Synthesis of poly-(ε)-caprolactone grafted starch co-polymers by ring-opening polymerisation using silylated starch precursors

Poly-(ε)-caprolactone grafted corn starch co-polymers were synthesized using a hydrophobised silylated starch precursor. The silylation reaction was performed using hexamethyl disilazane (HMDS) as the reagent in DMSO at 70 °C. Silylated starch with a degree of substitution (DS) between 0.45 and 0.7 was obtained. ε-Caprolactone is grafted to silylated starch by a ring-opening polymerisation catalysed by Al(OiPr)₃ in THF at 50 °C. The grafting efficiency varies between 28% and 58%, the remainder being homopolymers of ε-caprolactone. The DS of the polycaprolactone graft is between 0.21 and 0.72. The poly-(ε)-caprolactone side chains consist of 40–55 monomer units and is a function of the reagent intakes. Experiments with native starch under similar conditions do not result in the desired poly-(ε)-caprolactone grafted corn starch co-polymers and unreacted starch was recovered after work-up. Removal of the silyl groups of the poly-(ε)-caprolactone grafted starch co-polymers is possible using a mild acid treatment with diluted hydrochloric acid in THF at room temperature.     

Rapid shoot regeneration in industrial ‘high starch’ sweetpotato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> L.) genotypes

Sweetpotato (Ipomoea batatas L.) is an important crop in North Carolina with annual production of 0.33 million tons, accounting for 37% of total US supply (USDA, Louisiana Farm Reporter 8(12), August 2008). To target industrial use, novel high-starch industrial-type varieties that contain more than 30% dry matter were developed by conventional breeding methods. In vitro cultures from selected genotypes were established using meristem culture. To establish regeneration procedures that could be coupled with transformation experiments, conditions for the induction of rapid shoot-organogenesis in leaf explants were compared using varying concentrations of the auxins ‘NAA’, ‘IAA’, ‘2,4-D’, and ‘4-FA’ either alone or in combination with zeatin riboside. Regeneration efficiencies, defined as the number of explants developing shoots out of the total number tested, were as high as 57% for the best genotypes, with a significant genotype-dependent response observed in all the hormone regimes evaluated. In all treatments, shoot regeneration was observed within 2 months. Our results led to the establishment of optimized in vitro regeneration procedures for the novel high-starch sweetpotato (SP) genotypes ‘DM01-158’, ‘FTA94’, ‘FT489’, and ‘PDM P4’ that are rapid and reliable.     

Immobilization of porcine pancreatic <Emphasis Type="Italic">α</Emphasis>-amylase on magnetic Fe<Subscript>2</Subscript>O<Subscript>3</Subscript> nanoparticles: Applications to the hydrolysis of starch

Enzymes play a pivotal role in catalyzing diverse reactions. However, their instability upon repetitive/prolonged use, as well as their inhibition by high substrates and product concentration, remains an area of concern. In this study, porcine pancreatic α-amylase was immobilized on magnetic Fe₂O₃ nanoparticles (Fe₂O₃-NPs) in order to hydrolyze starch. The magnetic nanoparticle bound enzymes retained 94% of their initial enzyme activity. X-ray diffraction and atomic force microscopy analyses showed that the prepared matrix had advantageous microenvironment and a large surface area for binding significant amounts of protein. Functional groups present in enzyme and support were monitored by Fourier transform infrared spectroscopy. Immobilized enzyme exhibited lowered pH optimum (pH 6.0) to a greater degree than its soluble counterpart (pH 7.0). Optimum temperature for the immobilized enzyme shifted towards higher temperatures. The immobilized enzyme was significantly more resistant to inactivation caused by various metal ions and chemical denaturants. Immobilized α-amylase hydrolyzed 92% starch in a batch process, after 8 h at 40°C; while the free enzyme could hydrolyze only 73% starch under similar experimental conditions. A reusability experiment demonstrated that the immobilized enzyme retained 83% of its original activity even after its 8^th repeated use.     

