Biochemical characterization and technofunctional properties of bioprocessed wheat bran protein isolates.

The effect of three combinations of bioprocessing methods by lactic acid fermentation, cell wall hydrolyzing enzymes and phytase on the biochemical (protein, fat, carbohydrate composition) and technofunctional properties (protein solubility, emulsifying and foaming properties) of wheat bran protein isolates were evaluated. The bioprocessing increased the protein (up to 80%) and fat content (up to 22.8%) in the isolates due to the degradation of starch and soluble pentosans. Additional proteins, globulin 3A and 3C, chitinase, ß-amylase and LMW glutenins, were identified from the electrophoretic pattern of the protein isolate bioprocessed with added enzymes. Generally, the bioprocessed protein isolate had lower protein solubility and stronger net charge in pH below 7, when compared to the protein isolate made without bioprocessing. The emulsifying properties of the protein isolates were not affected by bioprocessing. However, the foaming stability of the protein isolates was nearly doubled by bioprocessing with cell wall hydrolyzing enzymes and phytase.

Biochemical and thermodynamic characterization of de novo synthesized ß-amylase from fenugreek.

ß-Amylase has been de novo synthesized from germinating fenugreek seeds. Enzyme has been isolated and purified from 36 h germinated seeds with 226-fold purification and specific activity of 763 U/mg. Homogeneity of the purified ß-amylase has been confirmed with size-exclusion chromatography, SDS-PAGE and MALDI MS/MS analysis. The isoelectric point, optimum pH and temperature of the enzyme were found to be pH 5.2, 5.7 and 57 °C, respectively. The enzyme was specific for soluble starch with Km and Vmax of 2.4 mg/mL and 833.3 U/mg, respectively. Maltose was found to be competitive inhibitor of the enzyme with inhibition constant (Ki) of 14 mM. However, metallic ions like Ag+ and Hg2+ were found to be non-competitive inhibitors of the enzyme. Thermodynamic parameters like Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes have further revealed that thermal denaturation of the enzyme has followed first-order with the enzyme unfolding rather an aggregation with the process being irreversible. The activation energy of ß-amylase during thermal activation and denaturation were 27.5 kJ/mol and 145.23 kJ/mol, respectively at R2 > 0.92. Thus, the enzyme was stable even at higher temperature with ability of undergoing catalysis making it commercially exploitable, particularly in food and pharmaceutical industries.

Prediction and analysis of GH14 family ß-amylases in oat seedling extract: Structure and function insights using in silico approaches.

Oat (Avena sativa L.) seedling extract exhibited a high degree of catalytic activities. Bioinformatics were used to identify ß-amylases as abundant enzymes in the oat seedling extract. These identified oat enzymes are a member of the GH14 family. Proteins in the Avena sativa seedling extract were separated by SDS-PAGE and 2 major protein bands with an apparent molecular weights of 53 and 42 kDa were the subject of this study. These materials were digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS and database searches. These sequences were used to identify cDNAs from expressed sequence tags (EST) and Transcriptome Shotgun Assembly (TSA) of Avena sativa. Based upon EST and TSA sequences, at least 6 predicted different sequences were identified and assigned as ß-amylases. Insights into structural characterization of the oat predicted ß-amylases were analyzed using in silico approaches. The identified ß-amylases conserved the two Glu residues assigned as the "putative" catalytic residues, which would act as an acid and base pair in the catalytic process. A similar core (ß/α)8-barrel architecture was found in the predicted oat ß-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (ß/α)8-barrel domain. This suggests an accessibility of the non-reducing end of the substrate towards the oat ß-amylases and thus confirming that are exo-acting hydrolases. The results provide a detailed view of the main residues involved in catalysis in this kind of enzyme.

Process optimization of the extraction condition of ß-amylase from brewer's malt and its application in the maltose syrup production.

