Effects of glucose as carbon catabolite repressor on alpha-amylase and glucoamylase production in Indonesian indigenous fungi

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.

Characterization and structure of a novel thermostable glucoamylase from Talaromyces leycettanus JCM12802 / 生物工程学报

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.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

Background: Zymomonas mobilis, as a novel platform for bio-ethanol production, has been attracted more attention and it is very important to construct vectors for the efficient expression of foreign genes in this bacterium. Results: Three shuttle vectors ( pSUZM 1, pSUZM2 and pSUZM3 ) were first constructed with the origins of replication from the chromosome and two native plasmids (pZZM401 and pZZM402) of Z. mobilis ZM4, respectively. The three shuttle vectors were stable in Z. mobilis ZM4 and have 3,32 and 27 copies, respectively. The promoter Ppdc (a), from the pyruvate decarboxylase gene, was clonedinto the shuttle vectors, generatingthe expressionvectors pSUZM1(2, 3)a. The codon-optimized glucoamylase gene from Aspergillus awamori combined with the signal peptide sequence from the alkaline phosphatase gene of Z. mobilis was cloned into pSUZM1(2, 3)a, resulting in the plasmids pSUZM1a-GA, pSUZM2a-GA and pSUZM3a-GA, respectively. After transforming these plasmids into Z. mobilis ZM4, the host was endowed with glucoamylase activity for starch hydrolysis. Both pSUZM2a-GA and pSUZM3a-GA were more efficientatproducingglucoamylase thanpSUZM1a-GA. Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Taxonomic Characterization, Evaluation of Toxigenicity, and Saccharification Capability of Aspergillus Section Flavi Isolates from Korean Traditional Wheat-Based Fermentation Starter Nuruk

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.

Caso familiar com diagnóstico para doença de Fabry / Familiar case with diagnostics for Fabry disease

A doença de Fabry é uma enfermidade genética ligada ao cromossomoX e de caráter progressivo, causada pela deficiênciaparcial ou total da enzima alfa galactosidase A (?-Gal A). Habitualmenteo diagnóstico é tardio em função das complicaçõespatológicas provocadas pela deficiência da enzima. OBJETIVO:Neste estudo, descrevemos os aspectos clínicos de um caso familiaratravés do acompanhamento ao longo de 3 anos, duranteo tratamentopela reposição enzimática. MÉTODOS: O métodoadotado foi indutivo, relacionado ao estudo de caso familiarde pacientes com doença de Fabry. Quanto à natureza das informações,a pesquisafoi qualitativa, utilizando-se, quanto aoseu objetivo, à pesquisa exploratória. Com relação as fonte deinformação e procedimento de coleta, a pesquisa caracteriza-secomo sendo bibliográfica e documental. A amostra foi compostapor três pacientesque realizamacompanhamento quinzenalpara aplicação de terapia de reposição enzimática. O critério deinclusão para a pesquisa partiu do pressuposto de se considerarque a doença de Fabry é uma afecção rara e que a família estudadacontempla com riqueza manifestações clínicas, capazesde caracterizar a doença de Fabry. RESULTADOS: Os principaissintomas clínicos relatados pelos pacientes foram: crisede dor generalizada, fadiga, acroparestesia, febre, mialgia, dorabdominal, hipohidrose, intolerância ao frio, calor e ao exercíciofísico. Esses sintomas segundo os pacientes surgiram nainfância e foram amenizados após o uso da terapia de reposiçãoenzimática, propiciando uma melhor qualidade de vida para osmesmos. Também, se observou sinais específicos desta patologianos pacientes, como córnea verticillata e angioqueratoma. Atravésda genotipagem se verificou a semelhança da mutação entre os pacientes do estudo, demonstrando padrão típico de herançarecessiva ligada ao cromossomo X. CONCLUSÃO: Os pacientesdeste estudo apresentaram quadro clínico semelhante,sendoque a sintomatologia iniciou na infância. Córnea verticillata eangioqueratoma umbilical foram sinais encontrados nos pacientes do sexo masculino e são considerados manifestações clínicas frequentes desta patologia. A herança encontrada nesta amostra tem um padrão típico de herança recessiva ligada ao cromossomo X. Desta forma, apesar de ser uma afecção rara na população em geral, o diagnóstico precoce e a terapia de reposição enzimática permitem a evolução clínica favorável e a melhoria da qualidade de vida do paciente.(AU)

