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Mechanized Huangjiu is a stable product, is not subject to seasonal production restrictions, and markedly reduces labor intensity compared to traditional manual Huangjiu. However, the bitterness of mechanized Huangjiu impedes its further development.Based on process optimization, when the fermentation temperature was 45 °C and the fermentation time was 122 h, the inoculation amount of Saccharopolyspora was 5%, the amount of added water was 26%, and the glucoamylase and amylase activities of wheat Qu increased by 27% and 40% respectively, compared with those before optimization. Huangjiu fermented by raw wheat Qu inoculated with Saccharopolyspora rosea F2014 showed a significant (P 0.05) decrease in bitter amino acid content (1.24 vs. 2.86 g LAn innovative fermentation process of inoculating Saccharopolyspora into raw wheat Qu was developed for the first time. Such a process could be used to control bitterness based on raw wheat Qu inoculated with Saccharopolyspora rosea F2014, instead of traditional wheat Qu in Huangjiu fermentation. © 2022 Society of Chemical Industry.

This study describes for the first time the purification and characterization of a glucoamylase from Aspergillus wentii (strain PG18), a species of the Aspergillus genus Cremei section. Maximum enzyme production (∼3.5 U/ml) was obtained in submerged culture (72 h) with starch as the carbon source, at 25°C, and with orbital agitation (100 rpm). The enzyme was purified with one-step molecular exclusion chromatography. The 86 kDa purified enzyme hydrolyzed starch in a zymogram and had activity against p-nitrophenyl α- d-glucopyranoside. The optimal enzyme pH and temperature were 5.0 and 60°C (at pH 5.0), respectively. The Tm of the purified enzyme was 60°C, at pH 7.0. The purified glucoamylase had a KM for starch of 1.4 mg/ml and a Vmax of 0.057 mg/min of hydrolyzed starch. Molybdenum activated the purified enzyme, and sodium dodecyl sulfate inhibited it. A thin layer chromatography analysis revealed glucose as the enzyme's main starch hydrolysis product. An enzyme's peptide sequence was obtained by mass spectrometry and used to retrieve a glucoamylase within the annotated genome of A. wentii v1.0. An in silico structural model revealed a N-terminal glycosyl hydrolases family 15 (GH15) domain, which is ligated by a linker to a C-terminal carbohydrate-binding module (CBM) from the CBM20 family.

BACKGROUND The general enzymatic method for producing reducing sugar is liquefaction followed by saccharification of starch. This method results in lower yields, consuming high energy and time. Therefore, the present study evaluated a new approach for producing reducing sugar from sweet potato starch (SPS), including simultaneous liquefaction (by α-amylase) and saccharification (by glucoamylase) of SPS pretreated with high power ultrasound. The effects of ultrasound parameters on the conversion rate of SPS and mechanism were investigated. RESULTS The optimum ultrasound pretreatment conditions were a frequency of 20 kHz, SPS concentration of 125 g L-1 , temperature of 30 °C, pulsed on-time of 3 s, pulsed off-time of 5 s, power density of 8 W mL-1 and sonication time of 15 min. The ultrasound assisted enzymolysis resulted in a SPS conversion rate of 59.10%, which was improved by 56.35% compared to the control. The results of pasting properties and thermal analysis showed that ultrasound pretreatment decreased the peak viscosity, breakdown temperature, setback viscosity, gelatinization range (TC - TO ) and enthalpy of gelatinization (ΔH) of SPS significantly (P

Glycogen storage disease type III (GSD III) is a rare autosomal recessive inborn error of glycogen degradation pathway due to deficiency or reduced activity of glycogen debranching enzyme (GDE) that results in accumulation of abnormal glycogen in the liver, muscle, and heart. The cardinal hallmarks are hepatomegaly, fasting hypoglycemia, seizures, growth retardation, progressive skeletal myopathy, and cardiomyopathy in few. To date, 258 mutations in amyloglucosidase (AGL) gene have been identified worldwide. However, the mutation spectrum in the Asian Indian region is yet to be well characterized. We investigated 24 patients of Asian origin from 21 unrelated families with a provisional diagnosis of GSD III based on clinical and biochemical criteria. Molecular diagnosis was assessed by bidirectional sequencing and the impact of novel missense variants on the tertiary (three-dimensional) structure of GDE was evaluated by molecular modeling approach. Eighteen different pathogenic variants were identified, out of which 78% were novel. Novel variants included five nonsense, three small duplications and two small deletions, a splice site variant, and three missense variants. Variations in Exons 4, 14, 19, 24, 27, and 33 accounted for 61% of the total pathogenic variants identified and Allele p.Gly798Alafs*3 showed a high allele frequency of 11%. Molecular modeling study of novel pathogenic missense variants indicated the probable underlying molecular mechanism of adverse impact of variations on the structure and catalytic function of human GDE. Our study is the first large study on GSD III from the Asian subcontinent, which further expands the mutation spectrum of AGL.

