Aims: The study aimed to investigate the effect of glucose on alpha-amylase and glucoamylase production in some Indonesian indigenous fungi. Methodology and results: Fungi were screened for their ability to produce alpha-amylase and glucoamylase in the presence of glucose. The strains were grown in a medium containing starch and glucose as carbon sources with glucose concentrations varying from 0 to 5% for four days, and the alpha-amylase and glucoamylase were analyzed at the end of the growth period. Most strains showed repression on the amylases production when glucose was added to the medium. However, some strains showed no repression on amylases production when glucose was supplemented to the medium. The addition of glucose repressed glucoamylase production, but no repression on alpha-amylase was noted for strain KKB4, vice versa, there was repression on alpha-amylase production but no repression on glucoamylase production for strain FIG1. Strains FNCC 6151 and MLT1J1 showed no repression on both alpha-amylase and glucoamylase production when glucose was added to the medium up to 5%. The occurrence of repression in the production of alpha-amylase and glucoamylase was strain-specific. Conclusion, significance and impact of study Out of the nine indigenous fungi strains examined, strains FNCC 6151 and MLT1J1 showed no repression on both alpha-amylase and glucoamylase production when glucose was added to the medium up to 5%. Those two strains have the potential to be improved further to produce both alpha-amylase and glucoamylase.
Glucosidases ; alpha-Amylases ; Glucan 1,4-alpha-Glucosidase

The fruit body of Ramaria botrytis DGUM 29001 was used to determine enzyme activities of fruit body. The specific activity of laccase was the highest(6.5 unit/mg.protein) and that of alpha-amylase and xylanase was relatively high. However, little or no enzyme activity of beta-glucosidase, CMCase, exo-beta-1,4-glucanase, chitinase, lipase and protease was found.
alpha-Amylases ; beta-Glucosidase ; Botrytis* ; Chitinase ; Fruit* ; Glucan 1,4-beta-Glucosidase ; Laccase ; Lipase

Glucoamylase overproducing A. niger T21 was mutated by UV mutagensis. An extracellular acid protease-deficient mutant, A. niger T21-201, which produced only 0.76% extracellular acid protease activity of the parent strain, was screened by casein-degradating plate and determination of protease activity. Moreover, the growth properties and the ability to secrete glucoamylase of A. niger T21-201 are identical to these of starting strain T21. The comparison of expression-secretion levels of heterologous gene in A. niger T21-201 and T21 was carried out with bacterial vhb as reporter, the level of expression-secretion of VHb in A. niger T21-201 was 6-7 times higher than that in T21, but the transcriptional levels of vhb gene in both strains were similar revealed by Northern blot. Therefore, it was demonstrated that the deficiency of acid protease of recipient T21-201 has significant effect on the protection of heterologous protein.
Aspergillus niger ; enzymology ; genetics ; Glucan 1,4-alpha-Glucosidase ; biosynthesis ; genetics ; Mutation ; Recombinant Proteins ; biosynthesis

The glucoamylase gene (glaA) of Aspergillus niger CICIM F0410 was cloned, sequenced and expressed. The integrated plasmid pBC-Hygro-glaA carrying the glaA was constructed and transformed into A. niger F0410. Transformants with multiple copies of glaA integrated in the chromosome were selected by 150 microg/mL hygromycin B and identified by real-time PCR. Two to three multiples of glaA in the chromosome were found to be optimal for higher expression of glucoamylase. Shake-flask fermentation under optimal conditions showed that glucoamylase secreted by the transformant GB0506 was 17.5% higher than parental strain F0410 at the end of fermentation. In conclusion, increasing copy number of glaA by chromosomal integration significantly improves the yield of glucoamylase in the industrial strain of A. niger.
Aspergillus niger ; genetics ; Cloning, Molecular ; Glucan 1,4-alpha-Glucosidase ; biosynthesis ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis

