y: C istr urr m en e t h R Mukerjea et al., Organic Chem Curr Res 2015, 4:3 C e c s i e n a a r c g DOI: 10.4172/2161-0401.1000142 r h Organic Chemistry O ISSN: 2161-0401 Current Research ResearchResearch Article Article OpenOpen Access Access In Vitro Synthesis of Ten Starches by Potato Starch-Synthase and Starch- Branching-Enzyme Giving Different Ratios of Amylopectin and Amylose Mukerjea R, Sheets RL, Gray AN and Robyt JF* Laboratory of Carbohydrate Chemistry and Enzymology, Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA Abstract The objectives of this study was the use of two highly purified enzymes, potato Starch-Synthase (SS) and Starch- Branching-Enzyme (SBE) to obtain in vitro the syntheses of ten starches, with different ratios of amylopectin and amylose. The amylose was first synthesized by the reaction of SS with ADP [14C] Glc to give ten identical amyloses; the amylopectin was then synthesized by the action of SBE to give ten starches with different amounts of amylopectin. Two of the 14C-amyloses were reacted with 1.0 mIU of purified Starch-Branching-Enzyme (SBE) for 75 and 130 sec, respectively. In a second synthesis, 3 of the amyloses were autoclaved and reacted with 1.0 mIU for 15, 45, and 75 sec. In the third synthesis, 5 amyloses were reacted with different amounts of SBE from 0.50 to 0.01 mIU for 130 sec each. The 10 synthesized starches were separated into amylopectin and amylose fractions. The amylopectin and amylose ratios ranged from 99.9% amylopectin and 0.1% amylose to 10% amylopectin and 90% amylose. The results show that 10 different starches were synthesized, using only two enzymes, SS and SBE. No primers were involved. Glycogen and debranching enzymes were also not involved. Keywords: Starch-Synthase; Starch-Branching-Enzyme; In vitro reactions and therefore decreased in the chase reactions. In the present synthesis of starch; ADPGlc; Amylose; Amylopectin; Ten kinds of study, the in vitro biosynthesis of starch was performed by using only starches two highly purified potato starch enzymes: Starch-Synthase (SS) and Starch-Branching-Enzyme (SBE). The amounts of synthesized amylose Introduction was approximately the same for each reaction and ten different kinds Starch usually is composed of a mixture of two polysaccharides: of starches were obtained by the conversion of different amounts of amylose and amylopectin. Amylose is a α-1, 4-linked D-glucopyranose amylose into amylopectin by the reaction of SBE with the amyloses for polymer and amylopectin has a number of α-1,4-linked D-glucopyranose different lengths of time or by different amounts of SBE. chains, joined to other α-1,4-linked D-glucopyranose chains by 5-6 % Experimental α-1,6 branch glycosidic linkages [1]. Most starches contain 75-80 % amylopectin with 25-20 % amylose, respectively. There are, however, Materials some starches that contain 100% amylopectin (waxy varieties) and Adenosine 5’-diphospho-α-D-glucose (ADPGlc); dithiothreitol some that are high amylose starches, containing 50-80% amylose and (DTT); polyvinyl alcohol 50K; PPO and PoPoP were obtained from 50-20% amylopectin, respectively. Amylopectins are branched 5-6 Sigma-Aldrich Chem. Co. (St. Louis, MO. USA). ADP-[14C]Glc (333 %, giving average branch chain lengths of 20-16 D-glucopyranose mCi/mmol) was obtained from American Radiolabeled Chemicals, units, [1] respectively. Using pulse and chase reactions of highly Inc. 101 Arc Drive, St. Louis, MO 63147, USA. Amylose was obtained purified Starch-Synthase (SS) with ADP-[14C] Glc and nonlabeled by fractionation of potato starch as given in section, Separation of ADPGlc, respectively, we recently have shown that starch chains are Amylose from Amylopectin, in the Methods Section [3], or obtained biosynthesized de novo from the reducing-end, without a primer from Sigma-Aldrich Chem. Co. Starch-Synthase (SS) used was requirement [2]. The synthesis starts by forming two D-glucopyranosyl Fraction 23 [3] a highly purified enzyme, with a specific activity of 944 covalent intermediates at the active-site of SS; the C-4-OH group of mIU/mg (1.0 IU is an International Unit that incorporates 1.0 µmole one of the glucopyranosyl intermediates then makes an attack on the of D-glucose into starch/min) and was shown to be primer-free [4]. C-1-carbon of the other glucopyranosyl intermediate to transfer the Starch-Branching-Enzyme (SBE) was Fraction 4 [3]. It had a specific glucose and make a α-1,4-glycosidic linkage. The free catalytic group activity of 198 mIU/mg, where 1.0 IU equal 1.0 µM of α-1,6 branch then attacks another ADPGlc unit and forms a D-glucopyranosyl- linkages formed/min and also was