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

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

The most economically important species used in a wide range of fermentation industries throughout Asia belong to Aspergillus section Flavi, which are morphologically and phylogenetically indistinguishable, with a few being toxigenic and therefore a major concern. They are frequently isolated from Korean fermentation starters, such as nuruk and meju. The growing popularity of traditional Korean alcoholic beverages has led to a demand for their quality enhancement, therefore requiring selection of efficient non-toxigenic strains to assist effective fermentation. This study was performed to classify the most efficient strains of Aspergillus section Flavi isolated from various types of traditional wheat nuruk, based on a polyphasic approach involving molecular and biochemical evaluation. A total of 69 strains were isolated based on colony morphology and identified as Aspergillus oryzae/flavus based on internal transcribed spacer and calmodulin gene sequencing. Interestingly, none were toxigenic based on PCR amplification of intergenic regions of the aflatoxin cluster genes norB-cypA and the absence of aflatoxin in the culture supernatants by thin-layer chromatography analysis. Saccharification capability of the isolates, assessed through α-amylase and glucoamylase activities, revealed that two isolates, TNA24 and TNA15, showed the highest levels of activity. Although the degrees of variation in α-amylase and glucoamylase activities among the isolates were higher, there were only slight differences in acid protease activity among the isolates with two, TNA28 and TNA36, showing the highest activities. Furthermore, statistical analyses showed that α-amylase activity was positively correlated with glucoamylase activity (p < 0.001), and therefore screening for either was sufficient to predict the saccharifying capacity of the Aspergillus strain.
Aflatoxins ; Alcoholic Beverages ; Amylases ; Asia ; Aspergillus* ; Calmodulin ; Chromatography, Thin Layer ; DNA, Intergenic ; Fermentation* ; Glucan 1,4-alpha-Glucosidase ; Mass Screening ; Polymerase Chain Reaction ; Triticum

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

OBJECTIVETo analyze and summarize the characteristics of glycogen storage disease type II (Pompe disease) patients according to the clinical description and prognosis.

METHODSeventeen Chinese patients diagnosed by acid alpha-glucosidase (GAA) enzyme activity test were reviewed. Clinical data tables were designed. Interviews were made via phone calls. Information was collected to reach the objective.

RESULTFour of 17 patients diagnosed by acid alpha-glucosidase are infantile-onset, symptoms started between 2 to 6 months after birth with increased serum creatine kinase and cardiac problems, with or without respiratory concerns. Other 13 patients were later-onset cases, and their symptoms started between 2 to 22 years of age with increased serum creatine kinase. Eleven later-onset patients started with muscle weakness, 2 patients developed respiratory insufficiency, 2 patients showed scoliosis, and 1 patient expressed increased serum creatine kinase with abnormal liver function. Just 3 of the later-onset patients were treated with mechanical ventilator and adjuvant therapy, others were not. All patients' acid alpha-glucosidase (GAA) enzyme activity analysis showed lower than 10% of normal. Fourteen patients were tested by muscle biopsy pathology, and 9 of them progressed to glycogen storage disease type II; 10 patients received genetic analysis, and 6 of them had two mutations which cause the disorder. Twelve of the 17 patients were interviewed successfully. In 3 of the infant-onset patients the disease resulted in death from respiratory failure, and 1 is still alive at the age of 1 year and 7 months. In 4 of 8 later-onset patients the disease resulted in death from respiratory failure between 3 to 5 years after onset of symptoms. Three of 4 survivors had increased muscle weakness, and 1 patient kept alive with ventilator without any changes. Seven of 12 interviewed patients died, the mortality rate was 58.3%.

CONCLUSIONGlycogen storage disease type II (Pompe disease) present differently in the clinic. Infant-onset Pompe disease is mainly characterized by generalized muscle weakness and obvious cardiac involvement. It's a dangerous disease, with high mortality rate. Later-onset Pompe disease is characterized by chronic proximal muscle weakness and respiratory insufficiency. GAA enzyme activity analysis, muscle biopsy and genetic analysis used to support the diagnosis of Pompe disease. Prognosis of the disease depends on age of onset and respiratory muscle involvement.

