Production of slowly digestible corn starch using hyperthermophilic Staphylothermus marinus amylopullulanase in Bacillus subtilis

Accepted Manuscript Short communication Production of slowly digestible corn starch using hyperthermophilic Staphylothermus marinus amylopullulanase in Bacillus subtilis Xiaolei Li, Jingying Pei, Teng Fei, Jiahui Zhao, Yong Wang, Dan Li PII: DOI: Reference:

S0308-8146(18)31867-3 https://doi.org/10.1016/j.foodchem.2018.10.092 FOCH 23753

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Food Chemistry

Received Date: Revised Date: Accepted Date:

9 July 2018 18 October 2018 19 October 2018

Please cite this article as: Li, X., Pei, J., Fei, T., Zhao, J., Wang, Y., Li, D., Production of slowly digestible corn starch using hyperthermophilic Staphylothermus marinus amylopullulanase in Bacillus subtilis, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.2018.10.092

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Production of slowly digestible corn starch using hyperthermophilic Staphylothermus marinus amylopullulanase in Bacillus subtilis Xiaolei Li a, c, Jingying Pei c, Teng Fei c, Jiahui Zhao c, Yong Wang c, Dan Li b, c *

Running title: Slowly digestible starch produced by an amylopullulanase

a

School of Biological Science and Biotechnology, Minnan Normal University, 36 Xianqianzhi

Street, Zhangzhou 363000, Fujian, People's Republic of China b

College of Food and Biological Engineering, Jimei University, 43 Yindou Road, Xiamen 361021,

Fujian, People's Republic of China c

Key Laboratory of Agro-products Processing Technology at Jilin Provincial Universities,

Changchun University, 6543 Weixing Road, Changchun 130022, Jilin, People’s Republic of China * Corresponding author. Tel.: +86 592 6181487; fax: +86 592 6180470. E-mail address: [email protected] (D. Li).

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/ToqCAB

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ABSTRACT We describe a novel method for enzymatic production of slowly digestible starch (SDS) at 100°C. A hyperthermophilic amylopullulanase from archaeon Staphylothermus marinus (SMApu) was expressed in a generally recognised as safe (GRAS) microorganism, Bacillus subtilis. Purified SMApu at 0–240 U/g substrate was incubated with 0.1 g/mL of corn starch in a boiling water bath for 2 h. SDS content of the SMApu-modified starch increased significantly from 17.4 to 42.7% (P < 0.05). Molecular weight and amylopectin branch chains A (DP 6–12) of SMApu-modified starch were significantly lower, and amylose content and amylopectin B1 chains significantly higher (P < 0.05), than those of natural corn starch. These changes in molecular structure partially explained the reduced digestibility. High optimal temperature of SMApu facilitated the simultaneous gelatinisation and hydrolysis of cereal starch. These results demonstrate that SMApu has potential applications in the production of SDS during thermal processing of cereal foods. Keywords: Slowly digestible starch; Corn; Debranching enzyme; GRAS microorganism; Archaea.

1. Introduction Starch, the major glycemic carbohydrate in cereal foods, has been classified into three nutritional types: rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS; Englyst, Kingman, & Cummings, 1992). SDS can deliver a slow and sustained release of postprandial blood glucose, resulting in low glycemic and insulinemic responses (Zhang & Hamaker, 2009; Miao, Jiang, Cui, Zhang, & Jin, 2015). SDS can also reduce daily food intake by downregulating appetite-stimulating neuropeptide genes (Hasek et al., 2018).

