicus SRT4 (pH 5.0) (Hernandez et al., 1995), B. licheniformis RN-01 (pH 6.0) (Nakapong et al., 2013), B. licheniformis 8-37-0-1 (pH 6.5) (Lu et al., 2014), B. megaterium (pH 6.6) (Homann et al. 2007), Bacillus sp. TH4-2 (pH 6.0) (Ben Ammar et al., 2002), G. stearothermophilus ATCC 7953 (pH 6.75) (Inthanavong et al., 2013), and L. sanfranciscensis TMW 1.392 (pH 5.4) (Tieking et al., 2005).

Effect of temperature on the various activities of the recombinant B. goodwinii levansucrase was also studied (Fig. 3B). The enzyme interestingly showed different optimum temperature for different activity. The optimum temperatures were measured to be 40, 35, and 45 °C for the total, transfructosylation, and hydrolysis activity, respectively. Levan is biosynthesized from sucrose by transfructosylation reaction of levansucrase, and thus higher transfructosylation activity favors to generate levan from sucrose. In previous works, it was indicated that lower temperatures favored the transfructosylation reaction of levansucrase to produce levan and higher temperatures favored the sucrose hydrolysis reaction (Li et al., 2015). The optimum temperatures of B. amyloliquefaciens levansucrase for sucrose hydrolysis and transfructosylation were 30 and 4 °C, respectively (Rairakhwada et al., 2010). Z. mobilis levansucrase showed the highest sucrose hydrolysis and levan formation at 50 and 30 °C, respectively (Sangiliyandi et al., 1999), and the one from M. laevaniformans ATCC 15953 at 45 and 30 °C, respectively (Park et al., 2003). P. syringae pv. phaseolicola levansucrase, which exhibited optimum temperature at 20 °C for transfructosylation to produce levan, almost lost all levan-producing activity at 50 °C, but showed the highest sucrose hydrolysis activity at 60 °C (Hettwer, Gross, & Rudolph, 1995).

14

The total specific activity was measured to be 620 U/mg at pH 6.0 and 40 °C, and the specific activities for sucrose hydrolysis and transfructosylation reaction were determined to be 260 U/mg at pH 6.0 and 45 °C and 385 U/mg at pH 6.0 and 35 °C, respectively.

3.4. Levansucrase thermostability The recombinant B. goodwinii levansucrase showed excellent thermostability at 35 °C and retained >80% of residual activity after incubation at 35 °C for 3 h. The half-life at 35 °C was approximately 11 h and the enzyme still had 45% of residual activity after incubation at 35 °C for 12 h. But the enzyme showed much less thermostability at higher temperatures and rapidly lost its activity at 50 °C within 0.5 h (Fig. 3C). The similar thermostability profile was also observed in Z. mobilis levansucrase, which had excellent thermostability at 35 °C but was easily inactivated at 50 °C (Sangiliyandi et al., 1999). Obviously, these two levansucrases were not thermostable. In previous studies, levansucrases from Bacillus sp. TH4-2 and G. stearothermophilus ATCC 7953 were identified to be highly thermostable. The levansucrase from Bacillus sp. TH4-2 was stable up to 50 °C (Ben Ammar et al., 2002), and G. stearothermophilus levansucrase almost retained all of initial activity after incubated for 6 h at temperatures ranging from 4 to 47 °C (Inthanavong et al., 2013). But both levansucrases from Bacillus sp. TH4-2 (Ben Ammar et al., 2002) and G. stearothermophilus (Inthanavong et al., 2013) required relatively high temperatures to display the highest transfructosylation activities, which were measured to be 60 and 57 °C, respectively. By comparison, the optimum temperatures for transfructosylation activities of other levansucrases were less than 40 °C.

15

3.5. Effect of metal ions on levansucrase activity The effect of various metal ions on the total activity of the recombinant B. goodwinii levansucrase was assayed at pH 6.0 and 40 °C, in which the metal ions were tested at the final concentration of 1 mM (Fig. 3D). The Ni2+ increased the catalytic activity to 153% of initial relative activity, Mn2+ and Ca2+ caused marginal increases in the enzyme activity (<25%), whereas other metal ions including Zn2+, Fe2+, Mg2+, and Cu2+ exhibited slight inhibitory effect (<20%) on the enzyme activity.

