Starch Binding Research Articles

Stbd1-deficient mice display insulin resistance associated with enhanced hepatic ER-mitochondria contact

Starch binding domain-containing protein 1 (STBD1) is an endoplasmic reticulum (ER)-resident, glycogen-binding protein. In addition to glycogen, STBD1 has been shown to interact with several proteins implicated in glycogen synthesis and degradation, yet its function in glycogen metabolism remains largely unknown. In addition to the bulk of the ER, STBD1 has been reported to localize at regions of physical contact between mitochondria and the ER, known as Mitochondria-ER Contact sites (MERCs). Given the emerging correlation between distortions in the integrity of hepatic MERCs and insulin resistance, our study aimed to delineate the role of STBD1 in vivo by addressing potential abnormalities in glucose metabolism and ER-mitochondria communication associated with insulin resistance in mice with targeted inactivation of Stbd1 (Stbd1KO). We show that Stbd1KO mice at the age of 24 weeks displayed reduced hepatic glycogen content and aberrant control of glucose homeostasis, compatible with insulin resistance. In line with the above, Stbd1-deficient mice presented with increased fasting blood glucose and insulin levels, attenuated activation of insulin signaling in the liver and skeletal muscle and elevated liver sphingomyelin content, in the absence of hepatic steatosis. Furthermore, Stbd1KO mice were found to exhibit enhanced ER-mitochondria association and increased mitochondrial fragmentation in the liver. Nevertheless, the enzymatic activity of hepatic respiratory chain complexes and ER stress levels in the liver were not altered. Our findings identify a novel important role for STBD1 in the control of glucose metabolism, associated with the integrity of hepatic MERCs.

Reducing the Fermentability of Wheat with a Starch Binding Agent Reduces Some of the Negative Effects of Heat Stress in Sheep.

Simple SummaryHeat stress is one of the major problems affecting the thermotolerance of livestock in Australia and in many parts of the world. Dietary interventions are considered to be one of the effective strategies to beat the heat. Hence, this study evaluated the benefits of reducing the fermentability of wheat grains (BioprotectTM) in the diet of Merino lambs. These results indicated that Bioprotect supplementation had a positive effect on the respiratory rate and heart rate of heat-stressed lambs.The objective of this study was to investigate the effects of reducing the fermentability of grains on thermoregulatory responses in heat stressed (HS) lambs. To achieve this, wheat grain treated with a commercial starch binding agent, Bioprotect, is compared to maize, which has already demonstrated effects in ameliorating heat stress-induced thermoregulation responses and untreated wheat grains. An initial in vitro experiment was conducted to examine cumulative gas production from the fermentation of wheat grain with different dosages of the commercial starch binding agent, Bioprotect. Based on the in vitro results, an in vivo lamb experiment was conducted using 24 Merino lambs (1 year old; 42.6 ± 3.6 kg BW). The lambs were offered one of three dietary treatments: a wheat-based diet (WD), a Bioprotect treated wheat-based diet (BD), and a maize-based diet (MD). Three successive 1-week experimental periods were conducted with lambs from all dietary groups (P1, P2, and P3). During P1, lambs were exposed to a TN environment and fed a 1.7× Maintenance feed intake (MF) level; in P2, lambs were kept in a HS environment and fed a 1.7× MF level; and in P3, animals were kept in a HS environment and fed a 2× MF level. The in vitro experiment revealed a reduction in cumulative gas production (p < 0.05) from the Bioprotect treated wheat compared to untreated wheat samples. In the in vivo component of the study, the replacement of wheat with maize or 2% Bioprotect-treated wheat reduced the respiration rate (p < 0.001) and heart rate (p ≤ 0.01) of lambs during HS. There was a reduction in the concentration of blood gas variables such as a base excess of blood (BE(b)) and extracellular fluid (BE(ecf)), bicarbonate (CHCO3−), the partial pressure of carbon dioxide (pCO2), the total concentration of carbon dioxide (ctCO2), and sodium (Na+) (p ≤ 0.001 for all) during the periods of HS compared to the thermoneutral conditions. Moreover, BD- and MD-fed lambs had a higher blood potassium concentration (K+) than the WD-fed lambs (p = 0.008). The results of the present study suggest that Bioprotect can be a viable feed treatment strategy for treating rapidly fermentable grains such as wheat to alleviate the effects of HS. Further, Bioprotect-treated wheat could be an option to replace maize in concentrate rations in jurisdictions where maize is cost-prohibitive or unavailable.