ß-Amylase is of important biotechnological aid in maltose syrup production. In this study, the extraction condition of ß-amylase from brewer's malt and the optimal dosage of ß-amylase in maltose syrup production were optimized using response surface methodology and uniform design method. The optimal extraction condition of ß-amylase from brewer's malt was composed of 1:17 (g/v) material/liquid ratio, 44°C extraction temperature, pH 6.4 buffer pH, 2.3 H extraction time, and 1.64 g L-1 NaSO3 dosage with a predicted ß-amylase activity of 1,290.99 U g-1 , which was close to the experimental ß-amylase activity of 1,230.22 U g-1 . The optimal dosages of ß-amylase used in maltose syrup production were 455.67 U g-1 starch and its application in maltose syrup production led to a 68.37% maltose content in maltose syrup, which was 11.2% and 28.9% higher than those using ß-amylases from soybean and microbe (P < 0.01). Thus, ß-amylase from brewer's malt was beneficial for production of high maltose syrup.

An antioxidant rich novel ß-amylase from peanuts (Arachis hypogaea): Its purification, biochemical characterization and potential applications.

ß-Amylase from un-germinated seeds of peanut (Arachis hypogaea) was purified to apparent electrophoretic homogeneity with final purification fold of 205 and specific activity of 361µmol/min/mg protein. The enzyme was purified employing simple purification techniques for biochemical characterization. The purified enzyme was identified as ß-amylase with Mr of 31kDa. The enzyme displayed its optimum catalytic activity at pH5.0 and 60°C with activation energy of 4.5kcal/mol and Q10 1.2. The enzyme displayed Km and Vmax values, for soluble potato starch of 1.28mg/mL and 363.63µmol/min/mg, respectively. Thermal inactivation of ß-amylase at 65°C resulted into first-order kinetics with rate constant 0.0126min-1 and t½ 55min. The enzyme was observed to act on native granular potato starch, which could minimize the high cost occurring from solubilization of starch in industries. Enzyme fractions scavenge 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical, indicating its antioxidative nature. In addition, the purified ß-amylase was successfully utilized for the improvement of antioxidant potential of wheat. These findings suggest that ß-amylase from peanuts have potential application in food and pharmaceutical industries.

Simplified recovery of enzymes and nutrients in sweet potato wastewater and preparing health black tea and theaflavins with scrap tea.

The industry discards generous organic wastewater in sweet potato starch factory and scrap tea in tea production. A simplified procedure to recover all biochemicals from the wastewater of sweet potato starch factory and use them to make health black tea and theaflavins from scrap green tea was developed. The sweet potato wastewater was sequentially treated by isoelectric precipitation, ultrafiltration and nanofiltration to recover polyphenol oxidase (PPO), ß-amylase, and small molecular fractions, respectively. The PPO fraction can effectively transform green tea extracts into black tea with high content of theaflavins through the optimized fed-batch feeding fermentation. The PPO transformed black tea with sporamins can be used to make health black tea, or make theaflavins by fractionation with ethyl acetate. This work provides a resource- and environment-friendly approach for economically utilizing the sweet potato wastewater and the scrap tea, and making biochemical, nutrient and health products.

Characterization of thermostable ß-amylase isozymes from Lactobacillus fermentum.

A strain of Lactobacillus fermentum producing two isozymes of a 20kDa ß-amylase was isolated from the faecal sample of a newborn. The starin was identified by sequencing its 16S rRNA gene. The two ß-amylase isozymes were resolved and visualized by two dimensional protein gel electrophoresis (2-D gel electrophoresis). Some of the physical and biochemical properties of the enzymes were characterized. The ß-amylase displayed two optimum pH s, 5.0 and 10.0 and two optimum temperatures, 45°C and 37°C, respectively. The isozymes hydrolyzed different substrates: glycogen at pH 5.0, and corn starch at pH 10.0. The activity did not require Ca2+, though the activity at pH 10.0 was enhanced in the presence of 5.0mM and 10.0mM CaCl2, 110% and 130%, respectively.

Screening, separating, and completely recovering polyphenol oxidases and other biochemicals from sweet potato wastewater in starch production.