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These functions are particularly beneficial for individuals with diabetes, obesity, or cardiovascular diseases. Small amounts of SDS are naturally present in cereals. Some processes are used to enhance the SDS content of natural starches, including freeze-thaw cycles and amylolytic hydrolysis (Magallanes-Cruz, Flores-Silva, & Bello-Perez, 2017). However, SDS produced by changes in temperature is subject to loss during cooking. Most desirable is to have the slow glucose release property structurally inherent to the material so that it is retained through processing or home cooking (Lee et al., 2013). Amylolytic enzymes have been used to produce SDS from cereal starches by changing the amylose to amylopectin ratio, or the ratio of short- to long-branch amylopectin. Barley βamylase (EC 3.2.1.2) of glycoside hydrolase (GH) family 14 (GH 14) and Bacillus subtilis maltogenic α-amylase (EC 3.2.1.133) of family GH 13 hydrolyse α-1,4-D-glucosidic linkages of amylopectin branch chains to remove maltose residues and reduce the chain length (Ao et al., 2007; Miao et al., 2014b & 2014c). Aspergillus niger transglucosidase (EC 2.4.1.161) of family GH 31 catalyses the hydrolysis and transfer of D-glucosyl units to convert α-1,4-D-glucosidic linkages to α-1,6-D-glucosidic linkages (Ao et al., 2007; Miao et al., 2014a; Shi et al., 2014). Neisseria polysaccharea amylosucrase (EC 2.4.1.4) of the family GH 13 hydrolyses α-1,2glycosidic linkages of sucrose and transfers D-glucosyl units to the ends of amylopectin branch chains (Kim et al., & Choi, 2014). Streptococcus mutans (Jo et al., 2016), Acidothermus cellulolyticus (Li et al., 2014b), Geobacillus thermoglucosidans (Li et al., 2018e), and Rhodothermus obamensis branching enzymes (EC 2.4.1.18; Sorndech et al., 2015) of families GH 13 and 57 catalyse the amylose branching process, shorten branch chain length, and increase the branching points of amylopectin. Acidothermus cellulolyticus 4-α-glucanotransferase (Jiang

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et al, 2014) and Thermus thermophilus amylomaltase (EC 2.4.1.25; Sorndech et al., 2015) of the family GH 77 decreases the amylose and increases the short (degree of polymerisation [DP] < 13) and long (DP > 30) chains of amylopectin. Bacillus acidopullulyticus pullulanase (EC 3.2.1.41; Li et al., 2017) and Pseudomonas sp. isoamylase (EC 3.2.1.68; Shin et al., 2004) of the family GH 13 specifically attack α-1,6-D-glucosidic linkages to rearrange the branch chain length and distribution of amylopectin. These amylolytic enzymes of microbial or plant origin can be incubated at 30–75°C with cereal starches to produce SDS. Amylopullulanase (EC 3.2.1.41), a type 2 pullulanase, occurs naturally in the thermophilic archaea Thermococcus and Pyrococcus. They catalyse reactions at elevated temperatures, which offers several advantages including higher substrate solubility, decreased viscosity, better bacterial decontamination, and increased reaction rates (Vieille & Zeikus, 2001). Recently, an amylopullulanase of the family GH57 from the hyperthermophilic archaeon Staphylothermus marinus (SMApu), was heterologously expressed in Escherichia coli in our laboratory (Li, Li, & Park, 2013). The purified SMApu is highly thermoactive at 95–110°C and extremely thermostable at 85–95°C. It can hydrolyse not only pullulan/starch but also cyclodextrins, a property never before seen in amylopullulanases of the family GH57 (Blesák & Janeček, 2013). The other two archaeal amylopullulanases expressed in E. coli, CMApu and TPApu, with similar amino acid sequences to SMApu, have been used in the debranching process of starch and the production of linear malto oligosaccharides, showing the potential applications of archaeal amylopullulanases in the food industry (Li & Li, 2015; Li et al., 2018d). However, the effects of archaeal amylopullulanase on the digestibility of starch are still unknown. In this study, SMApu was heterologously expressed in the generally recognised as safe (GRAS) microorganism B. subtilis to ensure the safety of origin for potential applications in

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foods. The purified SMApu was then used to produce slowly digestible corn starch. Finally, we propose the possible molecular mechanisms involved in these changes.