Cationic starch modified bentonite-alginate nanocomposites for highly controlled diffusion release of pesticides

To promote the controlled release efficacy of nanocomposites based on alginate and bentonite, (3-chloro-2-hydroxypropyl)trimethyl ammonium grafted starch was prepared and used as modifying agent of the clay. The nanocomposites were characterized by FTIR, XRD, SEM and TG analysis, to reveal the structural effects on the swelling property of the matrix and the release of alachlor, the model compound. Thermodynamics study indicated that the adsorption of alachlor on the bentonite was dominated by hydrophobic interaction with the siloxane surface of the clay and enhanced by the binding of the cationic starch. The electrostatic attraction between alginate and cationic starch bound on the surface also decreased the aggregation of bentonite platelets, leading to a more compact structure of the nanocomposites. The higher adsorption capability and lower permeability of the matrix resulted in a slower release of alachlor, which was dominated by Fickian diffusion mechanism. The release of alachlor first decreased and then increased with increasing content of bentonite and cationic starch modified bentonite in the nanocomposites, reaching a minimum around weight percentage 10%, at which the time taken for 50% of active ingredient to be released were 4.4 and 7.3 times that for the release from pure alginate hydrogel.

EVALUATION OF RECOMBINANT GLUCOAMYLASE EXPRESSION BY A NATIVE AND α-MATING FACTOR SECRETION SIGNAL IN PICHIA PASTORIS

Raw starch degrading enzyme specially glucoamylase with starch binding domain (SBD) has great values in the starch processing industry because it digests the starch particles below the gelatinization temperature by releasing glucose from the non-reducing ends sequentially. The purpose of the study was to measure the secretion levels of recombinant glucoamylase from Pichia pastoris, by using the α-mating factor secretion signal peptide (α-MF) and the native signal peptide of glucoamylase from Aspergillus flavus NSH9. The Aspergillus flavus NSH9 gene (with and without native signal sequences), encoding a pH and thermostable glucoamylase with an SBD, was successfully cloned and expressed in Pichia pastoris to produce recombinant glucoamylases. The constructed recombinant plasmids pPICZB_GA2 (having a native signal peptides) and pPICZαC_GA2 (having the α-MF) were 5144 and 5356 bp in length respectively. Recombinant pichia having α-MF signal sequence (plasmid, pPICZαC_GA2) gave the highest level of secretions of recombinant glucoamylase after 6 days of incubation period with 0.5% methanol. In conclusion, yeast expression vector signal peptide is more efficient for heterologous expression/secretions of recombinant glucoamylase compared to its native signal sequences.

"Nata Puree," a Novel Food Material for Upgrading Vegetable Powders, Made by Bacterial Cellulose Gel Disintegration in the Presence of (1,3)(1,4)-β-Glucan.

Pulverization is a potentially powerful solution for the resource management of surplus- and non-standard agricultural products, maintaining their nutritional values for long and ensuring their homogeneity, whereas their original textures could disappear to narrow the application ranges. Therefore, new technologies should be developed for reconstructing the powders to provide them with new physical characteristics. Herein, we developed a novel food material, nata puree (NP), by nata de coco (bacterial cellulose gel) disintegration with a water-soluble polysaccharide using a household blender. The process worked well with (1,3)(1,4)-β-glucan (BGL) as the polysaccharide, which could be substituted with barley extract. Lichenase treatment of the NP dramatically modified its physical properties, suggesting the importance of the BGL polymeric forms. NP exhibited distinct potato powder and starch binding activities, which would be attributed to its interactions with the cell wall components and a physical capture of powders by the NP network, respectively. NP supplementation into the potato paste improved its firmness and enabled its printable range shift for 3D food printing to a lower powder-concentration. NP also promoted the dispersion of powders in its suspension, and designed gelation could also be successfully performed by the laser irradiation of an NP suspension containing dispersed curdlan and turmeric powders. Therefore, NP could be applied as a powder modifier to a wide range of products in both conventional cooking, food manufacturing, and next generation processes such as 3D food printing.

