A gene encoding an amylopullulanase of the glycosyl hydrolase (GH) family 57 from
Staphylothermus marinus
(SMApu) was heterologously expressed in
Escherichia coli
. SMApu consisted of 639 amino acids ...with a molecular mass of 75.3 kDa. It only showed maximal amino acid identity of 17.1 % with that of
Pyrococcus furiosus
amylopullulanase in all identified amylases. Not like previously reported amylopullulanases, SMApu has no signal peptide but contains a continuous GH57N_Apu domain. It had the highest catalytic efficiency toward pullulan (
k
cat
/
K
m
, 342.34 s
−1
mL mg
−1
) and was extremely thermostable with maximal pullulan-degrading activity (42.1 U/mg) at 105 °C and pH 5.0 and a half-life of 50 min at 100 °C. Its activity increased to 116 % in the presence of 5 mM CaCl
2
. SMApu could also degrade cyclodextrins, which are resistant to the other amylopullulanases. The initial hydrolytic products from pullulan, γ-CD, and 6-
O
-maltooligosyl-β-CD were 6)-α-
d
-Glcp-(1 → 4)-α-
d
-Glcp-(1 → 4)-α-
d
-Glcp-(1→
n
, maltooctaose, and single maltooligosaccharide plus β-CD, respectively. The final hydrolytic products from above-mentioned substrates were maltose and glucose. These results confirm that SMApu is a novel amylopullulanase of the family GH57 possessing the cyclodextrin-degrading activity of cyclomaltodextrinase.
Enzyme technology has many potential applications in the baking industry because carbohydrate-active enzymes specifically react with carbohydrate components, such as starch, in complex food systems. ...Amylolytic enzymes are added to starch-based foods, such as baking products, to retain moisture more efficiently and to increase softness, freshness, and shelf life. The major reactions used to modify the structure of food starch include: (1) hydrolysis of α-1, 4 or α-1, 6 glycosidic linkages, (2) disproportionation by the transfer of glucan moieties, and (3) branching by formation of α-1, 6 glycosidic linkage. The catalytic reaction of a single enzyme or a mixture of more than two enzymes has been applied, generating novel starches, with chemical changes in the starch structure, in which the changes of molecular mass, branch chain length distribution, and the ratio of amylose to amylopectin may occur. These developments of enzyme technology highlight the potential to create various structured-starches for the food and baking industry.
Puerarin is an isoflavone derived from Kudzu roots and has antioxidant and hypocholesterolemic effects; however, its insolubility often limits its biological availability
in vivo. Using a novel ...transglycosylation process, the solubility of puerarin glycosides was increased >
100-fold, but it was not known whether these modified puerarin glycosides maintained biological activities. We found that water-soluble puerarin glycosides fully maintained antioxidant activities compared with puerarin assessed by radical scavenging activity, reducing power assay, superoxide dismutase activity, and non-site-specific hydroxyl radical scavenging activity. Both puerarin and its glycosides also significantly reduced low-density lipoprotein (LDL) oxidation. Mice fed with puerarin glycosides (0.1% w/w) showed significantly reduced plasma total cholesterol levels, thus, we further investigated their hypocholesterolemic mechanisms by assessing several key gene expressions both
in vitro and
in vivo. Puerarin and its glycosides induced multiple changes in hepatic cholesterol metabolism. The LDL receptor promoter activity was increased dose-dependently in puerarin glycosides-treated HepG2 cells. Accordingly, the expression of LDL receptor mRNA and protein were also significantly increased in HepG2 cells and mouse livers. The transcription and translation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were down-regulated both
in vitro and
in vivo. The cholesterol 7α-hydroxylase (CYP7A1) mRNA levels were not affected
in vitro but significantly up-regulated in the mouse livers. Collectively, our results show that puerarin and its glycosides are biologically fully active isoflavone and have antioxidant and hypocholesterolemic effects in HepG2 cells and in C57BL/6J mice. In the livers, hypocholesterolemic effects of puerarin glycoside may be achieved by multiple mechanisms including increasing LDL uptake, reducing cholesterol biosynthesis, and possibly enhancing cholesterol degradation.