Background During the last decade buckwheat was reported to have positive health effects. The present study investigated a high-polyphenol buckwheat protein (Fagopyrum esculentum Moench) prepared by enzyme-assisted processing, together with its physicochemical properties, in vitro digestibility, and antioxidant activity. Results Buckwheat protein prepared from the synergistic enzymatic action of α-amylase and amyloglucosidase (E-BWP) had much higher polyphenol content than buckwheat protein prepared by isoelectric precipitation (I-BWP) or salt extraction (S-BWP). Rutin degraded during the process, giving quercetin. The protein constituents and amino acid composition of E-BWP were very similar to those of native buckwheat and were able to meet the WHO/FAO requirements for both children and adults. During in vitro digestion, E-BWP showed anti-digestive behavior with a nitrogen release that was lower than that of I-BWP or S-BWP. The positive effect of the polyphenol content of E-BWP resulted in a higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) content and greater reducing activity. Conclusion Buckwheat protein with high polyphenol content was successfully developed by enzyme-assisted processing. It had a well-balanced amino acid profile, antidigestive behavior, and high antioxidant activities. The results suggest that enzyme-assisted processing is promising in the production of polyphenol-enriched cereal protein, contributing higher functionality with good nutritional and antioxidant properties. © 2018 Society of Chemical Industry.

Engineering near-infrared (NIR) light-sensitive enzymes remains a huge challenge. A photothermal effect-associated method is developed for tailoring the enzymatic activity of enzymes by exposure to NIR light. An ultrasmall platinum nanoparticle was anchored in an enzyme to generate local heating upon NIR irradiation, which enhanced the enzyme activity without increasing bulk temperature. Following NIR irradiation, the enzyme activity was tailored rapidly and reversibly, and was modulated by varying laser power density and irradiation time. Four enzymes were engineered, including glucoamylase, glucose oxidase, catalase, and proteinase K with NIR-light sensitivity, and demonstrated their utility in practical applications such as photolithography and NIR light-responsive antibacterial or anticancer actions. Our investigation suggests that this approach could be broadly used to engineer enzymes with NIR-light sensitivity for many biological applications.

Carbohydrate intolerance or malabsorption has been suggested as a cause of chronic abdominal pain (CAP) in a subset of patients. We aimed to evaluate disaccharidase deficiencies in children with functional CAP and to correlate deficiencies with clinical features.Patients presenting to the gastroenterology clinic at Children's Hospital of Wisconsin with abdominal pain prospectively completed a detailed demographic, history, and symptom questionnaire. The CAP cohort included those with at least 1 month of symptoms. Data on disaccharidase activity and histology of endoscopic biopsies were collected retrospectively. Only patients with normal histology were included in the study. The association between groups with low disaccharidases and clinical features was examined.A total of 203 pediatric patients with CAP were included. The mean (SD) age was 11.5 (3.1) years, and 32.5% were male. The percentages of abnormally low disaccharidase levels using the standard laboratory cutoffs were lactase, 37%; sucrase, 21%; glucoamylase, 25%; and palatinase, 8%. Thirty-nine percent of the patients with low lactase also had low sucrase, and 67% of the patients with low sucrase had low lactase. There was no significant difference in the activities of any of the disaccharidases or sucrase/lactase ratio in relation to age. Also, no association was found between stool consistency, stool frequency, or location of pain and low disaccharidase activity.A large proportion of patients with CAP have deficiencies in disaccharidases. Bowel frequency, vomiting, or location of pain was no different between groups, suggesting that these clinical features cannot be used to predict disaccharidase deficiencies.

Infantile Pompe disease is a rare, metabolic disorder due to deficiency of the enzyme acid α-glucosidase that degrades lysosomal glycogen. The deficiency leads to multisystem dysfunction. Neuromuscular weakness due to metabolic myopathy is present, which predisposes children to sleep-disordered breathing. With the advent of enzyme replacement therapy (ERT), children are living longer, and there is a new natural history that is emerging. In a prior paper on our cohort of infantile Pompe disease patients, we reported a high incidence of both hypoventilation and obstructive sleep apnea (OSA). In this retrospective study, we analyzed longitudinal nocturnal polysomnography results from 10 patients with infantile-onset Pompe disease, all of which were on enzyme replacement therapy for a mean of 34.9 months at the time of follow-up study. Patients demonstrated relative stability in sleep disordered breathing, with a trend towards improvement in both OSA and central sleep apnea. ERT may help in the treatment of sleep apnea in this cohort. © 2015 Wiley Periodicals, Inc.

Background Schizophyllum commune is one of the causative agents of basidiomycosis including disorders such as allergic bronchopulmonary mycosis, allergic fungal sinusitis, and mucoid impaction of bronchi, the incidence of those of which has been increasing. These mycoses are difficult to diagnose because only a limited number of diagnostic tools are currently available. The biggest problem is that no specific antigens of S. commune have been identified to enable serodiagnosis of the disease. Objective In this study, we attempted to identify a major antigen of S. commune to establish a reliable serodiagnostic method. Methods We used mass spectrometry to identify an antigen that reacted with the serum of a patient with allergic bronchopulmonary mycosis caused by S. commune. The protein was expressed in Escherichia coli, highly purified, and the patient sera IgG and IgE titres against the protein were determined by enzyme-linked immunosorbent assay. Results The protein identified as a major antigen of S. commune was named Sch c 1; it was a homolog of glucoamylase. The IgG and IgE titres against Sch c 1 in patient sera were significantly higher than those in healthy volunteer sera (P Conclusions and clinical relevance Sch c 1 is recognized by the host immune system of patients as an antigen/allergen. The purified glucoamylase Sch c 1 is a promising candidate antigen for the serodiagnosis of S. commune-induced mycosis.

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α-amylase from Aspergillus oryzae as well as α-amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date.