Understanding the Binding of Starch Fragments to Granule-Bound Starch Synthase.

Granule-bound starch synthase (GBSS) plays a major role, that of chain elongation, in the biosynthesis of amylose, a starch component with mostly (1 → 4)-α connected long chains of glucose with a few (1 → 6)-α branch points. Chain-length distributions (CLDs) of amylose affect functional properties, which can be controlled by changing appropriate residues on granule-bound starch synthase (GBSS). Knowing the binding of GBSS and amylose at a molecular level can help better determine the key amino acids on GBSS that affect CLDs of amylose for subsequent use in molecular engineering. Atomistic molecular dynamics simulations with explicit solvent and docking approaches were used in this study to build a model of the binding between rice GBSS and amylose. Amylose fragments containing 3-12 linearly linked glucose units were built to represent the starch fragments. The stability of the complexes, interactions between GBSS and sugars, and difference in structure/conformation of bound and free starch fragments were analyzed. The study found that starch/amylose fragments with 5 or 6 glucose units were suitable for modeling starch binding to GBSS. The removal of an interdomain disulfide on GBSS was found to affect both GBSS and starch stability. Key residues that could affect the binding ability were also indicated. This model can help rationalize the design of mutants and suggest ways to make single-point mutations, which could be used to develop plants producing starches with improved functional properties.

292 Reducing the Fermentability of Wheat with a Starch Binding Agent Has the Potential to Ameliorate Heat Stress in Sheep

Abstract Two experiments were conducted to determine whether a starch binding agent could (1) reduce the rate of fermentation of wheat grain and (2) improve heat tolerance in sheep. Firstly, fermentation kinetics and buffered rumen fluid pH variation were measured during in vitro incubation of wheat with 0, 1, 2, and 4% Bioprotect® (Feedworks Pty Ltd, Australia). Bioprotect® reduced gas production at 12 h in a dose-dependent manner (-6.4, -11 and -20% respectively; P &amp;lt; 0.001) and increased pH (P &amp;lt; 0.001), indicating slower ruminal fermentation. In a randomized control experiment, Merino lambs (n = 24) were fed either 50% wheat (WD, fast fermenting), corn (CD, slow fermenting), or wheat treated with 2% Bioprotect® (BD), with the balance of the diets being forage. Lambs were housed in climate-controlled rooms and exposed to 3 experimental periods: period 1 (P1: 7 days of thermoneutral conditions [18–21°C and 40–50% relative humidity (RH)] and fed 1.7×maintenance); period 2 [P2: 7 days of HS (28–40°C and 30–50% RH) and fed 1.7×maintenance]; and period 3 (P3: 7 days of HS as in P2, and 2×maintenance) with ad libitum water. Increases in respiration rate (RR), heart rate (HR), rectal temperature (RT) and left and right flank skin temperature (P &amp;lt; 0.001) occurred during HS, particularly during P3 (P &amp;lt; 0.001). Lambs fed CD and BD had lower RR (150, 137 and 140 breaths/min for WD, CD and BD, P &amp;lt; 0.001), HR (91.3, 85.3 and 85.9 beats/min, P &amp;lt; 0.001 and RT (39.7, 39.7 and 39.7°C P &amp;lt; 0.05). While there was no effect of diet, lambs reduced feed intake (-4%, P &amp;lt; 0.05) during P2 but consumed more (+16%) during P3. These data confirm that feeding corn or Bioprotect® treated wheat can be used as an ameliorative strategy to reduce the thermal load in lambs during summer.

Interplay Between the N-Terminal Domains of Arabidopsis Starch Synthase 3 Determines the Interaction of the Enzyme With the Starch Granule.