In this study a disproportionating enzyme, 4-α-glucanotransferase (4αGTase), was used to modify the structural properties of rice starch to produce a suitable fat substitute in reduced-fat (RF) ...mayonnaise. The mayonnaise fat was partially substituted with the 4αGTase-treated starch paste at levels up to 50% in combination with xanthan gum and the physical and rheological properties of the modified RF mayonnaise samples were investigated. All mayonnaises prepared in this study exhibited shear thinning behavior and yield stress. Viscoelastic properties of mayonnaise were characterized using dynamic oscillatory shear test and it was observed that mayonnaises exhibited weak gel-like properties. The magnitude of elastic and loss moduli was also affected by 4αGTase-treated starch concentration and presence of xanthan gum. In relation to microstructure, RF mayonnaise prepared with 3.8 or 5.6
wt% of 4αGTase-treated starch and xanthan gum showed smaller droplets. The use of 5.6
wt% of 4αGTase-treated starch and 0.1
wt% of xanthan gum produced a RF mayonnaise with similar rheological properties and appearances as FF mayonnaise with gum. This study demonstrated a high feasibility for using 4αGTase-treated rice starch as a viable fat replacer in mayonnaise.
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•A packed-bed enzyme reactor (PBR) to produce pure maltodextrin from cyclodextrin was developed.•Pyrococcus furiosus thermostable amylase (PFTA) was immobilized on hydrophobic ...resin.•The immobilized PFTA remained stable during continuous process up to 25h.•Maltodextrin at high-purity (92–97%) was continuously produced using PFTA–PBR system.•This study established an efficient bioconversion system for pure maltodextrin production.
Maltodextrin in specific length is of great interest because of its wide industrial applications. Previously, we have proven that Pyrococcus furiosus thermostable amylase (PFTA) produces high purity maltoheptaose (G7) from β-cyclodextrin (CD) at early reaction time. In this study, an optimal continuous process for pure maltodextrin production from CDs using PFTA with immobilization in a packed-bed reactor (PBR) was developed. For testing PBR, β-CD was used as a substrate to produce G7. The activity of immobilized PFTA in PBR retained 85% of the initial activity after 6 cycles, and the product purity was unchanged. The optimal conditions for the maximized G7-purity of 96.3% were predicted at 4.7mL/min of flow, 1.1% substrate and 48.7°C. Maltohexaose and maltooctaose, 92% and 97% purity respectively, were produced from CDs at the similar condition as well. Through this study, we successfully demonstrated the possibility of large-scale continuous production of pure maltodextrins from CDs.
•α-Amylase hydrolysis of long starch chains led to chain-cleavage of glutenins.•Insoluble starch significantly inhibited the unfolding of insoluble deamidated wheat gluten.•The interactions between ...amylopectin and gliadins were by α-pyran-glycosidic bonds.•Gluten-starch interactions likely caused the incomplete dissolution of wheat gluten.
After carboxylic acid deamidation upon heating (CADH), wheat gluten still contains a total of ∼10% insoluble fractions, of which ∼10% is starch, which depreciate the values of wheat gluten. To elucidate gluten-starch interactions and their role in the deamidation behavior of gluten, the macrostructural characteristics of gluten citric acid suspensions of different concentrations (1% and 10%, w/v) and with different types of residual starch chains (achieved by enzyme hydrolyzed by α-amylase and/or glucoamylase assisted by sonication) were investigated. We found the degradation of long starch chains and branched short chains induced dramatic bond-cleavages in insoluble glutenins and gliadins. FTIR and SDS-PAGE analyses indicated that without these two types of chains in the precipitates, the insoluble deamidated wheat gluten exhibited minimal changes in the molecular force and the conformation. Their glycosylation, hydrophobic force and hydrogen bonds between amylopectin and small proteins, such as LMW-GS and α, β, γ-gliadins, were detected. FTIR suggested that the associations between gliadins and amylopectin were covalent. Gluten-starch interactions were likely to cause an incomplete dissolution of wheat gluten during CADH. A simple model was proposed to clarify the aggregation state and the relationships between starch granules and wheat gluten components during CADH.