Glucoamylase is a critical ingredient for saccharification in the starch decomposition, and widely used in food, pharmaceutical and fermentation industries. Glucoamylases are usually thermostable and have peak activities at high temperature, as required for the industrial process of glucose production. In this study, a glucoamylase gene belonging to the glycoside hydrolase (GH) family 15, Tlga15A, was cloned from Talaromyces leycettanus JCM12802, and successfully expressed in Pichia pastoris GS115. Recombinant glucoamylase TlGA showed optimal activities at pH 4.5 and 75 °C. The result of thermostability analysis showed that TlGA retained above 70% activity after incubating for 1 h at 65 °C, and 43% residual activity after 30 min at 70 °C. Moreover, TlGA had high resistance to most metal ions and chemical reagents tested. Various starch substrates could be hydrolyzed by TlGA, including soluble starch (255.6±15.3) U/mg, amylopectin (342.3±24.7) U/mg, glycogen (185.4±12.5) U/mg, dextrin (423.3±29.3) U/mg and pullulan (65.7±8.1) U/mg. The primary, secondary and tertiary structures of glucoamylase were further analyzed. The low ratio of Gly in the primary structure and low exposed nonpolarity solvent accessible surface in the tertiary structure may be the main reasons for TlGA's thermostability. These results show that TlGA is great promising for potential use in the commercial production of glucose syrups. Moreover, this research will provide knowledge and innovating ideas for the improvement of glucoamylase thermostability.
Cloning, Molecular ; Enzyme Stability ; Glucan 1,4-alpha-Glucosidase ; Hydrogen-Ion Concentration ; Pichia ; Talaromyces ; Temperature

PURPOSE: Icodextrin in peritoneal cavity is absorbed via the lymphatics to the blood and metabolized to maltose and maltriose which may interfere with correct measurement of glucose. In an attempt to evaluate the effects of icodextrin on the erroneous results of blood glucose, we measured blood glucose by different methods. METHODS: Peripheral capillary blood and venous blood were obtained from 12 patients using icodextrin and from 12 patients not using icodextrin. Venous blood glucose was measured by using the laboratory technique (glucose oxidase method), and capillary blood glucose was measured by using a Surestep (glucose oxidase method) and an Acucheck (GDH-PQQ method). To estimate icodextrin and its metabolites indirectly, we calculated osmolal gap. We measured blood icodextrin and its metabolites with amyloglucosidase in icodextrin group. RESULTS: In icodextrin group, glucose was overestimated in the results of the GDH-PQQ method (delta= GDH-GOD=56.2+/-30 mg/dL [vein] 58+/-32 mg/dL [capillary]), but in the control group, there were no significant differences in the results between the glucose oxidase method and the GDH-PQQ method. There was a correlation between the osmolal gap and the differences in the results (delta=GDH-GOD) (r=0.741, p=.006 [vein], r=0.671, p=.017 [capillary]). Blood icodextrin and its metabolites were related with the differences in the results (delta=GDH-GOD) (p=.026, r=0.635), but there was no significant correlation between the osmolal gap and the icodextrin and its metabolites (p=0.086, r=0.515). CONCLUSION: Icodextrin and its metabolites may lead to erroneously high blood glucose levels when measured by GDH-PQQ method. It is necessary to be aware of this factor in order to prevent overlooking dangerous hypoglycemia.
Blood Glucose* ; Capillaries ; Glucan 1,4-alpha-Glucosidase ; Glucose ; Glucose Oxidase ; Humans ; Hypoglycemia ; Maltose ; Oxidoreductases ; Peritoneal Cavity ; Peritoneal Dialysis, Continuous Ambulatory*

Carbon-limited continuous culture was used to study the relationship between the growth of Aspergillus niger and the production of glucoamylase. The result showed that when the specific growth rate was lower than 0.068 h(-1), the production of glucoamylase was growth-associated, when the specific growth rate was higher than 0.068 h(-1), the production of glucoamylase was not growth-associated. Based on the result of continuous culture, the Monod dynamics model of glucose consumption of A. niger was constructed, Combining Herbert-Pirt equation of glucose and oxygen consumption with Luedeking-Piret equation of enzyme production, the black-box model of Aspergillus niger for enzyme production was established. The exponential fed-batch culture was designed to control the specific growth rate at 0.05 h(-1) by using this model and the highest yield for glucoamylase production by A. niger reached 0.127 g glucoamylase/g glucose. The black-box model constructed in this study successfully described the glucoamylase production by A. niger and the result of the model fitted the measured value well. The black-box model could guide the design and optimization of glucoamylase production by A. niger.
Aspergillus niger ; metabolism ; Batch Cell Culture Techniques ; Carbon ; Culture Media ; Glucan 1,4-alpha-Glucosidase ; biosynthesis ; Glucose ; Industrial Microbiology ; methods ; Oxygen