The elongation of the linear chains of starch is undertaken by starch synthases. class 3 of starch synthase (SS3) has a specific feature: a long N-terminal region containing starch binding domains (SBDs). In this work, we analyze in vivo the contribution of these domains to the localization pattern of the enzyme. For this purpose, we divided the N-terminal region of Arabidopsis SS3 in three domains: D1, D2, and D3 (each of which contains an SBD and a coiled-coil site). Our analyses indicate that the N-terminal region is sufficient to determine the same localization pattern observed with the full-length protein. D2 binds tightly the polypeptide to the polymer and it is necessary the contribution of D1 and D3 to avoid the polypeptide to be trapped in the growing polymer. The localization pattern of Arabidopsis SS3 appears to be the result of the counterbalanced action of the different domains present in its N-terminal region.

Asiatic acid alleviates ischemic myocardial injury in mice by modulating mitophagy- and glycophagy-based energy metabolism.

Myocardial infarction (MI) causes disturbances in myocardial energy metabolism, ultimately leading to a poor prognosis. Cytosolic glycogen autophagy (glycophagy) and mitochondrial autophagy (mitophagy) are upregulated in MI to optimize energy metabolism but to a limited extent. Asiatic acid (AA), a pentacyclic triterpene derived from the traditional Chinese herb Centella asiatica, displays anti-inflammatory, antioxidant, and antiapoptotic activities. AA has been found to alleviate focal cerebral and liver ischemic injury by reversing mitochondrial dysfunction. In this study, we investigated whether AA exerted cardioprotective effects against MI by activating glycophagy and mitophagy to improve the energy balance. In vitro cardioprotective effects were examined in neonatal mouse cardiomyocytes subjected to oxygen-glucose deprivation for 12 h. Treatment with AA (2-50 μM) significantly increased cell viability and improved the energy metabolism evidenced by increased ATP level and phosphocreatine/ATP ratio. In vivo cardioprotective effects were studied in a mouse model of MI. Administration of AA (5-125 mg·kg-1·d-1, ig) significantly reduced infarct size and ischemic myocardial injury, and improved cardiac function. AA treatment also promoted mitophagy and relieved mitochondrial edema evidenced by increased number of mitophagosomes in ischemic myocardium in vivo and increased mitochondria-light chain 3 (LC3)-II colocalization in ODG-treated cardiomyocytes in vitro. Mitophagy activation was accompanied by activation of the AMPK signaling pathway. Knockdown of AMPK abolished AA-activated mitophagy. Furthermore, we showed that glycophagy was upregulated in OGD cardiomyocytes evidenced by increased starch binding domain protein 1 (STBD1)-GABA type A receptor-associated protein-like 1(GABARAPL1) interaction and extracellular acidification rate, whereas AA treatment further promoted glycophagy accompanied by PI3K/Akt activation. PI3K inhibitor LY294002 or Akt inhibitor GSK690693 blocked the effects of AA on glycophagy and glycolysis. Finally, simultaneous inhibition of glycophagy and mitophagy abolished the cardioprotective effects and energy regulation of AA. These results demonstrate that AA protects ischemic cardiomyocytes by modulating glycophagy- and mitophagy-based energy metabolism through the PI3K/Akt and AMPK pathways.

Extraction and identification of phospholipids from whole grain kabog millet flour and predictive effects on starch binding and retrogradation

Kabog millet, an indigenous gluten-free cereal grain from the Philippines, is considered an ecotype of Panicum miliaceum L. (proso millet). Phospholipids (PLs) from kabog millet could improve the texture of kabog millet food products, such as breads and cookies, by their interaction with starch. Besides their influence on baking properties of flours, PLs have been studied for their health effects. No information exists regarding the PL content of kabog millet. In our study, we analysed the content and identity of PLs from whole grain kabog millet flour. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC) were found to be present in whole grain kabog millet flour. To test PL-starch interactions, rice, maize, and potato starches, and commercial amylopectin were mixed with commercial soy PC and the viscosity was measured with a rheometer. Models of PL-amylose/amylopectin complexes were deduced from rheology, iodine capacity, and isothermal titration calorimetry (ITC) experiments. PLs can bind with starch and inhibit its retrogradation by increasing the viscosity after gelatinisation. In summary, whole grain kabog millet flour contains PC, PE, PI, and LPC, which can potentially affect the retrogradation of baked products made from kabog millet flour.