2. Materials and methods 2.1. Cloning and expression of SMApu in Bacillus The gene (Smar_1407) encoding SMApu was amplified from the plasmid pSMApu6xH (Li et al, 2013) by PCR using PrimeSTAR HS DNA Polymerase (Takara Bio, Dalian, China; 30 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 150 s), forward primer (BLMA promoter 5′ATCGGATCCGAATTCGGAAGATGTCGC-3′), and reverse primer (T7 terminator 5′GCTAGTTATTGCTCAGCGGTGGCA-3′). The recombinant plasmid pUBRT-Smar1407 was constructed through ligation of the amplified fragment (2.4 kb) and the cloning vector pUBRT29 (Choi et al., 2010) at the restriction sites BamH I and Xho I.

2.2. Enzyme preparation and assays Bacillus subtilis ISW1214 (Takara Bio) was transformed by the pUBRT-Smar1407 plasmid, inoculated into six flasks containing 250 mL of medium A (3.3% Oxoid tryptone, 2% Oxoid yeast extract, 0.74% NaCl, 0.8% Na2HPO4, 0.4% KH2PO4, 2% Oxoid casamino acids, and 0.06 mmol L−1 MnCl2) supplemented with kanamycin (80 μg/mL), and then cultured on an orbital shaker at 250 rpm and 37°C for 30 h. SMApu was purified from the cells, and then its purity, molecular weight, and activity were assayed as described previously. One unit (1 U) of enzymatic activity is defined as the amount of enzyme required to produce 1 μmol of reducing sugars per minute under the defined assay conditions (Li et al, 2013).

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2.3. Hydrolysis of starch Enzymatic hydrolysis was performed in 5 mL of pH 5.0 and 50 mM sodium acetate buffer. Normal corn starch (0.5 g; Sigma-Aldrich, St. Louis, MO, USA) was boiled with stirring for 30 min, and then the reaction was initiated by adding 80–240 U SMApu. After incubation at 100°C for 2 h, the modified starch was precipitated by ethanol, dried in a convection oven overnight at 45°C, and then pulverised and passed through a 100-mesh sieve.

2.4. Determination of digestibility The in vitro digestibility of the starch was analysed according to the Englyst procedure (Englyst et al., 1992) with some modifications. Corn starch (20 mg) was mixed by vortexing with 250 μL sodium acetate buffer (0.2 M, pH 5.2) and 650 μL water in a 4 mL tube. Then one glass ball (2 mm diameter) was added. After the solutions were equilibrated to 37°C for 5 min, the digestion reactions were initiated with the addition of 320 U porcine pancreatic α-amylase (Sigma-Aldrich) and 0.3 U A. niger amyloglucosidase (Sigma-Aldrich). Subsequently, the samples were shaken in a 37°C water bath at 150 rpm. The glucose contents of the hydrolysate were determined at 20 min and 120 min using glucose GOD-POD assay kits (Huili, Changchun, China). The contents of RDS, SDS, and RS were calculated as described previously (Li, Wang, Lee, & Li, 2018b).

2.5. Determination of amylose and amylopectin The amylose content of the starches was determined by amylolytic hydrolysis to glucose after the amylopectin was precipitated with concanavalin-A. All procedures were performed

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according to the instructions of the Megazyme (Wicklow, Ireland) amylose/amylopectin assay kit.

2.6. High-performance size exclusion chromatography-multiangle laser-light scatteringrefractive index (HPSEC-MALLS-RI) analysis Two tandem columns (300 × 8 mm, Shodex OH-pak SB-806 and 805; Showa Denko K.K., Tokyo, Japan) at 55°C and distilled–deionised water (pH 6.8, 18.2 MΩ·cm) containing 0.02% NaN3 at a flow rate of 0.5 mL/min were used for the HPSEC-MALLS-RI (Wyatt Technology, Santa Barbara, CA, USA) analysis. A dn/dc value of 0.146 was used in the molecular weight calculations (Li et al., 2018a).