Based on the characteristics of polycations of chitosan and glucoamylase, which are oppositely charged, they were successfully alternatingly deposited onto the surface of aldehyde-modified Fe3O4 nanoparticles by using a layer-by-layer ion exchange method to form magnetic carriers to construct multilayer films (designated as Fe3O4@(CS/GA)n). The (CS/GA)n film systems were endowed with the pH-dependent properties of chitosan as well as the catalytic activity of glucoamylase. The changes in weight loss and surface chemistry, morphology, and magnetic sensitivity were monitored and verified by UV/Vis spectroscopy, zeta potential, TEM, and a vibrating sample magnetometer. Subsequently, the influence of the number of bilayers, storage stability, pH, temperature, and reusability of Fe3O4@(CS/GA)5 biocatalysts on catalytic activity were investigated. The results from characterization and determination remarkably indicate that Fe3O4@(CS/GA)5 presents excellent catalytic activity, storage stability, pH stability, and reusability in comparison with free enzyme. Fe3O4@(CS/GA)5 retained >60% of its initial activity at 65 °C over 6 h; the optimum temperature and pH also increased to the ranges of 45-65 °C and 2.5-3.5, respectively, and only 27% activity was lost after 10 cycles. This new strategy simplifies the reaction protocol and improves encapsulation efficiency and catalytic activity for new potential applications in biotechnology.

Baker's yeast, Saccharomyces cerevisiae, is a key component in bread baking. Total of 12 commercial baker's yeast and 2 hybrid strains were compared using traditional quality parameters. Total of 5 strains with high leavening power and the 2 hybrid strains were selected and evaluated for their alpha-amylase, maltase, glucoamylase enzymes, and compared using random amplified polymorphic DNA (RAPD). The results revealed that all selected yeast strains have a low level of alpha-amylase and a high level of maltase and glucoamylase enzymes. Meanwhile, the Egyptian yeast strain (EY) had the highest content of alpha-amylase and maltase enzymes followed by the hybrid YH strain. The EY and YH strains have the highest content of glucoamylase enzyme almost with the same level. The RAPD banding patterns showed a wide variation among commercial yeast and hybrid strains. The closely related Egyptian yeast strains (EY and AL) demonstrated close similarity of their genotypes. The 2 hybrid strains were clustered to Turkish and European strains in 1 group. The authors conclude that the identification of strains and hybrids using RAPD technique was useful in determining their genetic relationship. These results can be useful not only for the basic research, but also for the quality control in baking factories.

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is industrially produced by fermentation using Saccharomyces cerevisiae. Before the glutathione fermentation process with S. cerevisiae, a glucose extraction process from starchy materials is required. This glucose extraction is usually carried out by converting starchy materials to starch using high-temperature cooking and subsequent hydrolysis by amylases to convert starch to glucose. In this study, to develop an energy-saving glutathione production process by reducing energy consumption during the cooking step, we efficiently produced glutathione from low-temperature cooked rice using amylase-expressing S. cerevisiae. The combination of the amylase-expressing yeast with low-temperature cooking is potentially applicable to a variety of energy-saving bio-production methods of chemicals from starchy bio-resources.

For the first time, a semi-permanent phospholipid coating is utilized in capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS). Although phospholipids in free solution are generally avoided in ESI, they did not interfere with the detection of linear and branched oligosaccharides using ESI operated in negative mode. The CE and ESI were coupled using a coaxial sheath flow interface. The separation was operated in reversed polarity and the electroosmotic flow was effectively suppressed by the phospholipid coating. The method was characterized with linear oligosaccharides and used to monitor the enzymatic hydrolysis of maltooligosaccharides with α-amyloglucosidase. Branched oligosaccharides were separated and detected with the system. The enzyme β1-4 galactosidase was used to distinguish branched isomeric oligosaccharides derived from asialofetuin.

The market for glucoamylase is large and very competitive and the production process has been optimized through several decades. So far a thorough characterization of the process has not been published, but previous academic reports suggest that the process suffers from severe byproduct formation. In this study we have carried out a thorough characterization of a process as close as possible to the industrial reality. The results show that the oxygen-limited phases of the process have the highest glucoamylase yields on carbon and that the byproducts are efficiently reused in late phases of the process. An alternative process with low glucose concentration show that high osmolarity is beneficial for the process, and we conclude that oxygen limitation, high osmolarity, and the associated byproduct metabolism are important for the efficiency of the process.

BACKGROUND: To verify the viscous potential of rye bread made from population and hybrid cultivars, the overall extract viscosities (EVs) of endosperm and wholemeal breads (EBs and WMBs respectively) were assessed using extractants of different pH. Also, arabinoxylan-dependent EV (AX-EV) was determined after combined action of starch- and protein-degrading enzymes. RESULTS: The synergistic effect of a combination of α-amylase, amyloglucosidase and protease on EV was almost two times higher than the effects observed after single addition of each of them. However, water-extractable arabinoxylans (WE AXs) were the major contributors to EV. Unexpectedly, a decrease in the mean WE AX contents observed in the following order, WMB (31.7 g kg−1) and EB (29.3 g kg−1) of hybrid rye and WMB (28.8 g kg−1) and EB (23.8 g kg−1) of population rye, was accompanied by an increase in their AX-EVs (12, 21, 21 and 29 mPa s respectively). CONCLUSION: In some cases the structure of WE AXs is a more important determinant of AX-EV than their content in rye bread. Nevertheless, both factors influence AX-EV, which may reflect the viscosity of the human small intestine after rye bread consumption, related to some beneficial metabolic effects. Therefore AX-EV can be a preliminary parameter for selection towards high-viscosity bread. Copyright © 2010 Society of Chemical Industry