We enzymatically modified rice starch to produce highly branched amylopectin and amylose and analyzed the resulting structural changes. To prepare the highly branched amylopectin cluster (HBAPC), we ...first treated waxy rice starch with Thermus scotoductus α-glucanotransferase (TSαGT), followed by treatment with Bacillus stearothermophilus maltogenic amylase (BSMA). Highly branched amylose (HBA) was prepared by incubating amylose with Bacillus subtilis 168 branching enzyme (BBE) and subsequently treating it with BSMA. The molecular weight of TSαGT-treated waxy rice starch was reduced from 8.9 × 108 to 1.2 × 105 Da, indicating that the α-1,4 glucosidic linkage of the segment between amylopectin clusters was hydrolyzed. Analysis of the amylopectin cluster side chains revealed that a rearrangement in the side-chain length distribution occurred. Furthermore, HBAPC and HBA were found to contain significant numbers of branched maltooligosaccharide side chains. In short, amylopectin molecules of waxy rice starch were hydrolyzed into amylopectin clusters by TSαGT in the enzymatic modification process, and then further branched by transglycosylation using BSMA. HBAPC and HBA showed higher water solubility and stability against retrogradation than amylopectin clusters or branched amylose. The hydrolysis rates of HBAPC and HBA by glucoamylase and α-amylase greatly decreased. The k cat/K m value of glucoamylase acting on the amylopectin cluster was 45.94 s−1(mg/mL)−1 and that for glucoamylase acting on HBAPC was 11.10 s−1(mg/mL)−1, indicating that HBAPC was 4-fold less susceptible to glucoamylase. The k cat/K m value for HBA was 15.90 s−1(mg/mL)−1, or about three times less than that for branched amylose. The k cat/K m values of porcine pancreatic α-amylase for HBAPC and HBA were 496 and 588 s−1(mg/mL)−1, respectively, indicating that HBA and HBAPC are less susceptible to hydrolysis by glucoamylase and α-amylase. HBAPC and HBA show potential as novel glucan polymers with low digestibility and high water solubility.
Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1–4)-glycosyl linkages in cyclodextrins ...and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N′ domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N′ domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.
Maltogenic amylases that are known to date form dimers to perform hydrolysis.
The structure of maltogenic amylase from Staphylothermus showed a novel domain at the N terminus associated with the active site.
Staphylothermus amylase has all of its substrate-binding structural components in a single monomer.
This is the first report of the newly observed domain arrangement adopted by hyperthermophilic archaic maltogenic amylase.
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•A highly thermostable plant β-amylase was isolated with a temperature optimum of 65°C.•This plant leaf β-amylase represents a new resource for the food and bio- industries.•The ...thermal kinetic parameters for starch conversion and food processing were determined.•The enzyme can be purified from a textile industry byproduct.
We characterized ramie leaf β-amylase, and determined its thermostability and kinetic parameters. The enzyme was purified 53-fold using ammonium sulfate fractionation (40–60% saturation), anion exchange chromatography on DEAE-cellulose and gel permeation chromatography on Superdex-200. The purified enzyme was identified as β-amylase with molecular mass of 42kD. The enzyme displayed Km and kcat values for soluble potato starch of 1.1mg/mL and 7.8s−1, respectively. The enzyme had a temperature optimum of 65°C, and its activity at 70°C was 92% of that at the optimal temperature after a 15-min incubation. Furthermore, enzyme activity was stable during treatment at 55°C for 60min but was inactivated rapidly at >75°C. This thermal behavior indicates that ramie leaf β-amylase has excellent intermediate temperature-stable enzyme properties for the baking and bio-industries. Inactivation of the enzyme followed first-order kinetics in the range of 55–80°C. The enthalpy change of thermal inactivation (ΔH‡), ΔG‡, and ΔS‡ were 237.2kJ/mol, 107.7kJ/mol, and 0.39kJ/molK at 333K, respectively. The D-value at 65°C (=110min) and the z-value (=9.4°C) are given for food processing.
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