2.7. High performance anion exchange chromatography (HPAEC) analysis An anion exchange column (3 × 250 mm, CarboPac PA200; Dionex, Sunnyvale, CA, USA) at 30°C and an electrochemical detector containing Au working and Ag/AgCl reference electrodes in pulsed amperometry with standard quad waveform were used for HPAEC analysis (ICS-3000; Dionex). The mobile phases consisting of eluents A (100 mM NaOH) and B (1 M sodium acetate in 100 mM NaOH) were pumped at a flow rate of 0.4 mL/min. Eluent B was increased linearly from 0 to 60% over 60 min (Li et al., 2014a).

2.8. Statistical analyses SPSS software (ver. 22.0; IBM Corp., Armonk, NY, USA) was used to analyse the data. The final values were expressed as the means of triplicate determinations ± standard deviation.

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Significant differences between groups were assessed at the P < 0.05 level using the least squares method.

3. Results and discussion 3.1. Plasmid pUBRT-Smar1407 construct In our previous construct, Smar_1407 was inserted downstream of a constitutive BLMA promoter from a maltogenic amylase gene of B. licheniformis in the vector pTKNd6H to accomplish the expression of SMApu in E. coli (Li et al., 2013). To express SMApu in B. subtilis, a shuttle vector pUBRT29 with two origins of replication from pMB1 and pUB110 was used in this study. Compared with the vector pUBRTAMY used to express S. marinus maltogenic amylase in B. subtilis (Li et al., 2018c), pUBRT29 lacked the AmyR2 promoter. Therefore, the constitutive BLMA promoter and Smar_1407 of pSMApu6xH were amplified together by PCR. The PCR-amplified product and pUBRT29 were respectively digested with two restriction enzymes, BamH I and Xho I. Two long fragments were ligated into a new recombinant plasmid pUBRT-Smar1407. The gene Smar_1407 in this plasmid was also located downstream of the BLMA promoter. Recombinant SMApu expressed from the resulting construct pUBRT-Smar1407 in Bacillus had an extra sequence [-Leu-Glu-(His)6] at the carboxyl terminus, the same as previously expressed in E. coli (Li et al., 2013).

3.2. SMApu production in B. subtilis SMApu was expressed in B. subtilis with plasmid pUBRT-Smar1407. The data from the purification procedures are summarised in Table 1. The pullulan-hydrolysing activity of the

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crude SMApu in the cell extracts from Bacillus was determined to be 0.6 U/mL, almost onetenth of that from E. coli. pUB110-derived plasmids frequently exhibit segregational (plasmid loss from the cells) and structural (partial gene deletion in plasmids) instability. For B. subtilis harbouring pUBRTBL6×HBSMA, half transformants no longer contained this plasmid after 20 generations (Choi et al., 2010). Therefore, the activity of SMApu in the cell extracts from Bacillus was lower than that from E. coli, which may be due to the instability of pUBRT-Smar1407. A 32-fold purification of SMApu was achieved by subsequent heat treatment and Nisepharose affinity chromatography. After the final purification step, 31% of the initial pullulanhydrolysing activity was recovered, and the specific activity of purified SMApu from B. subtilis was calculated to be 31.9 U/mg, 76% of that from E. coli. The purified recombinant SMApu from B. subtilis appeared as a single band with an estimated molecular mass of 74 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (Fig. 1, lane 3), the same as that from E. coli.

3.3. In vitro digestibility of SMApu-modified corn starch The RDS, SDS, and RS contents of the natural corn starch were 85.2, 13.8, and 1.0%, respectively (Fig. 2). All of these values were within the ranges previously reported (Zhang, Ao, & Hamaker, 2008). The RDS content of the SMApu-modified starch decreased significantly, from 77.9 to 49.7% (P < 0.05), whereas the SDS content increased significantly, from 17.4 to 42.7% (P < 0.05), as the enzyme concentration increased from 80 to 240 U/g substrate during the hydrolysis of SMApu. The RS content increased slightly, with a maximum value of 7.5% at 240 U/g substrate.