Open or closed: N-Glycosyl oxyamine versus open-chain glycosyl oxime is the key to protein recognition on glyconanoparticles. Unprotected glycans are captured by oxime formation with a novel bifunctional reagent and the resulting glycan-linker conjugates are anchored to gold nanoparticles (AuNPs). These glyconanoparticles maintain the structural integrity of glycans in the study of protein–carbohydrate interactions (see figure). Nanoparticles functionalized with glycans are emerging as powerful solid-phase chemical tools for the study of protein–carbohydrate interactions using nanoscale properties for detection of binding events. Methods or reagents that enable the assembly of glyconanoparticles from unprotected glycans in two consecutive chemoselective steps with meaningful display of the glycan are highly desirable. Here, we describe a novel bifunctional reagent that 1) couples to glycans by oxime formation in solution, 2) aids in purification through a lipophilic trityl tag, and 3) after deprotection then couples to gold nanoparticles through a thiol. NMR studies revealed that these oximes exist as both the open-chain and N-glycosyl oxy-amine tautomers. Glycan-linker conjugates were coupled through displacement of ligands from preformed, citrate-stabilized gold nanoparticles. Recognition of these glycans by proteins was studied with a lectin, concanavalin A (ConA), in an aggregation assay and with a processing enzyme and glucoamylase (GA). We demonstrate that the presence of the N-glycosyl oxy-amines clearly enables functional recognition in sharp contrast to the corresponding reduced oxy-amines. This concept is then realized in a novel reagent, which should facilitate nanoglycobiology by enabling the operationally simple capture of glycans and their biologically meaningful display.

Total synthesis of naturally occurring casuarine (1) and the first total synthesis of casuarine 6-O-alpha-glucoside (2) were achieved through complete stereoselective nitrone cycloaddition, Tamao-Fleming oxidation and selective alpha-glucosylation as key steps. Biological assays of the two compounds proved their strong and selective inhibitory properties towards glucoamylase NtMGAM and trehalase Tre37A, respectively, which place them among the most powerful inhibitors of these enzymes. The structural determination of the complexes of NtMGAM with 1 and of Tre37A with 2 revealed interesting similarities in the catalytic sites of these two enzymes which belong to different families and clans.

The adsorption of glucoamylases I and II (GA I and GA II, respectively) from Aspergillus niger on the anion exchanger DEAE-Toyopearl 650 was studied in fixed-bed experiments, and the effect of temperature, flowrate, inlet concentration, bed length, and particle size on the process was characterized. The anion exchanger showed a higher adsorption capacity for the more active isoenzyme GA I in all experimental conditions studied. A mathematical model accounting for external and pore diffusion and nonlinear equilibrium isotherm (for GA I) was used to fit the experimental breakthrough curves, showing very accurate fittings in all of the operating conditions. The values of the pore diffusion coefficient at 15, 20, and 25 degrees C were, respectively, 1.25 x 10(-)(11), 1.46 x 10(-)(11) and 1.83 x 10(-)(11) (for GA I) and 1.82 x 10(-)(11), 2.44 x 10(-)(11) and 2.73 x 10(-)(11) (for GA II) m(2)/s. Bicomponent adsorption experiments showed no significant interference effects between GA I and GA II, and so the mathematical model was again used to fit these experiments, yielding very satisfactory results.

Prenatal diagnosis of glycogenosis type II was performed by direct assay of acid alpha-glucosidase (EC 3.2.1.20) in chorionic villous biopsy obtained by transcervical cannula aspiration from a pregnancy at risk in the 10th week of gestation. The exact value of the enzyme activity estimated by the use of antibody preparations for purified human liver acid alpha-glucosidase was in the heterozygous range, and so the homozygous enzyme deficiency could be excluded. The subsequent analysis of cells cultured from amniocentesis sampling in the 18th week of gestation resulted in a similar outcome. The study with antibodies showed that in 23 control chorionic villi obtained during gestational ages between 7-13 weeks, 1-15% of the total alpha-glucosidase activity at pH 4.0 were due to renal or neutral enzyme. This indicates that it may be important to employ antibodies for prenatal diagnosis using chorionic villous sampling. A healthy and unaffected boy was born. The biochemical values obtained from an umbilical blood specimen were in accordance with the results of the prenatal diagnosis.

Textural profile, pasting behavior, gelatinization characteristics, sedimentation volume, and gel consistency of acetylated (Ac) and enzyme (glucoamylase)-modified (EM) potato and sweet potato flours have been investigated to determine their suitability in products such as baked goods, soup, and pudding. Dough hardness of Ac and EM samples was significantly higher than their native samples (P < 0.01). Dough cohesiveness of modified potato did not change, while it decreased in modified sweet potato. With increase in moisture, textural properties of modified samples, in general, showed reduced values. Rapid Visco Analyser showed least pasting viscosities of Ac flours due to restricted swelling of starch granules while EM flours exhibited high viscosities. Acetylated samples showed reduced gelatinization temperature (GT), and enthalpy (DeltaH) compared to native samples, whereas enzyme-treated samples showed no significant changes in GT, indicating their comparable crystallinity values with those of native samples. Modified flour samples had lower sediment volumes and gel consistency, and the gel consistency of EM flour correlated with its cold paste viscosity.