3.6. Effect of enzyme dosage on levan biosynthesis The effect of levansucrase dosage was studied when levan was biosynthesized by the recombinant B. goodwinii levansucrase from 20% (w/v) sucrose at pH 6.0 and 35 °C for 12 h (Fig. 4). Both levan formation and fructose production were obviously improved when increasing the enzyme amount from 1 to 4 U/g sucrose, but the increase of levan formation was much higher than that of fructose production, indicating that transfructosylation activity improvement was higher than that of sucrose hydrolysis at this range of enzyme dosage. Levan synthesis reached the maximum at the amount of 6 U/g sucrose and then dropped slowly when further increasing the enzyme amount. However, fructose production was consistently and slowly improved with the increase of levansucrase amount, indicating that the sucrose hydrolysis activity consistently increased. In a previous work, a similar result that higher amount of levansucrase preferred hydrolysis reaction was shown in the study of B. subtilis levansucrase (Raga-Carbajal et

al.,

2016;

Porras-Domínguez,

Ávila-Fernández,

Miranda-Molina,

Rodríguez-Alegría, & Munguía, 2015). B. subtilis levansucrase showed equal hydrolysis and transfer activity when 1 U/mL enzyme was used. A lower amount (0.1

16

U/mL) of enzyme showed significantly lower hydrolysis activity in the total activity (27%), whereas the increase of the enzyme amount (10 U/mL) improved the ratio of hydrolysis activity (59%).

3.7. Effect of sucrose concentration on levan biosynthesis Sucrose concentration generally plays an important role in levan biosynthesis and high concentration favors to the transfructosylation reaction to produce levan (Li et al., 2015; Zhang et al., 2014a). Herein, the effect of sucrose concentration was investigated on levan biosynthesis at pH 6.0 and 35 °C by the recombinant B. goodwinii levansucrase with enzyme dosage fixed at 6 U/g sucrose (Fig. 5). When 10% (w/v) sucrose was used to biosynthesize levan for 12 h, 31 g/L levan and 17 g/L fructose, with the ratio of approximately 1.8 : 1, were produced by transfructosylation and sucrose hydrolysis reactions, respectively. With the sucrose concentration increased, levan formation was significantly improved but the fructose production was only slightly changed, indicating that the transfructosylation reaction held a prominent position and sucrose hydrolysis was significantly inhibited by high concentration of sucrose. The ratio of levan biosynthesis to fructose production was approximately 6 : 1 when initial sucrose concentration was improved to 60% (w/v) after reaction for 12 h.

3.8. Levan biosynthesis by the recombinant B. goodwinii levansucrase Levan biosynthesis was carried out from 50% (w/v) sucrose at pH 6.0 and 35 °C, using the recombinant B. goodwinii levansucrase of 6 U/g sucrose (Fig. 6). The production of levan increased with increasing reaction time up to 6 h. The highest levan yield reached 185 g/L after reaction for 6 h and then the levan concentration

17

dropped very slightly, probably because of levan hydrolysis. During the levan biosynthesis process, fructose was steadily and slowly released, indicating the constant occurrence of the hydrolysis reaction (data not shown).

The molecular weight of levan products was measured using HPSEC-MALLS-RI. The molecular weight distribution profiles of levan products produced at different time intervals all showed single symmetrical peaks in the HPSEC chromatograms, indicating homogeneous polysaccharides (Fig. S2). The weight-average molecular weight of levan products was measured to be 0.920, 1.478, 1.338, 1.333, and 1.330 × 108 Da after reaction for 2, 4, 6, 8, and 12 h, respectively. The weight-average molecular weight reached the maximum at 4 h and then reduced very slightly. Obviously, the molecular weight of levan produced by the recombinant B. goodwinii levansucrase was much higher than most of the reported ones (Table 2). In previous studies, the levansucrase from B. methylotrophicus SK 21.002 only produced levan polysaccharides with molecular weight between 4 – 5 × 103 Da (Zhang et al., 2014a), the enzyme from B. licheniformis RN-01 produced levan with a wide range of molecular weights from 1 × 103 to 6 × 105 Da (Nakapong et al., 2013), the molecular weight of the levan produced by the immobilized Bacillus natto levansucrase was estimated to be 2.5 × 106 Da (Iizuka, Yamaguchi, Ono, & Minamiura, 1993), and the molecular weight of the levan produced by the purified recombinant B. licheniformis 8-37-0-1 reached 9.6 × 106 Da (Lu et al., 2014).

Conclusions In this study, the recombinant B. goodwinii levansucrase was purified and

18

characterized. Base on the FTIR and NMR measurements, the polysaccharide produced by the recombinant B. goodwinii levansucrase from sucrose was determined to be levan. High concentration of sucrose significantly favored to the polymerization reaction to produce levan. The enzyme produced 185 g/L levan with average molecular weight of approximately 1.3 × 108 Da from 50% (w/v) sucrose after reaction at pH 6.0 and 35 °C for 12 h. Therefore, the B. goodwinii levansucrase could be considered as a good candidate for levan production in an industrial enzymatic process.