Evaluation of temperature and concentration on the development of rice hydrogel and 2D xerogel

The gelification behavior of starch in rice flour was investigated for its application on food structuring to produce rice xerogel. The effect of hydrogel nature in drying was studied for the xerogel surface design. The impact of changes in rice flour was found by the least gelation concentration and pasting properties. It exhibited 4% (w/v) gelatinized rice flour dispersion and can form a 3D polymer network during cooling. Assessment of hydrogel preparation temperature was examined by swelling power, solubility, surface tension, contact angle, and transmittance. An increase in swelling power, solubility, and density is due to the effective binding of starch with water molecules. The decline in absorbance specifies loss of crystallinity and birefringence because of starch granules disruption. Surface tension increased, and contact angle decreased at a higher temperature because of gelling nature. Various concentrations (2%, 4%, and 6%) of rice flour dispersion were exposed to heat at temperatures (60, 70, 80, 90, and 100°C) for developing rice xerogel. It was observed that 4% (w/v) of rice flour suspension gellified at 90°C possesses better structural integrity without deformation and cracks. Novelty Impact Statement The obtained 2D rice xerogel with the addition of flavor could be utilized in complex-shaped foods like chips, pasta, and noodles. As 2D flour-based materials are capable of undergoing shape change into a 3D form during frying/cooking, this is an arising innovation that could encourage diminishing packaging and shipping costs as well as to design user-customized attractive materials.

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

α-Amylase from Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

A highly divergent α-amylase from Streptomyces spp.: An evolutionary perspective

The present study deals with the genetic changes observed in the protein sequence of an α-amylase from Streptomyces spp. and its structural homologs from Pseudoalteromonas haloplanktis, invertebrates and mammals. The structural homologs are renowned for their important features such as chloride binding triad and a serine-protease like catalytic triad (a triad which is reported to be strictly conserved in all chloride-dependent α-amylases). These conserved regions are essential for allosteric activation of enzyme and conformational stability, respectively. An evaluation of these distinctive features in Streptomyces α-amylases revealed the role of mutations in conserved regions and evolution of chloride-independent α-amylases in Streptomyces spp. Besides, the study also discovers a highly divergent α-amylase from Streptomyces spp. which varies greatly even within the homologs of the same genus. Another very important feature is the number of disulfide bridges in which the structural homologs own eight Cys residues to form four disulfide bridges whereas Streptomyces α-amylases possess only seven Cys to form three disulfide bridges. The study also highlights the unique evolution of carbohydrate binding module 20 domain (CBM20 also known as raw starch binding domain or E domain) in Streptomyces α-amylases which is completely absent in α-amylases of other structural homologs.

Produção e caracterização da amilase obtida de Rhizopus microsporus var. oligosporus

As amilases possuem a capacidade de catalisar a ligação α-1,4 do amido liberando glicose e dextrina, com destaque em diversos campos industriais. A pesquisa buscou caracterizar a enzima amilase obtida do fungo Rhizopus microsporus var. oligosporus, através da metodologia de Doehlert, avaliando o comportamento da enzima frente as variações de pH e temperatura. A produção da amilase ocorreu com a utilização do amido como indutor, e a caracterização foi realizada através da metodologia de superfície de resposta, com a análise do pH em 5 níveis (3,0, 4,0, 5,0, 6,0 e 7,0) e temperatura em 3 níveis (30, 50 e 70 °C). A avaliação da termoestabilidade da amilase ocorreu à 60, 70 e 80ºC. A aplicação do modelo experimental indicou que a amilase obtida de R. microsporus var. oligosporus apresenta melhor desenvolvimento catalítico em temperaturas entre 40ºC e 55ºC e pH entre 2,5 e 3,2. A avaliação da termoestabilidade indicou que o aumento da temperatura influencia negativamente na atividade catalítica da amilase. O modelo experimental conduziu à compreensão das condições favoráveis à produção de amilases de R. microsporus var. oligosporus.