Polyphenol oxidase (PPO) has multiple functions, and the lack of commercially available enzyme sources limits its widespread application in various industries. An accurate PPO assay was developed by HPLC determination of the substrate oxidation. Resources screening indicated that sweet potato (Ipomoea batatas L.) wastewater in starch production has high PPO activity. A procedure was developed for separately recovering PPO, ß-amylase, sporamins, and small molecular nutrients (SMNs) from sweet potato wastewater. The wastewater was adjusted to pH 3.5 to precipitate PPO, and then adjusted to 50 % acetone to precipitate ß-amylase and further to 80 % acetone to precipitate sporamins. The SMNs were obtained after acetone recovery. Purified powders of 4.3 × 10(5) units of PPO, 4.0 × 10(6) units of ß-amylase, 8.70 g sporamins, and 20.2 g SMNs were obtained from the wastewater of 1 kg sweet potato. More than 50 million tons of sweet potato is used for starch production annually around the world. Through this simple procedure, huge amount of biochemical resources can be recovered from the wastewater, which greatly increases the economic value of the crop and saves the environment.

Β-amylase from starchless seeds of Trigonella foenum-graecum and its localization in germinating seeds.

Fenugreek (Trigonella foenum-graecum) seeds do not contain starch as carbohydrate reserve. Synthesis of starch is initiated after germination. A ß-amylase from ungerminated fenugreek seeds was purified to apparent electrophoretic homogeneity. The enzyme was purified 210 fold with specific activity of 732.59 units/mg. Mr of the denatured enzyme as determined from SDS-PAGE was 58 kD while that of native enzyme calculated from size exclusion chromatography was 56 kD. Furthermore, its identity was confirmed to be ß-amylase from MALDI-TOF analysis. The optimum pH and temperature was found to be 5.0 and 50°C, respectively. Starch was hydrolyzed at highest rate and enzyme showed a Km of 1.58 mg/mL with it. Antibodies against purified Fenugreek ß-amylase were generated in rabbits. These antibodies were used for localization of enzyme in the cotyledon during different stages of germination using fluorescence and confocal microscopy. Fenugreek ß-amylase was found to be the major starch degrading enzyme depending on the high amount of enzyme present as compared to α-amylase and also its localization at the periphery of amyloplasts. A new finding in terms of its association with protophloem was observed. Thus, this enzyme appears to be important for germination of seeds.

Unexpected mode of action of sweet potato ß-amylase on maltooligomer substrates.

ß-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato ß-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme-product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54mM) in the early phase of the reaction.

Purification and characterization of novel organic-solvent-tolerant ß-amylase and serine protease from a newly isolated Salimicrobium halophilum strain LY20.

A halophilic isolate Salimicrobium halophilum strain LY20 producing extracellular amylase and protease was isolated from Yuncheng, China. Production of both enzymes was synchronized with bacterial growth and reached a maximum level during the early-stationary phase. The amylase and protease were purified to homogeneity with molecular weights of 81 and 30 kDa, respectively. Optimal amylase activity was observed at 70 °C, pH 10.0% and 10% NaCl. Complete inhibition by EDTA, diethyl pyrocarbonate (DEPC), and phenylarsine oxide (PAO) indicated that the amylase was a metalloenzyme with histidine and cysteine residues essential for its catalysis. Maltose was the main product of starch hydrolysis, indicating an ß-amylase activity. The purified protease from LY20 showed highest activity at 80 °C, pH 10.0% and 12.5% NaCl. Complete inhibition was shown by phenylmethylsulfonyl fluoride, DEPC, and PAO, indicating that the enzyme probably belonged to the subclass of the serine proteases with histidine and cysteine residues essential for catalysis. Furthermore, both enzymes were highly stable over broad temperature (30-80 °C), pH (6.0-12.0) and NaCl concentration (2.5-20%) ranges, showing excellent thermostable, alkalistable, and halotolerant nature. The surfactants (SDS, Tween 80, and Triton X-100) did not affect their activities. In addition, both enzymes from LY20 displayed remarkable stability in the presence of water-soluble organic solvents with log P(ow) ( ) ≤ -0.24.

Characterization of recombinant ß-amylases from Oryza sativa.

Four putative ß-amylase genes found in the Oryza sativa cDNA sequence database (KOME) were expressed in Escherichia coli. Recombinant proteins from two of these genes showed ß-amylase activity. Similarly to ß-amylases from other plants, the optimum pH of the recombinant rice ß-amylases was about 5.5-6.0, but they exhibited inferior heat stability to soybean ß-amylase.

Recent advances in microbial raw starch degrading enzymes.

Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.

Purification and characterization of camel (Camelus dromedarius) milk amylase.