This study describes the course of enzymatic hydrolysis of the native corn starches Maritena 100 and Maritena 300. Hydrolyses were carried out with glucoamylase Glm produced by Saccharomycopsis fibuligera IFO 0111, which degrades also native starch, with the purpose to substitute a two-step hydrolysis (amylase followed by glucoamylase) by a one-step process (glucoamylase only). Hydrolysis generally became more effective by adding the pullulanase Promozyme D, which cleaves alpha-1,6-glycosidic bonds more effectively than glucoamylase Glm does. The time course (kinetics) of hydrolysis was followed by determination of the glucose concentration and calculation of dextrose equivalents.

During silylation of porous glass with 3-aminopropyltriethoxysilane, the properties of the carrier affected the concentration of bound amino groups, the formation of aminopolysiloxane mono- or multilayer on the surface, and the hydrolytic stability of the layer formed. The influence of the carrier depended on the specific surface area and the size of pores. In contrast to silylation performed in organic solvents, in aqueous solutions a monolayer of aminopolysiloxane of high hydrolytic stability was formed on the surface of porous glass. Tetraethoxysilane modification of porous glass prior to silylation with aminosilane yields carriers of increased hydrolytic stability. Glucoamylase immobilised on carriers, that were modified in aqueous solutions, exhibit higher enzymatic activity.

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).

This work evaluated the use of high power ultrasonic energy to treat corn slurry in dry corn milling ethanol plants to enhance liquefaction and saccharification for ethanol production. Corn slurry samples obtained before and after jet cooking were subjected to ultrasonic pretreatment for 20 and 40 s at amplitudes of vibration ranging from 180 to 299 microm(pp) (peak to peak amplitude in microm). The resulting samples were then exposed to enzymes (alpha-amylase and glucoamylase) to convert cornstarch into glucose. A comparison of scanning electron micrographs of raw and sonicated samples showed the development of micropores and the disruption of cell walls in corn mash. The corn particle size declined nearly 20-fold following ultrasonic treatment at high power settings. The glucose release rate from sonicated samples increased as much as threefold compared to the control group. The efficiency of ultrasound exceeded 100% in terms of energy gain from the sugar released over the ultrasonic energy supplied. Enzymatic activity was enhanced when the corn slurry was sonicated with simultaneous addition of enzymes. This finding suggests that the ultrasonic energy did not degrade or denature the enzymes during the pretreatment.

Restructuring the traditional fermentation industry into viable biorefineries for the production of fuels, chemicals and plastics is essential in order to replace (petro)chemical processing. This work presents engineering aspects of Aspergillus awamori submerged fermentation for on-site production of an enzymatic consortium that contains glucoamylase, protease and phosphatase. The crude broth filtrate was used for the production of wheat flour hydrolysates. Improvements on traditional starch hydrolysis carried out in two stages (liquefaction and saccharification) were attempted through integration of unit operations and reduction of processing temperature and reaction duration. An initial increase of temperature to 68 degrees C and a subsequent decrease to 60 degrees C for the rest of the enzymatic hydrolysis resulted in a starch to glucose conversion yield of 94 and 92% when a wheat flour concentration and commercial starch concentration of 225 g L(-1) was used, respectively. The use of crude broth filtrates resulted in the simultaneous hydrolysis of wheat protein and phytic acid, as was indicated by the increase in free amino nitrogen and phosphorus concentration, respectively.

We have a limited understanding of the details of molecular recognition of carbohydrates by proteins, which is critical to a multitude of biological processes. Furthermore, carbohydrate-modifying proteins such as glycosyl hydrolases and phosphorylases are of growing importance as potential drug targets. Interactions between proteins and carbohydrates have complex thermodynamics, and in general the specific positioning of only a few hydroxyl groups determines their binding affinities. A thorough understanding of both carbohydrate and protein structures is thus essential to predict these interactions. An atomic-level view of carbohydrate recognition through structures of carbohydrate-active enzymes complexed with transition-state inhibitors reveals some of the distinctive molecular features unique to protein-carbohydrate complexes. However, the inherent flexibility of carbohydrates and their often water-mediated hydrogen bonding to proteins makes simulation of their complexes difficult. Nonetheless, recent developments such as the parameterization of specific force fields and docking scoring functions have greatly improved our ability to predict protein-carbohydrate interactions. We review protein-carbohydrate complexes having defined molecular requirements for specific carbohydrate recognition by proteins, providing an overview of the different computational techniques available to model them.

For many years, high broth viscosity has remained a key challenge in large-scale filamentous fungal fermentations. In previous studies, we showed that broth viscosity could be reduced by pulsed addition of limiting carbon during fed-batch fermentation. The objective in this study was to determine how changing the frequency of pulsed substrate addition affects fungal morphology, broth rheology, and recombinant enzyme productivity. To accomplish this, a series of duplicate fed-batch fermentations were performed in 20-L fermentors with a recombinant glucoamylase producing strain of Aspergillus oryzae. The total cycle time for substrate pulsing was varied over a wide range (30-2,700 s), with substrate added only during the first 30% of each cycle. As a control, a fermentation was conducted with continuous substrate feeding, and in all fermentations the same total amount of substrate was added. Results show that the total biomass concentration remained relatively unaltered, while a substantial decrease in the mean projected area of fungal elements (i.e., average size) was observed with increasing cycle time. This led to reduced broth viscosity and increased oxygen uptake rate. However, high values of cycle time (i.e., 900-2,700 s) showed a significant increase in fungal conidia formation and significantly reduced recombinant enzyme productivity, suggesting that the fungi channeled substrate to storage compounds rather than to recombinant protein. In addition to explaining the effect of cycle time on fermentation performance, these results may aid in explaining the discrepancies observed on scale-up to larger fermentors.