shown to be primer-free, along with intermediate. The synthesis then proceeds processively, going back- and-forth between the covalent D-glucopyranosyl-C-4-OH groups and the growing D-glucopyranose-chains. The D-glucopyranosyl units are transferred to the reducing-ends of the growing chains. The synthesis *Corresponding author: John F Robyt, Laboratory of Carbohydrate Chemistry is called a “two catalytic-site insertion mechanism” and results in the and Enzymology, Department of Biochemistry, Biophysics and Molecular processive addition of D-glucopyranose from ADPGlc to the reducing- Biology, Iowa State University, Ames, IA 50011, USA, Tel: 5152941964; E-mail: ends of growing starch chains [2] (Figure 1) During the pulse reaction, [email protected] the reducing-ends become labeled with 14C-D-glucose and that when Received: June 25, 2015; Accepted: July 13, 2015; Published: July 20, 2015 the pulsed-polysaccharides are isolated, reduced with NaBH4, and Citation: Mukerjea R, Sheets RL, Gray AN, Robyt JF (2015) In Vitro Synthesis acid hydrolyzed, 14C-D-glucitol is obtained; and in the chase reaction, of Ten Starches by Potato Starch-Synthase and Starch-Branching-Enzyme Giving the 14C-D-glucose is chased from the reducing-ends into the chain, Different Ratios of Amylopectin and Amylose. Organic Chem Curr Res 4: 142. and in reduction and acid hydrolysis, the 14C-D-glucitol is decreased, doi:10.4172/2161-0401.1000142 because nonlabeled D-glucose units are being added to the reducing- Copyright: © 2015 Mubarak S, et al. This is an open-access article distributed ends in the chase. If the synthesis had been to the nonreducing-ends under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the of primers, no D-glucitol would ever have been formed in the pulsed original author and source are credited. Organic Chem Curr Res ISSN:2161-0401 OCCR an open access journal Volume 4 • Issue 3 • 1000142 Citation: Mukerjea R, Sheets RL, Gray AN, Robyt JF (2015) In Vitro Synthesis of Ten Starches by Potato Starch-Synthase and Starch-Branching- Enzyme Giving Different Ratios of Amylopectin and Amylose. Organic Chem Curr Res 4: 142. doi:10.4172/2161-0401.1000142 Page 2 of 5 containing 200 units of isoamylase (where 1 unit=1.0 nmole of α-(1→6) bonds hydrolyzed/min), and the isoamylase reaction was allowed to proceed for 30 min at 37°C. Three 100-µL aliquots were added to the wells of a microplate, containing 100 µL of copper bicinchoninate reagent, and the reducing value was determined, using maltose (20-200 µg/mL), as a standard [4]. The number of nmoles of α-(1→6) branched linkages formed/min by SBE was calculated from the reducing value obtained from the isoamylase reaction. A control blank was prepared by adding the Standard Buffer with the substrate, substituting water for the enzyme sample, and then carrying it through the isoamylase reaction, giving the exact reaction conditions used for the samples. One unit of SBE = 1.0 nmol of α-(1→6) branch linkages formed/min. Separation of Amylose from Amylopectin in the synthesized starches [5]: Potato starch (1.0 g in 100 mL) was added to each of the enzyme digests as a carrier when the reaction was over. The pH was adjusted to 7.0 and stirred for 30 min at 21-22°C; 22 mL of 1-butanol was added and stirred for 24 hr at 22°C to precipitate the amylose/1- butanol complexes and were then centrifuged at 20K rpm for 30 min, washed with 20 mL of water; and the amylose was then suspended in 40 mL of water, and the 1-butanol was removed by heating to boiling with the addition of water to maintain the volume. The amylose was then precipitated by the addition of 3 volumes of ethanol and placed at 4°C for 24 hr. The amylopectin that remained in the supernatant Figure 1: Distributions of amylopectin and amylose in the ten in vitro synthesized from the precipitation of the amylose by butanol-1 was precipitated ° starches by highly purified potato Starch-Synthase and Starch-Branching- by the addition of 3 volumes of ethanol and placed at 4 C for 24 hr. Enzyme. The amylose and amylopectin were centrifuged and their pellets were washed with 40 mL of acetone/water, 80/20 (v/v), and then treated with the Standard Buffer [2]. The Standard Buffer was pH 8.4 and contained 100 mL of acetone, and dried under vacuo for 24 hr. 10 mM glycine, 2 mM EDTA, 0.04% (w/v) polyvinyl alcohol 50K, and In vitro synthesis of starch by SS reacting with ADP-[14C] 1 mM DTT. Liquid Scintillation Cocktail was prepared, containing 5.0 g PPO and 0.1 g PoPoP in 1 L of toluene (Table 1). Glc and SBE reacting with the synthesized amylose to give amylopectin Methods Synthesis type I: Two reactions were performed.