Skimmed camel milk contains 59,900 U/L amylase, which is 39,363 times less than serum and plasma amylase. Camel milk beta-amylase was purified as a 61 KDa band using DEAE-Sepharose and Sephadex G-100 and yielded 561 U/mg. The optimum working pH, Km and temperature were 7.0, 13.6 mg/Lstarch, 30-40 degrees C, respectively. The enzyme has been shown higher affinity toward amylose and soluble starch than glycogen, amylopectin, dextrin, or pullulan. Magnesium chloride, CaCl(2) and NaCl activated the amylase, while EDTA and EGTA decreased its activity. While its activity was increased in the presence of Triton X-100 and Triton X-114. Phenylmethanesulfonyl fluoride did not show any effect on enzyme activity. However, the enzyme activity was inhibited by urea, SDS, DTNB, iodoacetamide, N-ethylmalimide, aprotinin, and trypsin inhibitor. It worked on starch to yield a maltose. Scanning electron microscope images demonstrated a nano-degrading ability on starch granules from various sources (potato, corn, cassava, and rice).

Continuous extraction of alpha- and beta-amylases from Zea mays malt in a PEG4000/CaCl2 ATPS.

In the present work, alpha- and beta-amylase enzymes from Zea mays malt were recovered by continuous extraction in a PEG/CaCl2 aqueous two-phase system (ATPS). The influences of the flux rate (RQ), free area of vane (A(free)) and vane rotation (RV) on enzyme recovery were studied by optimization using response surface methodology (RSM). The protein content and enzyme activity were measured from time to time in the extract and refined fluxes. RSM curves showed a squared dependence of recovery index with the RQ, A(free) and RV. The best system for recovering the maize malt enzymes was with low vane rotation and flux rate and high free area of vane. Alpha- and beta-amylases were purified 130-fold in the salt-rich phase.

Beta-amylase in germinating millet seeds.

Beta-amylase (EC 3.2.1.2) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on DEAE-cellulofine and CM-cellulofine, and preparative isoelectric focusing. The enzyme was homogeneous by SDS-PAGE. The M(r) of the enzyme was estimated to be 58,000 based on its mobility on SDS-PAGE and gel filtration with TSKgel G4000SW(XL), which showed that it is composed of a single unit. The isoelectric point of the enzyme was 4.62. The enzyme hydrolyzed malto-oligosaccharides more readily as their degree of polymerization increased, this being strongest for malto-oligosaccharides larger than 13 glucose residues and very weakly for maltotriose. Amylose, amylopectin and soluble starch were the most suitable substrates for the enzyme. While the enzyme showed some activity against native starch by itself, starch digestion was accelerated 2.5-fold using alpha-amylase, pullulanase and alpha-glucosidase. This enzyme appears to be very important for the germination of millet seeds.

Phenylboronate-chitosan resins for adsorption of beta-amylase from soybean extracts.

Isolation and purification of bioproducts from crude extracts can be obtained by affinity methods based on reversible binding of a specific molecule to ligand immobilized in a porous matrix. In the present work, nicrospheres based on chitosan matrix, which incorporated aminophenylboronic acid as a derivative, were prepared and characterized, aimed at developing a beta-amylase adsorption process. Kinetic curves and adsorption isotheriru of the crude extracts as well as the breakthrough curves for a frontal chromatographic separation method of a commercial sample of beta-amylase from soybean are presented. These results were compared to similar data obtained with a comercial microspheres gel based-on agarose.

Magnetite-alginate beads for purification of some starch degrading enzymes.

Starch degrading enzymes, viz., beta-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. beta-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.

Purification, characterization, immunolocalization and structural analysis of the abundant cytoplasmic beta-amylase from Calystegia sepium (hedge bindweed) rhizomes.

An abundant catalytically active beta-amylase (EC 3.2.1.2) was isolated from resting rhizomes of hedge bindweed (Calystegia sepium). Biochemical analysis of the purified protein, molecular modeling, and cloning of the corresponding gene indicated that this enzyme resembles previously characterized plant beta-amylases with regard to its amino-acid sequence, molecular structure and catalytic activities. Immunolocalization demonstrated that the beta-amylase is exclusively located in the cytoplasm. It is suggested that the hedge bindweed rhizome beta-amylase is a cytoplasmic vegetative storage protein.