Cloning, expression, homology modelling and molecular dynamics simulation of four domain-containing α-amylase from Streptomyces griseus

In the present study, α-amylase from Streptomyces griseus TBG19NRA1 was amplified, cloned and successfully expressed in E. coli BL21/DE3. Sequence analysis of S. griseus α-amylase (SGAmy) revealed the presence of four domains (A, B, C and E). Alpha-amylases with E domain (also known as carbohydrate binding module 20 (CBM20)) are capable of degrading raw starch and this property holds great potential for application in starch processing industries. Though α-amylase is a well-studied and characterized enzyme, there is no experimental structure available for this four domain-containing α-amylases. To gain more insight about SGAmy structure and function, homology modelling was performed using a multi-template method. The template α-amylase from Pseudoalteromonas haloplanktis (PDB ID 1AQH) and E domain of Cyclodextrin glucanotransferase from Bacillus circulans (PDB ID 1CGY) was found to have significant similarity with the complete target sequence of SGAmy. Therefore, homology model for SGAmy was generated from the crystal structure of 1AQH and 1CGY and the resulting structure was subjected to 10 ns molecular dynamics (MD) simulation. Remarkably, CBM20 domain of SGAmy showed greater flexibility in MD simulation than other three domains. This observation is highly rational as this part of SGAmy is strongly implicated in substrate (raw starch) binding. Thus, conformational plasticity at CBM20 is functionally beneficial. Communicated by Ramaswamy H. Sarma

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2 fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant's phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Functional study of C-terminal domain of the thermoacidophilic raw starch-hydrolyzing α-amylase Gt-amy.

Since the thermoacidophilic raw-starch hydrolyzing α-amylase Gt-amy can effectively hydrolyze corn starch under starch liquefaction conditions, it has potential for many industrial applications. To identify the raw starch-binding domain of Gt-amy, a C-terminal domain (CTD)-truncated mutant (Gt-amy-T) was constructed, and its enzymatic properties were compared with Gt-amy. In comparison to CTD of Gt-amy, which could effectively bind corn starch, the Gt-amy-T could not bind to and hydrolyze corn starch under similar conditions. In addition, Gt-amy-T showed significantly lower thermal activity and thermal stability. Using soluble starch as the substrate, the k cat of Gt-amy-T at 80°C was approximately 77.9% of that of Gt-amy. The half-life of Gt-amy at 80°C was 3h, while that of Gt-amy-T was 2h. These results reveal that the CTD plays a vital role in raw starch binding and degradation by Gt-amy and helps Gt-amy maintain thermal activity and stability.

Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9

The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.

Analysis of the phylogenetic sequence relationships between plant alpha amylases

Enzymes are highly valuable in the industry, such industries are food, beverage industry and the biofuel sector. Amylases are very important in starch processing such as liquefaction, hydrolysis and saccharification. Alpha amylase has liquefying ability by hydrolysing the glycosidic bonds at internal positions. This paper aims to present a brief overview of the sequence comparison between plant alpha amylases. The protein sequences were obtained from the NCBI, aligned using the clustal W tool and the phylogenetic trees were constructed using the Mega 7.0 tool. The proteins can be classified into three distinct families; one, two and three. Each family has some peculiar features, family one amylases are proteins of about 450 amino acids long with signal peptides at their N-terminus, family two proteins are similar in size to those of family one but are cytosolic with no targeting peptide. In contrast, family three amylase are proteins targeted to the chloroplast with transit peptides at their N-terminus, however, they are 900 amino acids long. In addition, the last family of protein has an additional domain that is yet to be fully characterized. Across the three families, there is a high homology and thus conservation of the catalytic domain which is at the C-terminus. This implied that the proteins are evolutionary related and perform similar functions. The unknown domain in family three amylases is also conserved across members of this family. The domain may be a starch binding domain that is required for the family three amylases to digest storage starch in the plastid.Keywords: Alpha amylases, Cytosol, Plastid, Family one, Unknown domain