IMO - American Chemical Society

(1) Kwon, K. S.; Auh, J. H.; Choi, S. K.; Kang, G. J.; Kim, J. W.;. Park, K. H. Characterization of branched oligosaccharides produced by Bacillus lic...
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J. Agric. Food Chem. 2002, 50, 2812−2817

Cooperative Action of r-Glucanotransferase and Maltogenic Amylase for an Improved Process of Isomaltooligosaccharide (IMO) Production HYUN-SOO LEE,† JOONG-HYUCK AUH,† HYUN-GEUN YOON,† MYO-JEONG KIM,‡ JIN-HEE PARK,‡ SEUNG-SUH HONG,† MIN-HYUNG KANG,† TAE-JIP KIM,‡ TAE-WHA MOON,‡ JUNG-WAN KIM,§ AND KWAN-HWA PARK*,‡ Samyang Genex Biotech Research Institute, 63-2 Hwaam-Dong, Yusung-Gu, Taejeon 305-348, Korea; Department of Food Science and Technology and Research Center for New Bio-Materials in Agriculture, College of Agriculture and Life Sciences, Seoul National University, Suwon 441-744, Korea; and Department of Biology, University of Incheon, Incheon 402-749, Korea

Maltogenic amylase and R-glucanotransferase (R-GTase) were employed in an effort to develop an efficient process for the production of isomaltooligosaccharides (IMOs). Bacillus stearothermophilus maltogenic amylase (BSMA) and R-GTase from Thermotoga maritima were overexpressed in Escherichia coli using overexpression vectors. An IMO mixture containing 58% of various IMOs was produced from liquefied corn syrup by the hydrolyzing and transglycosylation activities of BSMA alone. When BSMA and R-GTase were reacted simultaneously, the IMO content increased to 68% and contained relatively larger IMOs compared with the products obtained by the reaction without R-GTase. Time course analysis of the IMO production suggested that BSMA hydrolyzed maltopentaose and maltohexaose most favorably into maltose and maltotriose and transferred the resulting molecules simultaneously to acceptor molecules to form IMOs. R-GTase transferred donor sugar molecules to the hydrolysis products such as maltose and maltotriose to form maltopentaose, which was then rehydrolyzed by BSMA as a favorable substrate. KEYWORDS: Bacillus stearothermophilus maltogenic amylase; isomaltooligosaccharides (IMOs); Thermotoga maritima; r-glucanotransferase (r-GTase)

INTRODUCTION

Production of isomaltosaccharides (IMOs) with various compositions and useful properties is in great demand in the starch industry. High-IMO syrups are characterized by low viscosity, resistance to crystallization, and reduced sweetness (1-3). They have been developed to prevent dental caries, as substitute sugars for diabetics, or to improve the intestinal microflora. Chemically, IMOs are glucosyl saccharides consisting solely of R-(1,6)-glycosidic linkages, but IMOs available commercially include all glucosyl saccharides with R-(1,6)glycosidic linkages such as isomaltose, panose and isopanose. Transglycosylation catalyzed by amylases and their related enzymes has been utilized in the industry for the production of various oligosaccharides (4). Many bacterial saccharifying R-amylases catalyze R-(1,4)-transglycosylation in addition to the hydrolysis of R-(1,4)-glycosidic linkages. Maltogenic amylases (EC 3.2.1.133) from various bacteria exhibited both R-(1,6)* Corresponding author (telephone 82-31-290-2582; fax 82-31-293-4789; e-mail [email protected]). † Samyang Genex Biotech Research Institute. ‡ Seoul National University. § University of Incheon.

transglycosylation and R-(1,4)-hydrolysis activities (5-8). Action modes of maltogenic amylases have been studied in detail, which were mainly composed of three steps: maltosyl transfer, glucosyl transfer, and condensation (9-12). The coupled transglycosylation and hydrolysis activities of maltogenic amylases (13, 14) were used to produce IMOs from liquefied starch in a more efficient way than the traditional process, which usually required more enzymes and longer time. The maximum concentration of IMOs accumulated in the final reaction mixture was 58% using maltogenic amylase, whereas it was only 40% in the traditional process. Enzymic synthesis of a wide variety of oligosaccharides has been attained in vitro using the transfer reactions between a segment of donor and various acceptors (13, 15). Usually the transfer takes place from a specific donor to a relatively large number of structurally different acceptors. Specificity of the transfer is dependent on the enzyme used, which usually determines the configuration of the glycosidic bond that is formed. The structure of the acceptor also often plays a role in determining the position of transfer for the formation of a glycosidic bond.

10.1021/jf011529y CCC: $22.00 © 2002 American Chemical Society Published on Web 04/18/2002

IMO Production Using Cooperative Action of R-GTase and BSMA

J. Agric. Food Chem., Vol. 50, No. 10, 2002

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Figure 1. Restriction maps of pGNX3 and pGNX4. A 1.7 kb NcoI−HindIII fragment containing the BSMA gene was inserted at the BspLU11I/HindIII

sites, and a 1.3 kb NdeI−HindIII fragment containing the R-GTase gene was inserted at the corresponding sites on the vectors.

