GBSS mutations in an SBE mutated background restore the potato starch granule morphology and produce ordered granules despite differences to native molecular structure.

Potato starch with mutations in starch branching enzyme genes (SBEI, SBEII) and granule-bound starch synthase gene (GBSS) was characterized for molecular and thermal properties. Mutations in GBSS were here stacked to a previously developed SBEI and SBEII mutation line. Additionally, mutations in the GBSS gene alone were induced in the wild-type variety for comparison. The parental line with mutations in the SBE genes showed a âˆ¼ 40 % increase in amylose content compared with the wild-type. Mutations in GBSS-SBEI-SBEII produced non-waxy, low-amylose lines compared with the wild-type. An exception was a line with one remaining GBSS wild-type allele, which displayed ∼80 % higher amylose content than wild-type. Stacked mutations in GBSS in the SBEI-SBEII parental line caused alterations in amylopectin chain length distribution and building block size categories of whole starch. Correlations between size categories of building blocks and unit chains of amylopectin were observed. Starch in GBSS-SBEI-SBEII mutational lines had elevated peak temperature of gelatinization, which was positively correlated with large building blocks.

Expression and characterization of 1,4-α-glucan branching enzyme from Microvirga sp. MC18 and its application in the preparation of slowly digestible starch.

Nutraceuticals containing modified starch with increased content of slowly-digestible starch (SDS) may reduce the prevalence of obesity, diabetes and cardiovascular diseases due to its slow digestion rate. Enzymatic methods for the preparation of modified starch have attracted increasing attention because of their low environmental impact, safety and specificity. In this study, the efficient glucan branching enzyme McGBE from Microvirga sp. MC18 was identified, and its relevant properties as well as its potential for industrial starch modification were evaluated. The purified McGBE exhibited the highest specificity for potato starch, with a maximal specific activity of 791.21 U/mg. A time-dependent increase in the content of α-1,6 linkages from 3.0 to 6.0% was observed in McGBE-modified potato starch. The proportion of shorter chains (degree of polymerization, DP < 13) increased from 29.2 to 63.29% after McGBE treatment, accompanied by a reduction of the medium length chains (DP 13-24) from 52.30 to 35.99% and longer chains (DP > 25) from 18.51 to 0.72%. The reduction of the storage modulus (G') and retrogradation enthalpy (ΔHr) of potato starch with increasing treatment time demonstrated that McGBE could inhibit the short- and long-term retrogradation of starch. Under the optimal conditions, the SDS content of McGBE-modified potato starch increased by 65.8% compared to native potato starch. These results suggest that McGBE has great application potential for the preparation of modified starch with higher SDS content that is resistant to retrogradation.

Synergistic effects of branching enzyme and transglucosidase on the modification of potato starch granules.

Potato starch displayed high viscosity, low hydroscopicity and dispersity, and acid susceptibility leading to the limited application of potato starch. To expand the potato starch utility with appropriate processing characteristics, potato starch granules were modified with branching enzyme (BE) and transglucosidase (TG). The results indicated that the susceptibility of potato starch granules to TG was higher than BE. Moreover, the two enzymes showed the synergistic effect in enzymatic modification of potato starch granules. They cooperatively attacked the external and interior of potato starch granules. The crystal forms of potato starch changed from B to C-type after double enzyme treatments, and enzyme-treated starches exhibited homogeneous crystal distribution. Compared to BE or TG alone, the combined action of BE and TG increased significantly the ratio of α-1,6-glycosidic linkage and the amounts of short chains of potato starch, which led to the significant reduction in degree of crystallinity, viscosity, gelatinization temperature and enthalpy, and a remarkable increase in solubility. Especially, the physicochemical characteristics of modified starch largely depended on the treatment time of TG. Thus, through the combination of BE and TG, the appropriate treatment time of TG may be chosen to improve the physicochemical properties of potato starch in processed starch-based products.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

BACKGROUND: Starch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers. RESULTS: A hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6-12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern. CONCLUSION: This work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.