Background Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice. Methods An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated. Results After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles. Conclusions Overall, the previously reported ‘resistance’ of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA. Copyright © 2004 John Wiley & Sons, Ltd.

Recombinant proteins are often produced as isoforms with different kinds and amounts of post-translational modifications that alter their function. Isoelectric focusing in shallow pH gradients, less than 0.5 pH/cm, might be capable of fractionating these isoforms. The synthetic carrier ampholyte mixtures typically used to generate these pH gradients are expensive and may adversely interact with proteins. Using defined buffers instead of synthetic carrier ampholytes reduces these problems. We tested two defined buffer systems in a vortex-stabilized electrophoresis device to see if they could form shallow pH gradients useful for separating isoforms. These pH gradients were formed by pouring a two-component concentration gradient. The poured gradients were smooth, reproducible, and stable for at least 1.5 h at 5 kV. One poured gradient focused 20 mg of cytochrome c. A second poured gradient separated glucose oxidase from amyloglucosidase. The breadth of the amyloglucosidase band indicates that the shallow, poured pH gradients can only partially separate protein isoforms at 10 kV. Proteins with pI < 0.2 pH units apart will have overlapping bands in these shallow, poured pH gradients.

The sequence of the STA1-encoded glucoamylase of amylolytic Saccharomyces cerevisiae (var. diastaticus) strains shows two well-defined regions: an amino-terminal part rich in serine and threonine residues and a carboxy-terminal part very similar to the catalytic domain of other fungal glucoamylases. A version of the enzyme in which most of the amino-terminal region was deleted still has glucoamylase activity, indicating that the remaining carboxy-terminal part forms a functional catalytic domain. Homology-based models of the two parts of the protein have been obtained. As expected, the shortened form of the enzyme is very similar to the catalytic domain of related glucoamylases of known structure. However, the amino-terminal part yielded a structure revealing an unexpected similarity to bacterial invasins, suggesting functional connections between several yeast proteins homologous to STA1-encoded glucoamylase and invasins. A characteristic of Saccharomyces glucoamylase in its native form is its extreme degree of glycosylation. Despite its high molecular mass (about 300 kDa), and in contrast with what occurs with other extracellular glycoproteins produced by yeast, the enzyme does not remain attached to the cell wall, being fully and efficiently secreted into the medium, even when it is produced in large amounts by overexpression of its gene. Copyright © 2004 John Wiley & Sons, Ltd.

We present an automated docking protocol specifically optimized to predict the structure and affinity of a protein-carbohydrate complex. A scoring function was developed based on a training set of 30 protein-carbohydrate complexes of known structure and affinity. Combinations of several models for hydrogen bonding, torsional entropy loss, and solvation were tested for their ability to fit the training set data, and the best model was used with AutoDock. The electrostatic empirical coefficient is larger than in a previously obtained model using a training set comprised of various types of protein-ligand complexes, indicating that electrostatic interactions play a more important role in determining the affinity between a carbohydrate and a protein. The differences in the relative weighting of the empirical coefficients in the model yields predicted free energies for the training set with a standard error of 1.403 kcal/mol. The new scoring function was tested on 17 Aspergillus niger glucoamylase inhibitors for which binding energies had been determined experimentally. Free energies of complex formation were predicted with a residual standard error of 1.101 kcal/mol. The new scoring function therefore provides a robust method for predicting free energies of formation and optimal conformations of carbohydrate-protein complexes.

Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source. Eight constructs containing the L. kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S. fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared. The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators. The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators. These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b). Southern-blot analysis confirmed the stable integration of the different gene constructs into the S. cerevisiae genome and plate assays revealed amylolytic activity. The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media. This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation. The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity. It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S. cerevisiae. This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S. cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries.

The unfolding mechanism of the 13 alpha-helices in the catalytic domain of Aspergillus awamori var. X100 glucoamylase was investigated by 200 ps molecular dynamics simulations in explicit water with temperature jump technique. Rather than a simultaneous event, the unfolding of these 13 alpha-helices followed a random ordered mechanism as alpha8-->alpha1-->alpha11-->alpha7-->alpha10-->alpha3-->alpha12-->alpha13-->alpha4-->alpha5-->alpha9-->alpha6-->alpha2. No significant relationships were found between the unfolding order and the length and the hydrophobicity of the helix. alpha-Helix 8 located in the inner region of the catalytic domain was predicted to be the first helix to unfold, indicating that the destruction of the secondary structure motif was initiated from the inner region of the catalytic domain. The dynamic behavior of these alpha-helices induced by increased kinetic energy during the unfolding process is considered to be similar to the expansion and compression of a series of springs under the influence of mechanical stress.

Productivity in many fungal fermentations is detrimentally affected by high broth viscosity and consequent reduced oxygen mass transfer capacity. The goal here was to determine whether pulsed feeding of limiting carbon in a fungal fermentation could lead to reduced viscosity and improved oxygen mass transfer. As a model, an industrially relevant recombinant strain of Aspergillus oryzae was grown in carbon-limited, fed-batch mode. Maltodextrin was used as a carbon source and was added either continuously or in 1.5-min pulses, 3.5 min apart. In both feeding modes the same total amount of carbon was added, and carbon feed rate was at sufficiently low levels to ensure cultures were always carbon-limited. Compared to continuous feeding, pulsed addition of substrate led to smaller fungal elements, which resulted in a significant reduction in broth viscosity. This in turn led to higher dissolved oxygen concentrations and increased oxygen uptake rates during pulsed feeding.