Direct production of cadaverine from soluble starch using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> coexpressing α-amylase and lysine decarboxylase

Here, we demonstrated the one-step production of cadaverine from starch using a Corynebacterium glutamicum strain coexpressing Streptococcus bovis 148 α-amylase (AmyA) and Escherichia coli K-12 lysine decarboxylase (CadA). We constructed the E. coli–C. glutamicum shuttle vector, which produces CadA under the control of the high constitutive expression (HCE) promoter, and transformed this vector into C. glutamicum CSS secreting AmyA. The engineered C. glutamicum expressed both CadA and AmyA, which retained their activity. We performed cadaverine fermentation using 50 g/l soluble starch as the sole carbon source without pyridoxal-5’-phosphate, which is the coenzyme for CadA. C. glutamicum coexpressing AmyA and CadA successfully produced cadaverine from soluble starch and the yield of cadaverine was 23.4 mM after 21 h. CadA expression levels under the control of the HCE promoter were assumed to be sufficient to convert l-lysine to cadaverine, as there was no accumulation of l-lysine in the culture medium during fermentation. Thus, we demonstrated that C. glutamicum has great potential to produce cadaverine from biomass resources.     

A single nucleotide polymorphism in the “<Emphasis Type="Italic">Fra</Emphasis>” gene results in fractured starch granules in barley

Key message

We report here that the mutation causing fractured starch granules in the barley line “Franubet” results from a point mutation in the barley gene corresponding to the rice FLO6 gene.

Abstract

The “fra” mutation in barley, which was originally isolated and characterized over 30 years ago, results in fractured starch granules and an opaque phenotype. This mutation has been used in breeding programs, since it appears to be useful in the production of pearled barley for human consumption. However, selection for this phenotype is difficult, since wild-type and heterozygous kernels cannot be distinguished phenotypically, and until now, the gene involved in this mutation has not been determined. Here, we used a map-based cloning approach using nanopore sequencing to obtain long reads from a BAC clone carrying markers on either side of the fra locus. By fine mapping followed by aligning RNA-seq reads to four genes within the mapped region, we were able to determine that the fra mutation is caused by the introduction of a stop codon in the barley homologue of the rice FLOURY ENDOSPERM 6 (FLO6) gene. This gene has a CBM48 domain that binds to starch, and may act through interactions with isoamylase1 (ISA1), assisting in the binding of ISA1 to starch granules. Perfect markers able to distinguish all genotypes were designed and tested in several large populations; in all cases, the markers were able to distinguish wild-type, heterozygous, and mutant genotypes.

     

HRP-mediated synthesis of starch–polyacrylamide graft copolymers

Starch was reacted with acrylamide in water in the presence of horseradish peroxidase (HRP) catalyst/H₂O₂/2,4 pentanedione to give starch–polyacrylamide graft copolymers.     

High day–night transition temperature alters nocturnal starch metabolism in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transitory starch plays a vital role in maintenance respiration as its degradation products provide substrate for the night respiration. A study was conducted with two contrasting rice cultivars: Vandana (high night temperature susceptible) and Nagina 22 (high night temperature tolerant) by subjecting them to increase in transition temperature from anthesis to physiological maturity. Night respiration on plant area basis increased by 35% in Vandana at 5 days after anthesis but was unaffected in tolerant cultivar. A simultaneous 18% decrease in starch content was observed in the susceptible cultivar. An analysis of the starch-metabolizing enzymes showed that activity of β-amylase increased markedly in Vandana whereas both β and α-amylase decreased in Nagina 22 following high day to night transition temperature exposure. The level of starch breakdown product, maltose, increased in the susceptible cultivar but glucose levels declined in both the cultivars. Concurrently, expression of chloroplastic isoforms α-amylase OsAMY1, OsAMY2 and β-amylase OsBAM2 increased in Vandana. A lower accumulation of dry matter was recorded in the susceptible than the tolerant cultivar. Our study elucidated the regulatory role of transitory starch in supporting the high day to night transition temperature-induced night-time respiration which is mediated by the increased activity of β-amylase through enhanced expression of OsBAM2 in flag leaves of susceptible cultivar.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-Lysine from starch by <Emphasis Type="BoldItalic">Corynebacterium glutamicum</Emphasis> displaying α-amylase on its cell surface