Structural and physical properties of granule stabilized starch obtained by branching enzyme treatment.

Chemical cross-linking of starch is an important modification used in the industry for granule stabilization. It has been demonstrated that treatment with branching enzyme (BE) can stabilize the granular structure of starch and such treatment thereby provides a potential clean alternative for chemical modification. This study demonstrates that such BE-assisted stabilization of starch granules led to partial protection from BE catalysis of both amylose (AM) and amylopectin (AP) in their native state as assessed by triiodide complexation, X-ray diffractometry (XRD) and differential scanning calorimetry (DSC). The granule stabilizing effects were inversely linked to hydration of the starch granules, which was increased by the presence of starch-phosphate esters and suppressed by extreme substrate concentration. The data support that the granule stabilization is due to the intermolecular transglycosylation occurring in the initial stages of the reaction prior to AM-AP phase separation. The enzyme activity needed to obtain granule stabilization was therefore dependent on the hydration capability of the starch used.

Synthesis of branched polysaccharides with tunable degree of branching.

An in vitro enzyme-catalyzed tandem reaction using the enzymes phosphorylase b from rabbit muscle and Deinococcus geothermalis glycogen branching enzyme (Dg GBE) to obtain branched polyglucans with tunable degree of branching (2% ÷ 13%) is presented. The tunable degree of branching is obtained by varying the reaction conditions such as pH value, the choice of reducing agent and its concentration and reaction time. Linear amylose is formed by the phosphorylase-catalyzed propagation of glucose-1-phosphate while Dg GBE introduces branching points on the α-(1→6) position by relocating short oligosaccharide chains. Our results show that the best way to obtain different degrees of branching with this set of enzymes is by regulation of the reaction time.

Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes: starch synthase and branching enzyme from potato tubers.

Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.

Proteins from multiple metabolic pathways associate with starch biosynthetic enzymes in high molecular weight complexes: a model for regulation of carbon allocation in maize amyloplasts.

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.

A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A.

Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers. The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids. Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein. Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE. Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves. This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E. coli expressed SBE A. SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid. Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed. SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected. Even so, the composition and structure of tuber starch from these plants was greatly altered. The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding. In addition, the starch had much higher levels of phosphorous.

The multiple forms of starch-branching enzyme I in Solanum tuberosum.

Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the sbeI gene showed that four sbeI alleles exist in the cultivar studied. Two of these four alleles, sbeia and sbeIb, had slightly longer 3' ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbeIc and sbeId of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.

Evidence for essential arginine residues at the active sites of maize branching enzymes.

Alignment of 23 branching enzyme (BE) amino acid sequences from various species showed conservation of two arginine residues. Phenylglyoxal (PGO) was used to investigate the involvement of arginine residues of maize BEI and BEII in catalysis. BE was significantly inactivated by PGO in triethanolamine buffer at pH 8.5. The inactivation followed a time- and concentration-dependent manner and showed pseudo first-order kinetics. Slopes of 0.73 (BEI) and 1.05 (BEII) were obtained from double log plots of the observed rates of inactivation against the concentrations of PGO, suggesting that loss of BE activity results from as few as one arginine residue modified by PGO. BE inactivation was positively correlated with [14C]PGO incorporation into BE protein and was considerably protected by amylose and/or amylopectin, suggesting that the modified arginine residue may be involved in substrate binding or located near the substrate-binding sites of maize branching enzymes I and II.

Characterization of the 97 and 103 kDa forms of starch branching enzyme from potato tubers.

N-Terminal analysis, peptide mapping and partial peptide sequencing of the 97 and 103 kDa forms of starch branching enzyme from potato tubers showed that the two forms are highly related. A comparison with sequence data in the literature showed that these forms belong to the starch branching enzyme isoform I family. An internal cDNA fragment was obtained using PCR technology on potato tuber RNA with two oligonucleotide primers constructed from the peptide sequence data. Southern blot analysis using the PCR fragment as probe showed that there is only one gene locus encoding this isoform of the enzyme in Solanum tuberosum as well as in Solanum commersonii.