Fungal morphology in many filamentous fungal fermentations leads to high broth viscosity which limits oxygen mass transfer, and often results in reduced productivity. The objective in this study was to determine if a simple, fed-batch, process strategy-pulsed addition of limiting-carbon source-could be used to reduce fungal broth viscosity, and increase productivity of an industrially relevant recombinant enzyme (glucoamylase). As a control, three Aspergillus oryzae fed-batch fermentations were carried out with continuous addition of limiting-carbon. To determine the effect of pulse-feeding, three additional fermentations were carried out with limiting-carbon added in 90-second pulses, during repeated five-minute cycles. In both cases, overall carbon feed-rate was used to control dissolved oxygen concentration, such that increased oxygen availability led to increased addition of limiting-carbon. Pulse-fed fermentations were found to have smaller fungal mycelia, lower broth viscosity, and improved oxygen mass transfer. As a result, more carbon was added to pulse-fed fermentations that led to increased enzyme productivity by as much as 75%. This finding has significant implications for the bioprocessing industry, as a simple process modification which is likely to cost very little to implement in most production facilities, has the potential to substantially increase productivity.

Background Previously, in murine models of acid maltase deficiency (AMD), we demonstrated that intravenous administration of an improved adenovirus (Ad) vector encoding human acid alpha glucosidase (hGAA) resulted in liver transduction, followed by high-level hepatocyte-mediated secretion of hGAA into the plasma space. The hGAA secreted by the liver was taken up and targeted to muscle cell lysosomes. The levels of hGAA achieved by this approach resulted in clearance of lysosomal glycogen accumulations; in some muscle tissues the effect was prolonged (>6 months). We next wished to demonstrate whether this approach could be generalized across divergent species. To accomplish this goal, we determined whether a similar approach would also result in efficacy, but in a quail model of AMD. Methods An [E1-, E2b-]Ad vector encoding hGAA was intravenously injected into AMD quails. At several time points thereafter, plasma, liver, and multiple muscle tissues were assayed for evidence of hGAA gene expression, liver-mediated hGAA secretion, uptake of hGAA by skeletal muscles, and evidence of glycogen correction in AMD skeletal muscles. These results were compared with those obtained from mock-injected AMD or wild-type quails. Results Intravenous [E1-, E2b-]Ad/hGAA vector injection resulted in high-level liver transduction and hepatic secretion of precursor forms of hGAA. The hepatically secreted hGAA was found to not only be efficiently taken up by cardiac and skeletal muscles, but was also proteolytically cleaved and processed equivalently to the quail-GAA protein detected in wild-type quails. The observations suggest that the signals regulating muscle cell uptake (but not proteolytic cleavage) of lysosomal enzymes are conserved and recognized across divergent species of vertebrates. Importantly, once localized to skeletal muscle lysosomes, the hGAA was able to effectively clear the glycogen accumulations present in AMD quail muscles. Conclusions Adenovirus-mediated transduction of the hGAA gene, followed by hepatic secretion, uptake, and cross-correction of the pathologic glycogen accumulation noted in multiple muscles of both the AMD mouse and AMD quail, adds support to the notion that gene transfer strategies (Ad-mediated or other agents) targeting liver tissues with the hGAA gene are likely to be highly efficacious in humans affected by AMD. Copyright © 2002 John Wiley & Sons, Ltd.

A novel porous ceramics support, named "Toyonite," for the immobilization of enzymes was prepared from the minerals of kaolinite under acidic conditions. Modification of the porous surface of Toyonite with two different organic coating agents gave Toyonite 200-M (TN-M), and Toyonite 200-A (TN-A), possessing methacryloyloxy and amino groups on the respective surfaces. Compared with other solid supports, TN-M and TN-A supports exhibited high selectivity for lipase PS (Pseudomonas cepacia, Amano) and glucoamylase (Gluczyme AF 6, Amano) proteins, respectively. The activities of both the transesterification of rac-1 with TN-M PS lipase and the hydrolysis of starch with TN-A glucoamylase were greater than those of similar reactions with these two enzymes immobilized on other solid supports. Further, TN-M PS lipase showed higher reactivity toward synthetic substrates, including aromatic and aliphatic secondary alcohols, than the free enzyme powder.

The hydrolysis of maltodextrins (10 DE) by glucoamylase was studied in a batch reactor at temperatures between 40 and 80 degrees C and substrate concentration range from 17 to 300 kg/m(-3). The experimental data were fitted to a model including thermal deactivation of the enzyme. In the model, the reaction rate was correlated with an extended Michaelis-Menten equation including inhibition by product, and the thermal deactivation of glucoamylase was fitted with a first-order reaction. The dependence of rate parameters on temperature was correlated using the Arrhenius equation. The differential equation of the model was integrated and the optimal enzyme demand and temperature were determined for isothermal operation.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase-GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 x 10(6) spores/mL. It resulted in a specific protease activity of 158 units/L, only one-third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0. 29 mg/g for free mycelial growth with an inoculum of 10(7) spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.

The mutation Gly467--Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.

Mass spectrometric identification of cysteinsulfinic acid resulting in restoration of activity of chemically modified Glu400 Cys catalytic-base glucoamylase (GA) mutants is described. This oxidation unexpectedly occurred during attempts to carboxyalkylate the Cys400 GA mutant using three different alkylation reagents. However, mass spectrometric peptide mapping did not show the presence of carboxyalkylation of the Cys400 residue but suggested an oxidation to cysteinsulfinic acid based on the observed mass increment. The presence of cysteinsulfinic acid was confirmed by employing matrix-assisted laser desorption/ionization mass spectrometry combined with post-source decay analysis. Furthermore, strong enhancement of metastable fragmentation was observed for peptides containing oxidized Cys in comparison with non-oxidized peptide. Copyright © 1999 John Wiley & Sons, Ltd.