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A similar phenomenon of low digestibility has been observed in moderately debranched rice starch (Lee, Lee, & Lee, 2010; Guraya, James, & Champagne, 2001), tigernut starch (Li et al., 2017), waxy corn starch (Liu et al., 2017), and waxy sorghum starch (Shin et al., 2004). Partially ordered crystalline structures in the debranched starches have been considered the probable reason for low digestibility. The thermoactive feature of SMApu facilitated the simultaneous gelatinisation and hydrolysis of cereal starch at 95–110°C. Accordingly, it seems possible to introduce SDS into some cereal foods by adding SMApu during thermal processing of these foods. However, the optimal temperatures of most amylolytic enzymes, including the commercial R. obamensis branching enzyme (Branchzyme) and B. acidopullulyticus pullulanase (Promozyme), are lower than the temperatures of complete gelatinisation of normal cereal starch. During the production of SDS, the starch slurries were often heated to 100°C for further gelatinisation. Then, an extra cooling step was required to obtain 40–65°C for the enzymatic reactions (Li et al., 2017; Sorndech et al., 2015).

3.4. Amylose and amylopectin contents of SMApu-modified corn starch An amylose content of 23.2%, a value close to the range of 26.6–28.7% previously reported in natural corn starch (Nalin et al., 2015), was observed (Table 2). During SMApu hydrolysis, the amylose content of SMApu-modified starch increased significantly, from 48.4 to 59.6% (P < 0.05), whereas the amylopectin content decreased significantly from 47.2 to 35.3% (P < 0.05) as the enzyme concentration increased from 80 to 240 U/g substrate. The results demonstrated that amylose content increased as the low digestible fraction, especially SDS content, increased. Similar phenomena have been observed in type I pullulanase

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modified rice starch (Lee et al., 2010), tigernut starch (Li et al., 2017), and waxy corn starch (Liu et al., 2017).

3.5. Molecular weight of SMApu-modified corn starch The number-averaged molecular weight (Mn) of 2.08 × 107 g/mol and the weight-averaged molecular weight (Mw) of 4.10 × 107 g/mol were observed in the native corn starch (Table 3). During SMApu hydrolysis, the Mn and Mw of SMApu-modified starch decreased from 3.59 × 105 g/mol to 1.83 × 105 g/mol, and from 1.92 × 106 g/mol to 1.42 × 106 g/mol, respectively, as the enzyme concentration increased from 80 to 240 U/g substrate. The results demonstrated that the SMApu-modified starch with lower molecular weight had properties supporting low digestibility. Similar phenomena have been observed in other enzymemodified starches (Jo et al., 2016; Lee et al., 2013; Li et al., 2018b).

3.6. Branch chain length distribution of SMApu-modified corn starch Branch chains B1 (DP 13–24) with a maximum content of 45.66% and the branch chains DP < 6 with a minimum content of 1.46% (Table 4) were observed in natural corn amylopectin. The other branch chains A (DP 6–12), B2 (DP 25–36), and B3 (DP ≥ 37) occupied 29.89, 14.27, and 8.72% of all branch chains, respectively. This mode of branch chain length distribution for natural corn amylopectin was nearly the same as that previously reported (Jane et al., 1999). After hydrolysis using SMApu at 0–240 U/g substrate, amylopectin branch chains A (DP 6–12) significantly decreased from 29.89 to 20.85% (P < 0.05), whereas the amylopectin branch chains B1 (DP 13–24) increased from 45.66 to 48.80% (P < 0.05). The branch chains DP < 6, B2, and B3 increased, but were not statistically significant. In general, the short branch chains (DP < 6, A) 11

significantly decreased from 31.35 to 23.53%, whereas the long branch chains (B1, B2, and B3) significantly increased from 68.67 to 76.45% (P < 0.05). The results demonstrated that the increased ratio of short chains to long chains led to the higher SDS content in SMApu-modified corn starch. Similar phenomena have been observed in pullulanase-modified sweet potato starch (Huang et al., 2015) and maize mutant flour (Zhang et al., 2008).