The gelatinization process of the starch is replaced by unpolluted steam-pretreatment on the base of the Radix Puerariae rich in fiber and isoflavones. The production of ethanol and isoflavones by simultaneous saccharification and solid state fermentation (SSF) of steam-pretreatment Radix Puerariae is presented. The optimal technological conditions were obtained: Radix Puerariae being steam-pretreated at a saturated vapor pressure of 0.8 MPa for 3.5 min, glucoamylase(65 u/g), cellulase(1.5 u/g), 0.1%(NH4)2SO4, 0.1%KH2PO4 and activated yeasts being added in, and fermentation at 35-37 degrees C for 60 h. Under these conditions, the yield of ethanol and isoflavones from 100 g Radix Pureriae (dry basis) were 27.47 g and 4.43 g, respectively, the starch utilization rate was 95%. In comparison with the traditional fermentation technology, the simultaneous saccharification and SSF of steam-pretreatment Radix Puerariae is clean and energy-saving. It provides new way of the production of ethanol from the non-food starch material, and worthwhile to be explored and implemented in industry.
Cellulase ; metabolism ; Ethanol ; metabolism ; Fermentation ; physiology ; Glucan 1,4-alpha-Glucosidase ; metabolism ; Isoflavones ; biosynthesis ; Pueraria ; metabolism ; Steam ; Yeasts ; metabolism

Extracellular amylase properties were examined with the mycelium of Tricholoma matsutake isolated from ectomycorrhizal roots of Pinus densiflora. The molecular weights of alpha-amylase and glucoamylase were estimated as 34.2 kD and 11.5 kD, respectively, after eluted through Superdex 75 column. The optimum pH of the purified enzyme was found in a range of pH 5.0~6.0, with a peak at pH 5.0. The activities of these enzymes were stable from 4degrees C to 30degrees C. The alpha-amylase of T. matsutake readily hydrolyzed soluble starch and amylose-B, while it weakly hydrolyzed glycogen, dextrin, amylose and amylose-A. The main products of hydrolysis were confirmed to be glucose, maltose and maltotriose on the basis of the similarities in the thin layer chromatographic mobility.
alpha-Amylases ; Amylases* ; Amylose ; Fungi* ; Glucan 1,4-alpha-Glucosidase ; Glucose ; Glycogen ; Hydrogen-Ion Concentration ; Hydrolysis ; Maltose ; Molecular Weight ; Mycelium ; Pinus ; Starch ; Tricholoma*

OBJECTIVEGlycogen storage disease type II (GSD II, Pompe disease) is caused by the deficiency of acid alpha-glucosidase (GAA) that leads to lysosomal glycogen accumulation. Early diagnosis and treatment of GSD II are considered to be critical for maximum efficacy of the enzyme replacement therapy. The aim of this study was to introduce two reliable methods and to generate the reference range of GAA activity.

METHODThe assay of GAA activity was performed in dried blood spots (DBS) and mixed leukocytes with acarbose to eliminate isoenzyme interference and to generate the reference range. GAA activity was assayed in 700 specimens for DBS from normal subjects and 100 specimens for mixed leukocytes from normal subjects to set up reference range. GAA activity in the samples of 4 patients who were clinically suspected of GSD II and their parents were also assayed.

RESULTThe intra-run and inter-run precision of the DBS method was less than 10%. GAA activity tested by DBS was stable for 28 days between room temperature and -80 degrees C. The reference range of newborns and children-adults in DBS samples was 8.92 - 60.03 pmol/(punch x h) and 8.00 - 37.43 pmol/(punch x h), respectively. The reference range in mixed leukocytes samples was 12.56 - 50.26 nmol/(mg protein x h). Four patients were diagnosed as GSD II with the above-mentioned two methods.

CONCLUSIONThe determination of GAA activity in DBS is sensitive and time-saving, and is suitable for high throughput analysis and newborn screening for GSD II. The assay of GAA activity in mixed leukocytes is accurate, fast and specific, and is suitable for final diagnosis of GSD II.

Adolescent ; Adult ; Child ; Child, Preschool ; Glucan 1,4-alpha-Glucosidase ; metabolism ; Glycogen Storage Disease Type II ; blood ; diagnosis ; enzymology ; Humans ; Infant ; Leukocytes ; enzymology ; Middle Aged ; Reference Values ; Young Adult