We engineered a Corynebacterium glutamicum strain displaying α-amylase from Streptococcus bovis 148 (AmyA) on its cell surface to produce amino acids directly from starch. We used PgsA from Bacillus subtilis as an anchor protein, and the N-terminus of α-amylase was fused to the PgsA. The genes of the fusion protein were integrated into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. l-Lysine fermentation was carried out using C. glutamicum displaying AmyA in the growth medium containing 50 g/l soluble starch as the sole carbon source. We performed l-lysine fermentation at various temperatures (30–40°C) and pHs (6.0–7.0), as the optimal temperatures and pHs of AmyA and C. glutamicum differ significantly. The highest l-lysine yield was recorded at 30°C and pH 7.0. The amount of soluble starch was reduced to 18.29 g/l, and 6.04 g/l l-lysine was produced in 24 h. The l-lysine yield obtained using soluble starch as the sole carbon source was higher than that using glucose as the sole carbon source after 24 h when the same amount of substrates was added. The results shown in the current study demonstrate that C. glutamicum displaying α-amylase has a potential to directly convert soluble starch to amino acids.     

Hydrolysis of starch particles using immobilized barley α-amylase

Barley α-amylase has been immobilized on silica particles with diameters between 0.5 and 10 μm using a covalent binding method. Immobilization procedures were adjusted to optimize enzyme activity. The effects of product inhibition, thermal stability and operational stability have been determined. The feasibility of using the immobilized enzyme to hydrolyze wheat starch particles at temperatures below the gelatinization temperature (<55 °C) was proven. The optimal conditions for the hydrolysis were found to be: pH 4.5, 40 °C, calcium ion concentration 0.002 M and immobilized enzyme loading of 30 mg/ml. At these conditions, the immobilized enzyme was able to hydrolyze wheat starch particles at concentrations as high as 100 mg/ml with a final conversion of 90% after 24 h of operation. Maltose and glucose were found to inhibit the immobilized enzyme in a similar manner as reported previously using soluble enzyme. Although the thermostability of the immobilized enzyme was superior to the soluble enzyme, the immobilized enzyme degraded at the same rate as the soluble enzyme during cold wheat starch hydrolysis (operational stability unchanged). Model equations are presented for product inhibition, hydrolysis kinetics and enzyme degradation. Using best-fit parameters, the equations are shown to fit the experimental data well.     

Development of new α-amylases for raw starch hydrolysis

This paper describes the discovery of a new 4 domain α-amylase from Anoxybacillus contaminans which very efficiently hydrolyses raw starch granules. Compared to traditional starch liquefying α-amylases, this new 4 domain α-amylase contains a starch binding domain. The presence of this starch-binding domain enables the enzyme to efficiently hydrolyse starch at a temperature below the gelatinisation temperature. At a reaction temperature of 60°C and in combination with a glucoamylase from Aspergillusniger it was possible to liquefy 99% of the starch obtaining a DX value of 95%.

Furthermore, we describe how the current HFCS process can be turned into a low temperature simultaneous liquefaction and saccharification process by using this new 4 domain α-amylase in combination with a glucoamylase.     

Osmotic regulation of starch synthesis in potato tubers?

Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 mM but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300–400 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 mM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.     

<Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> – a producer for simultaneous saccharification and fermentation of starch waste materials to l(+)-lactic acid

Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharification and fermentation process using starch waste effluents. Lactic acid at 19.5-44.3 g l(-1) with a yield of 0.85-0.96 g g(-1) was produced in 40 h using 20-60 g starch l(-1). Supplementation of nitrogen source may be unnecessary if potato or corn starch waste effluent was used as a production medium.     

Why does the establishment of the starch preferring<Emphasis Type="Italic">Entodinium caudatum</Emphasis> in the rumen decrease the numbers of the fibrolytic ciliate<Emphasis Type="Italic">Eudiplodinium maggii</Emphasis>?