The glaA gene encoding glucoamylase I (GAI) of Aspergillus awamori var. kawachi was heterologously expressed in mannosyltransferase mutants of Saccharomyces cerevisiae, in which the pmt1 gene and the kre2 gene were disrupted. The GAI enzymes expressed in these yeast mutant cells exhibited a lesser extent of O-glycosylation. Secretion of GAI expressed in the pmt1-disruptant and in the kre2-disruptant, respectively, was almost the same as that of GAI expressed in wild type (wt) strains. The number of O-linked mannose in GAI from wt yeast strain ranged in size from one (Man1) to five (Man5). On the other hand, the O-linked oligosaccharides of GAI from the pmt1-disruptant ranged in size from Man1 to Man4. Man5 was not detected and Man2-Man4 were reduced in proportion to the reduction of Man1. The O-linked oligosaccharides of GAI from the kre2-disruptant ranged from Man1 to Man4, and the molar amount of Man4 was reduced to 27.3%, compared to that of the wt strain. The hydrolyzing abilities for soluble starch and the adsorbing abilities on raw starch were comparable between both disruptants and wt strains. However, the digesting abilities for raw starch of the disruptants were decreased to 70% of those of the wt strains. Stabilities of GAI of the disruptants were reduced toward extreme pH and high temperature, compared to those of the wt strains. These results demonstrate that the O-linked oligosaccharides of GAI are responsible for the enzyme stability and activity toward insoluble substrates but not for secretion.

Summary Genotyping was carried out for glycogen storage disease type II and type V in seven cattle breeds. The analysis was carried out using the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) method. In the breeds analysed [Charolais, Czech Spotted (Czech Simmental), Belgian Blue, Limousine, Blonde d'Aquitaine, Aberdeen Angus, and Beef Simmental sires reared in the Czech Republic], the recessive allele was not found in the PYGM (phosphorylase glycogen, muscle) responsible for the glycogen storage disease type V. In the same panel, the recessive allele in exon 7, exon 9 and exon 13 of the GAA (glucosidase alpha, acid), causing the glycogen storage disease type II was not found. Therefore, we have not revealed the recessives outside previous reported breeds. The knowledge of the breed-specific occurrence of inherited disorders facilitates focusing and reduces the costs of detecting the heterozygous carriers of recessive inherited disorders.

Capillary electrophoresis (CE) of sodium dodecyl sulfate (SDS)-protein complexes using a nongel sieving matrix (CE-SDS) has been applied to the simultaneous analysis of alpha-amylase and glucoamylase activity in sake rice koji which is employed for the brewing of sake. Alpha-amylase and glucoamylase in sake rice koji extracts were successfully analyzed by CE-SDS. Alpha-amylase and glucoamylase were found to have molecular masses of 53000 and 63000 Da, respectively, as determined by the migration times of eight standard proteins. These values agree with those determined by SDS-polyacrylamide gel electrophoresis (PAGE). The results of CE-SDS method were compared with those achieved by the official method. The relative standard deviations (RSD) of the alpha-amylase and glucoamylase activities by CE-SDS were less than 5.0% in both intra-day and inter-day experiments. An electrophoretic analysis of products of an enzyme reaction of a substrate by in-capillary reaction was also useful for the activity measurement of glucoamylase in sake rice koji. p-Nitrophenyl-beta-D-maltoside (PNP-Mal) was employed as a substrate and p-nitrophenyl-beta-D-glucopyranoside (PNP-Glu) was the product of the enzyme reaction. The glucoamylase activity of sake rice koji samples gave the good linear relationship with the peak area observed in the in-capillary enzyme reaction method. The glucoamylase activity in sake rice koji was measured by either CE-SDS or the in-capillary enzyme reaction more easily than by the official method. Both methods can be applied to the routine quality control of alpha-amylase and glucoamylase activities in sake rice koji.

Release of product from the active site is the rate-limiting step in a number of enzymatic reactions, including maltose hydrolysis by glucoamylase (GA). With GA, an enzymatic conformational change has been associated with the product release step. Solvent characteristics such as viscosity can strongly influence protein conformational changes. Here we show that the rate-limiting step of GA has a rather complex dependence on solvent characteristics. Seven different cosolvents were added to the GA/maltose reaction solution. Five of the cosolvents, all having an ethylene glycol base, resulted in an increase in activity at low concentration of cosolvent and variable decreases in activity at higher concentrations. The increase in enzyme activity was dependent on polymer length of the cosolvent; the longer the polymer, the lower the concentration needed. The maximum increase in catalytic activity at 45 degrees C (40-45%) was obtained with the three longest polymers (degree of polymerization from 200 to 8000). A further increase in activity to 60-65% was obtained at 60 degrees C. The linear relationship between ln(kcat) and (viscosity)2 obtained with all the cosolvents provides further evidence that product release is the rate-limiting step in the GA catalytic mechanism. A substantial increase in the turnover rate of GA by addition of relatively small amounts of a cosolvent has potential applications for the food industry where high-fructose corn syrup (HFCS) is one of the primary products produced with GA. Since maltodextrin hydrolysis by GA is by far the slowest step in the production of HFCS, increasing the catalytic rate of GA can substantially reduce the process time.