4. Conclusions An archaeal SMApu was expressed in a GRAS microorganism, B. subtilis, using a constitutive BLMA promoter to ensure the safety of origin for its potential applications in foods. SDS was produced by incubating purified SMApu and corn starch in boiling water. The slow digestion property was attributed to decreased molecular weight and amylopectin, and an increased ratio of amylopectin short chains to long chains. The high optimal temperature of SMApu facilitated the simultaneous gelatinisation and hydrolysis of corn starch. SMApu shows potential applications in the production of SDS during thermal processing of cereal foods.

Acknowledgements: This work was sponsored in part by National Natural Science Foundation of China (NSFC-31671801, 31371749) and Jilin Provincial Government (Department of science and technology; Department of Education, JJKH20170502KJ).

Conflict of interest: The authors declared no conflict of interest.

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Table 1 Purification of SMApu from a 1.5-L culture of B. subtilis Purification

Total volume

Total

Total protein

Specific activity

Yield

Purification

step

(mL)

activity (U)

(mg)

(U/mg)

(%)

(fold)

Cell extract

150.0

90.3

86.8

1.0

100

1.0

Heat treat

148.0

73.0

35.8

2.0

80.8

2.0

Ni-sepharose

22.0

27.8

0.9

31.9

30.8

31.9

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Table 2 Amylose and amylopectin contents of SMApu modified corn starch Amylose

Amylopectin

(%)

(%)

Natural

23.24±0.74a

71.85±0.74a

80 U/g

48.42±1.10b

47.21±1.10b

160 U/g

53.23±1.79c

42.59±1.79c

240 U/g

59.60±2.10d

35.29±2.10d

Starches

*Significant differences between values in the same column are indicated by different letters (P < 0.05).

Table 3 Structural characteristics of SMApu modified corn starch Starches

Mn (g/mol)

Mw (g/mol)

Natural

2.08±0.64×107a

4.10±1.07×107a

80 U/g

3.59±0.58×105b

1.92±0.28×106b

160 U/g

2.22±0.15×105b

1.81±0.02×106b

240 U/g

1.83±0.8×105b

1.42±0.19×106b

*Significant differences between values in the same column are indicated by different letters (P < 0.05).

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Table 4 Amylopectin branch chain length of SMApu modified corn starch Starches

DP (<6)

DP (6-12)

DP (13-24)

Natural

1.46±0.17

a

29.89±1.82

80 U/g

1.55±0.43

a

160 U/g

2.56±0.75

240 U/g

2.68±0.21

a

45.66±1.18

25.13±1.86

b

47.48±0.15

a

21.89±1.70

b

48.30±0.65

a

20.85±0.14

b

48.8±0.26

DP (25-36)

DP (≥37)

DPW

b

14.27±1.15

a

8.72±1.77

ab

15.02±0.51

a

10.84±1.77

a

15.56±0.84

a

15.26±0.31

a

a

a

19.20±0.90

a

a

20.51±0.88

a

11.63±1.72

a

20.94±0.92

a

12.40±0.17

a

21.28±0.85

a

*Significant differences between values in the same column are indicated by different letters (P < 0.05).

Figure captions: Figure 1. SDS-PAGE of SMApu at different stages of purification from the B. subtilis lysate. Lane 1, cellular proteins from the crude extract; lane 2, proteins after heat treatment (70°C, 15 min); lane 3, purified SMApu after Ni-sepharose column chromatography; lane M, protein molecular weight markers.

Figure 2. RDS, SDS, and RS contents of SMApu-modified corn starch. Significant differences between values in the same component are indicated by different letters (P < 0.05).

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Figure 1.

20

100 90

RDS（%）

a

RS（%）

b

80

Relative content (%)

SDS（%）

70

c

60

d

50

b

40

a

30 20 10 0

c

d c Natural

b 80 U/g

Figure 2.

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b 160 U/g

a 240 U/g

Highlights • An amylopullulanase from archaeon S. marinus (SMApu) was expressed in B. subtilis. • Slowly digestible starch (SDS) of SMApu modified starch at 100 °C increased by 25.3%. • Structural changes of starch by SMApu partially explained the reduced digestibility. • Production of SDS by SMApu during thermal processing of cereal foods was proposed.

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