The effect of the establishment of Entodinium caudatum on the population of Eudiplodinium maggii was examined in the rumen of three sheep fed a hay/ground barley diet. The cell concentration of E. maggii were 15.9-38.5 and 11.7-12.4 x 10(3) cells per g of the rumen contents in the absence and presence of E. caudatum, respectively. Microscopic analysis showed that starch was the only material engulfed by eudiplodinia irrespective of the time after feeding and the presence or absence of E. caudatum. Up to 82-93% of individuals contained starch grains when E. maggii was the only ciliate species in the rumen; the proportion was 70-77% after entodinia had been established. The largest quantity of starch engulfed by E. maggii ciliates was 12.4-19.0 and 6.7-7.6 mg per 100 mg protozoal dry mass in the absence and presence of entodinia, respectively. No visible engulfment of hay was observed in vivo in spite of the fact that hay particles up to 42 microns in length were dominating in rumen fluid. Ingestion of fresh particles of hay separated from the rumen digesta was found when they were added in the proportion of 1 g per 40 mL suspension of ciliates. No preferential intake of starch was observed when E. maggii ciliates were incubated in vitro with a mixture of hay and barley starch. It is suggested that competition for starch between the two ciliate species was responsible for the drop in the numbers of E. maggii. This could result from a too low concentration of small particles of hay in the rumen fluid.     

Biochemical characterization of a raw starch degrading <Emphasis Type="Italic">α</Emphasis>-amylase from the Indonesian marine bacterium <Emphasis Type="Italic">Bacillus</Emphasis> sp. ALSHL3

An Indonesian marine bacterial isolate, which belongs to genus of Bacillus sp. based on 16S rDNA analysis and was identified as Bacillus filicolonicus according to its morphology and physiology, produced a raw starch degrading α-amylase. The partially purified α-amylase using a maize starch affinity method exhibited an optimum pH and temperature of 6.0 and 60°C, respectively. The enzyme retained 72% of its activity in the presence of 1.5 M NaCl. Scanning electron micrographs showed that the α-amylase was capable of degrading starch granules of rice and maize. This α-amylase from Bacillus sp. ALSHL3 was classified as a saccharifying enzyme since its major final degradation product was glucose, maltose, and maltotriose.     

Cloning and expression of acidstable,high maltose-forming,Ca<Superscript>2+</Superscript>-independent α-amylase from an acidophile <Emphasis Type="Italic">Bacillus acidicola</Emphasis> and its applicability in starch hydrolysis

The α-amylase encoding gene from acidophilic bacterium Bacillus acidicola was cloned into pET28a(+) vector and expressed in Escherichia coli BL21 (DE3). The recombinant E. coli produced a 15-fold higher α-amylase than B. acidicola strain. The recombinant α-amylase was purified to homogeneity by one-step nickel affinity chromatography using Ni²⁺-NTA resin with molecular mass of 62 KDa. It is active in the pH range between 3.0 and 7.0 and 30 and 100 °C with optimum at pH 4.0 and 60 °C. The enzyme is Ca²⁺-independent with K _m and k _cat values (on soluble starch) of 1.6 mg ml⁻¹ and 108.7 s⁻¹, respectively. The α-amylase of B. acidicola is acidstable, high maltose forming and Ca²⁺-independent, and therefore, is a suitable candidate for starch hydrolysis and baking.     

Raw starch fermentation to ethanol by an industrial distiller’s yeast strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing glucoamylase and α-amylase genes

Industrial strains of a polyploid, distiller’s Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l⁻¹) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.     

Structure and properties of starch/α-zirconium phosphate nanocomposite films

Glycerol-plasticized pea starch/α-zirconium phosphate (PS/ZrP) nanocomposite films with different loading levels of α-zirconium phosphate (α-ZrP) were prepared by a casting and solvent evaporation method. The effects of the α-ZrP on the structure and properties of the PS/ZrP films were characterized by Fourier transform infrared (FT-IR) spectroscopy, wide-angle X-ray diffraction (XRD), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and tensile testing. The results indicated that hydrogen bonds formed between pea starch (PS) and α-ZrP, which improved the compatibility between PS and α-ZrP. Compared with the neat PS, the tensile strength (σ_b) and elongation at break (ε_b) of the PS/ZrP nanocomposite films were significantly enhanced with an increase in α-ZrP content. The maximum values of σ_b and ε_b reached 9.44 MPa and 47.5%, respectively, at 0.3% α-ZrP and 25% glycerol as plasticizer. The moisture uptake of the nanocomposite films, measured in an environment with 92% relative humidity, was reduced by the addition of α-ZrP. The structure and properties of pea starch-based films were modified and improved by the incorporation of α-ZrP.     