Adolescent ; Biopsy ; Cardiomyopathies ; epidemiology ; etiology ; Child ; Child, Preschool ; Clinical Enzyme Tests ; Creatine Kinase ; blood ; Female ; Follow-Up Studies ; Glucan 1,4-alpha-Glucosidase ; genetics ; metabolism ; Glycogen Storage Disease Type II ; diagnosis ; genetics ; pathology ; Humans ; Infant ; Male ; Muscle Weakness ; epidemiology ; etiology ; Prognosis ; Respiratory Insufficiency ; epidemiology ; Retrospective Studies ; Young Adult

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

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

According to the reported gene sequence of Rhizopus oryzae glucoamylases, the glucoamylase gene containing four introns was cloned from the total DNA of the natural Rhizopus arrhizu. Specific primers were designed to delete introns by overlapping PCR and a new cDNA sequence of Rhizopus arrhizu glucoamylase was obtained. The accession number in gene bank is DQ903853. This gene is successfully expressed in the Picha pastoris, producing a new protein with a high activity of glucoamylase.
Biocatalysis ; Blotting, Western ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Fungal ; Glucan 1,4-alpha-Glucosidase ; genetics ; metabolism ; Molecular Sequence Data ; Pichia ; genetics ; Recombinant Proteins ; metabolism ; Rhizopus ; enzymology ; genetics ; Sequence Analysis, DNA

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*

OBJECTIVEGlycogen-storage disease type II (GSD II, Pompe's disease) is an autosomal recessive disorder caused by a functional deficiency of acid alpha-glucosidase (GAA) that leads to glycogen accumulation within lysosomes in most tissues. The GAA gene is located to human chromosome 17q25 and contains 20 exons, 19 of which are coding. Clinically, patients with the severe infantile form of GSD II have muscle weakness and cardiomyopathy eventually leading to death before the age of two years. Patients with the juvenile or the adult form of GSD II present with myopathy with a slow progression over several years or decades. A broad genetic heterogeneity has been described in GSD II in Europe, South Africa, USA, Japan and Korea, however, the investigation has not been performed in the patients from the mainland of China. In this study, clinical analysis and mutation detection were done on Chinese patients.

METHODSTwo unrelated juvenile patients with late onset GSD II (one boy, 3 years old and one girl, 9 years old) were included in the study with the informed consents. The diagnosis was confirmed by alpha-glucosidase determination in cultured fibroblasts. In addition, their clinical presentation, laboratory findings, electrophysiologic studies and muscle biopsy findings were analyzed in detail. Genomic DNA samples were extracted from fibroblasts of the probands, from peripheral blood of their parents and 50 unrelated, normal individuals. All the coding 19 exons and exon-intron boundaries of GAA were detected in the proband by polymerase chain reaction (PCR) and direct sequencing.

RESULTSOne patient presented decrease of muscle strength, limb-girdle hypotonia, the other patient presented reduced muscle volumes and respiratory problems. Both had increased CPK value, myopathic pattern on EMG; vacuoles on muscle biopsy, and deficiency of 1, 4-alpha-glucosidase activity. After 1 year follow up, the girl died after pneumonia at 10 years of age. One patient was found to be compound heretozygote for the novel mutation Arg702His, and the previously reported mutation Pro266Ser, which was reported in Korean population, with the late-onset phenotype. Two novel missense mutations Thr711Arg, Val723Met were found on the other patients.

CONCLUSIONSThree mutations identified in the patient were new missense mutations causing late onset GSD II, which had not been reported elsewhere before.

Child ; China ; Female ; Glucan 1,4-alpha-Glucosidase ; deficiency ; genetics ; Glycogen ; genetics ; metabolism ; Glycogen Storage Disease Type II ; enzymology ; genetics ; Humans ; Male ; Mutation ; Mutation, Missense ; Phenotype ; Young Adult