In this study, R-glucanotransferase (R-GTase; EC 2.4.1.25) from Thermotoga maritima with R-(1,4)-transferring and liquefying activities was introduced in an effort to improve the IMO productivity using liquefied starch and maltogenic amylase from Bacillus stearothermophilus ET1 (BSMA). For that purpose, both enzymes were overexpressed in Escherichia coli from the corresponding genes subcloned on expression vectors, pGNX3 and pGNX4, and used to determine the most appropriate conditions for effective IMO production. The cooperative action mode of the two enzymes to enhance the production of IMOs from liquefied starch was also investigated. MATERIALS AND METHODS Bacterial Strains and Plasmids. E. coli MC1061 [F-, araD139, recA13, ∆(araABC-leu)7696, galU, galK, ∆lacX74, rpsL, thi, hsdR2, mcrB] and HB101 [F-, supE44, ara-14, proA2, galK2, lacY1, rpsL20, xyl-5, mtl-1, hsdS20(rB- mB-)] were used as hosts for DNA manipulation and transformation. E. coli TG1 [supE, hsd∆5, thi, ∆(lac-proAB), F′(traD36, proAB+, lacIq, lacZ∆M15)] and BL21 [F-, ompT, hsdSB(rBmB-), gal, dcm(DE3)] were used as hosts for overexpression of the clones constructed in this study. E. coli MC1061 and HB101 were grown in LB medium (1% Bacto-tryptone, 0.5% yeast extract, and 0.5% NaCl) at 37 °C. E. coli transformants were cultured in LB medium containing ampicillin (100 µg/mL) or kanamycin (100 µg/mL). E. coli vectors, pBR322, pUC119, and pBluescript II SK (Stratagene, La Jolla, CA), were used for cloning and subcloning (16). Overexpression and Purification of Enzymes. To overexpress BSMA or R-GTase in E. coli, the corresponding genes were subcloned into pGNX3 and pGNX4 under the control of the T7 and tac promoters, respectively. The resulting transformants were screened for starch hydrolyzing activity by the iodine test after treatment with D-cycloserine (5). E. coli transformants harboring the genes were cultured in LB broth containing kanamycin. Expression of the gene cloned on pGNX4 was induced by the addition of IPTG (1 µmol/mL) when the culture reached mid-log phase. The cells harvested by centrifugation (5000g, 4 °C, 20 min) were resuspended in 4 mL of 50 mM Tris-HCl buffer (pH 7.5) per gram of wet cells. Cells were homogenized (Microfluidizer; Microfluidics Co., Newton, MA), and polyethylenimine (PEI) was carefully added to a final concentration of 0.5% (w/v) to remove nucleic acids. To purify BSMA, the precipitate was removed by centrifugation (12000g, 4 °C, 30 min) and washed once more. The supernatant was dialyzed against 50 mM Tris-HCl buffer (pH 7.5) and concentrated

using a Pellicon ultrafiltration kit (Millipore Co., Bedford, MA; MW cutoff 10000). Other proteins were precipitated in 20% (w/v) ammonium sulfate at 4 °C for 2 h in an ice bath. Proteins in the supernatant were precipitated in saturated ammonium sulfate (60%, w/v) at 4 °C for 2 h. The pellet harvested by centrifugation (10000g, 4 °C, 15 min) was resuspended in 50 mM Tris-HCl buffer (pH 7.5) and dialyzed against the same buffer (pH 7.5) before being concentrated using a Pellicon ultrafiltration kit (Millipore Co.; MW cutoff 10000). The partially purified BSMA solution was used for enzymatic reactions in this study. To purify R-GTase, the cells were incubated at 80 °C for 20 min in a water bath, and then PEI was carefully added to a final concentration of 0.5% (w/v). The precipitate was removed by centrifugation (12000g, 4 °C, 30 min) and washed once more. The supernatant was dialyzed against 50 mM Tris-HCl buffer (pH 7.5) and concentrated using a Pellicon ultrafiltration kit (Millipore Co.; MW cutoff 10000). Enzyme Assay. BSMA activity was assayed according to the dinitrosalicylic acid (DNS) method (17) by determining the amount of reducing sugar produced by the enzyme. The enzyme reaction mixture was composed of 250 µL of 1% (w/v) substrate solution (β-CD or soluble starch) in 50 mM sodium acetate buffer (pH 6.0), 160∼220 µL of reaction buffer (50 mM sodium acetate buffer, pH 6.0), and 30∼90 µL of enzyme solution. A reaction mixture was preincubated at 55 °C for 5 min before diluted enzyme solution was added and incubation continued for 30 min. The reaction mixture was stopped by adding 1.5 mL of DNS and boiling for 5 min. The reaction was cooled immediately by placing the tube under running water. Absorbance was measured at 575 nm using a spectrophotometer (UV-1601, Shimadzu, Kyoto, Japan). One unit of β-CD hydrolyzing activity (CU) was defined as the amount of BSMA that formed reducing sugars to give 1.0 unit of ∆Abs575. The activity of R-GTase was measured by the change in iodinestaining properties during the conversion of amylose in the presence of maltose (18). The assay mixture containing 0.05% amylose, 0.05% maltose, 50 mM Tris-HCl buffer (pH adjusted to 7.5 at 60 °C), and the enzyme was incubated at 60 °C. Samples (0.1 mL) taken at 0 and 15 min were mixed with 1 mL of 0.02% iodine/potassium iodide solution (Lugol’s solution diluted 1:50 in 50 mM Tris/HCl buffer, pH 7.5), and absorbance at 620 nm was measured immediately with a spectrophotometer. The absorbance at 0 min was ∼1.0. The difference between the absorbances of each sample taken at 0 and 15 min was used to estimate the enzyme unit. One unit of R-GTase activity (AU) was arbitrarily defined as the amount of enzyme that caused a change of absorbance by 1 in 15 min under the conditions described above. The assay was reproducible and linear for the range of enzyme concentrations causing absorbance differences up to 0.5. Protein

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Table 1. Expression Levels of BSMA and R-GTase from Various Constructs R-GTase

BSMA constructs host plasmids promoters expression levela a

pSG18 TG1 pUC18 BSMA