Acknowledgements This work was supported by the NSFC Project (No. 21276001), the 863 Project (No. 2013AA102102), the Support Project of Jiangsu Province (No. BK20130001 and 2015-SWYY-009), and the project of outstanding scientific and technological innovation group of Jiangsu Province (Jing Wu).

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Fig. 1. SDS-PAGE analysis of the recombinant B. goodwinii levansucrase. Lane 1, molecular standard marker; lane 2, the soluble crude enzymes extracted from the recombinant E. coli cells after IPGT induction; and lane 3, the recombinant B. goodwinii levansucrase purified by nickel affinity chromatography.

27

Fig. 2. Structure determination of polysaccharide produced from sucrose by the purified recombinant B. goodwinii levansucrase. A shows the FTIR spectrum of the produced polysaccharide. B and C show the 13C and 1H NMR spectrum, respectively.

28

Fig. 3. Enzymatic properties of the recombinant B. goodwinii levansucrase. A represents the effect of pH on the catalytic activity. Three different buffer systems were used the analysis including acetate buffer (50 mM, pH 4.0 – 6.0), sodium phosphate buffer (50 mM, pH 6.0 – 7.5), and Tris-HCl (50 mM, pH 7.5 – 9.0). B represents the effect of temperature. C represents the effect of temperature on the stability of the enzyme. D shows the effect of various metal ions on the total activity. Values are means of three replications ± standard deviation.

29

Fig. 4. Effect of enzyme dosage on levan biosynthesis. The enzyme quantification is based on the total catalytic activity. The levan biosynthesis was performed from 50% (w/v) sucrose at pH 6.0 and 35 °C for 12 h. Values are means of three replications ± standard deviation.

30

Fig. 5. Effect of sucrose concentration on levan biosynthesis. The levan biosynthesis was performed by 6 U/g sucrose of B. goodwinii levansucrase at pH 6.0 and 35 °C for 12 h. Values are means of three replications ± standard deviation.

31

Fig. 6. Levan production from sucrose by the purified recombinant B. goodwinii levansucrase. The enzymatic reaction was performed by 6 U/g sucrose of the purified recombinant levansucrase from 50% (w/v) sucrose at pH 6.0 and 35 °C. Values are means of three replications ± standard deviation.

32

Table 1. 13C-NMR chemical shifts of produced polysaccharide and reported standard levan. Chemical shifts (ppm) of 13C-NMR resonance of levans formed by levansucrase from:

Carbon

B.

B. subtilis

L.

B.

(Natto)

reuteri

licheniformis

Takahashi

121

RN-01

59.8

60.1

59.6

62.9

104.66

104.1

104.4

104.0

106.9

77.273

77.51

76.2

76.5

76.0

79.3

C-4

75.914

76.10

75.13

75.4

74.9

78.1

C-5

80.675

80.77

80.2

80.5

80.0

83.0

C-6

63.830

63.94

63.2

63.6

63.2

66.1

atom

B.

number

goodwinii

C-1

60.953

61.20

C-2

104.568

C-3

methylotrophicus SK 21.002

B. amyloliquefaciens

van Reference

This

Zhang et al.,

study

2014a

Tian et al., 2011

Shih et

Hijum

Nakapong et

al., 2005

et al.,

al., 2013

2001

Table 2. Comparison of molecular weight of levan produced from sucrose by various levansucrase. Molecular weight Microbial source for Bioctatalyst

(Da) of the produced

Reference

levansucrase levan 1.4 × 108

This study

1 × 103 – 6 × 105 9.6 × 106

Nakapong et al., 2013 Lu et al., 2014

Crude enzyme

4 - 5 × 103

Zhang et al., 2014a

Immobilized purified enzyme

2.5 × 106

Iizuka et al., 1993

6.6 × 105 and 6 × 103

Ben Ammar et al.,

(two fractions)

2002

B. goodwinii

Purified recombinant enzyme

B. licheniformis RN-01

Purified enzyme

B. licheniformis 8-37-0-1 B. methylotrophicus SK

Purified recombinant enzyme

21.002 B. natto

Bacillus sp. TH4-2

Purified enzyme

33

Abdel-Fattah, B. subtilis NRC 33a

5 - 6 × 104

Crude enzyme

Mahmoud, & Esawy, 2005

1.5 × 105 and ≥ 2 ×

van Hijum et al.,

L. reuteri 121

Purified enzyme

106 (two fractions)

2001

L. sanfranciscensis TMW 1.392 P. syringae pv. phaseolicola

Purified recombinant enzyme

≥ 5 × 106

Tieking et al., 2005

1 × 105 - 1 × 107

Hettwer et al., 1995

Purified enzyme

34