Monte Carlo simulation of the α-amylolysis of amylopectin potato starch

The branched structure of potato amylopectin (degree of polymerization ~200,000) was modeled in a computer matrix. The chain-length distribution and the length and width of a cluster of the amylopectin molecule were used as input variables in the model. Independent literature values related to the structure of amylopectin (percentage #-hydrolysis and ratio of A- to B-chains) were used for evaluation of the branching characteristics (length of branch area and chance of branching) of the modeled amylopectin. The structural parameters predicted by the model agreed very well with data from the literature. The chain-length distribution and values for the percentage of #-hydrolysis were the two most important parameters required to model the structure of amylopectin. This computer-generated model of potato amylopectin in solution can be used to simulate various enzymatic (i.e., !-amylase, #-amylase, glucoamylase, pullunanase) or chemical reactions (i.e., acid hydrolysis, hypochlorite oxidation). The modeling approach described in this paper is also suitable for starches from other botanical sources (i.e., corn, wheat, tapioca).     

The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos

Mutations that reduced the rate of starch synthesis in pea (Pisum sativum L.) embryos through effects on enzymes on the pathway from sucrose to adenosine 5′-diphosphoglucose (ADPglucose) also led to a reduction in the amylose content of the starch of developing embryos. Evidence is presented that this relationship between rate of synthesis and the composition of starch is due to the fact that amylopectin-synthesising isoforms of starch synthase have higher affinities for ADPglucose than the amylose-synthesising isoform. First, developing mutant embryos (rb, rug3 and rug4 mutants) displayed both reduced amylose contents in their starches and reduced ADPglucose contents relative to wild-type embryos. Second, incubation of detached, wild-type embryos for 6 h at high and low glucose concentrations resulted in differences in both ADPglucose content and the relative rates of amylose and amylopectin synthesis. At 0.25 M glucose both ADPglucose content and the proportion of synthesised starch that was amylose were about twice as great as at 25 μM glucose. Third, S _0.5 values for soluble (amylopectin-synthesising) starch synthases in developing embryos were several-fold lower than that for granule-bound (amylose synthesising) starch synthase. Estimates of the expected amylose contents of the starch of the mutant embryos, based on the reduction in their ADPglucose contents and on the S _0.5 values of the starch synthases, were very similar to the measured amylose contents. The implications of these results for the determination of starch composition are discussed. Received: 6 February 1999 / Accepted: 22 May 1999     

Display of α-amylase on the surface of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> cells by using NCgl1221 as the anchoring protein,and production of glutamate from starch

We developed a new cell surface display system in Corynebacterium glutamicum based on the C-terminally truncated NCgl1221 anchor protein to increase l-glutamate production from starch directly. The C-terminally truncated NCgl1221 protein is a mutant NCgl1221 and leads to the constitutive export of l-glutamate. The N terminus of α-amylase (AmyA) was fused to truncated NCgl1221, and the resulting fusion protein was expressed on the cell surface by IPTG induction. Localization of the fusion protein was confirmed by immunofluorescence microscopy and flow cytometric analysis. The results of l-glutamate fermentation showed that the soluble starch was utilized to grow and produce l-glutamate by the recombinant strain displaying AmyA. The amount of soluble starch was reduced from 30.0 ± 2.8 to 4.5 ± 0.7 g/l under non-inducing condition and from 50.0 ± 2.4 to 12.5 ± 1.1 g/l under biotin limitation in 36 h. The glutamate concentration in the medium was transiently increased in 14 h under no induction, while under biotin-limiting condition, glutamate production was continuously elevated during fermentation. The amount of glutamate reached 19.3 ± 2.1 g/l after 26 h of fermentation with biotin limitation, which was greater than that produced by the strain using PgsA, one of the poly-γ-glutamate synthetase complexes, as the anchor protein under the same condition. Therefore, the truncated NCgl1221 anchor protein has more advantages than the PgsA anchor protein in glutamate fermentation because truncated NCgl1221 leads to the constitutive export of l-glutamate without any treatments.