Fabry disease is a genetic disorder linked to the X chromosomeand progressive, caused by partial or total deficiency of alphagalactosidase A (?-Gal A). Usually the diagnosis is delayed dueto the pathological complications caused by deficiency of theenzyme. OBJECTIVE: In this study, we describe the clinicalaspects of a family case by monitoring for over three years,during the treatment by enzyme replacement. METHODS:The method adopted was inductive, related to the study of afamily case with patients with Fabry disease. About the nature ofthe information, the research was qualitative, using, as its goal,the exploratory research. Regarding the source of informationand collection procedure, the research is characterized asbibliographical and documentary. The sample was composed ofthree patients submitted to biweekly monitoring for applicationof enzyme replacement therapy. The inclusion criterion forthe research assumed to consider that Fabry disease is a raredisease and that the studied family contemplates with wealththe clinical manifestations, able to characterize the Fabrydisease. RESULTS: The main clinical symptoms related bypatients were: generalized pain crisis, fatigue, acroparesthesia,fever, myalgia, abdominal pain, hypohidrosis, intolerance tocold, heat and exercise. These symptoms according to patientsemerged in childhood and were alleviated after the use ofenzyme replacement therapy, providing a better quality of lifefor them. Also, we found specific signs of this disease in patients,as verticillata cornea and angiokeratoma. By genotyping, it wasfound the similarity of the mutation among patients in thestudy, showing typical pattern of recessive inheritance linkedto chromosome X. CONCLUSION: The patients in this study showed similar clinical condition, and the symptoms began inchildhood. Verticillata cornea and umbilical angiokeratomasigns were found in male patients and are considered commonclinical manifestations of this pathology. The heritage found inthis sample has a typical pattern of recessive inheritance linkedto chromosome X. Thus, despite being a rare disease in generalpopulation, early diagnosis and enzyme replacement therapyallow favorable clinical evolution and improved patient qualityof life.(AU)

Construction and application of black-box model for glucoamylase production by Aspergillus niger / 生物工程学报

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.

Isolation and characterization of different strains of bacillus licheniformis for the production of commercially significant enzymes

Utilization of highly specific enzymes for various industrial processes and applications has gained huge momentum in the field of white biotechnology. Selection of a strain by efficient plate-screening method for a specific purpose has also favored and boosted the isolation of several industrially feasible microorganisms and screening of a large number of microorganisms is an important step in selecting a potent culture for multipurpose usage. Five new bacterial isolates of Bacillus licheniformis were discovered from indigenous sources and characterized on the basis of phylogeny using 16S rDNA gene analysis. Studies on morphological and physiological characteristics showed that these isolates can easily be cultivated at different temperatures ranging from 30°C to 55°C with a wide pH values from 3.0 to 11.0 All these 05 isolates are salt tolerant and can grow even in the presences of high salt concentration ranging from 7.0 to 12.0%. All these predominant isolates of B. licheniformis strains showed significant capability of producing some of the major industrially important extracellular hydrolytic enzymes including alpha-amylase, glucoamylase, protease, pectinase and cellulase in varying titers. All these isolates hold great potential as commercial strains when provided with optimum fermentation conditions

Clinical sequelae of 17 cases with glycogen storage disease type II/Pompe disease / 中华儿科杂志

<p><b>OBJECTIVE</b>To analyze and summarize the characteristics of glycogen storage disease type II (Pompe disease) patients according to the clinical description and prognosis.</p><p><b>METHOD</b>Seventeen 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.</p><p><b>RESULT</b>Four 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%.</p><p><b>CONCLUSION</b>Glycogen 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.</p>

Establishment and clinical application of dried blood spots and mixed leukocytes for determination of acid alpha-glucosidase activity / 中华儿科杂志

<p><b>OBJECTIVE</b>Glycogen 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.</p><p><b>METHOD</b>The 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.</p><p><b>RESULT</b>The 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.</p><p><b>CONCLUSION</b>The 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.</p>

Production of ethanol and isoflavones from steam-pretreated Radix Puerariae by solid state fermentation / 生物工程学报

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.

Cloning and expression of a new glucoamylase gene / 生物工程学报

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.

Analysis of Erroneous Overestimation of Blood Glucose in Patients on Continuous Ambulatory Peritoneal Dialysis with Icodextrin / 대한신장학회잡지

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.

Clinical and molecular genetic study on two patients of the juvenile form of Pompe disease in China / 中华儿科杂志

<p><b>OBJECTIVE</b>Glycogen-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.</p><p><b>METHODS</b>Two 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.</p><p><b>RESULTS</b>One 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.</p><p><b>CONCLUSIONS</b>Three mutations identified in the patient were new missense mutations causing late onset GSD II, which had not been reported elsewhere before.</p>

Overproduction of glucoamylase by recombinant Aspergillus niger harboring multiple copies of glaA / 生物工程学报

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.

Estudio de cepas nativas amilolíticas / Study of amilolytic native microbial stocks

En el presente trabajo se realiza el estudio de cepas microbianas aisladas de diferentes nichos ecológicos, productoras de las enzimas amilolíticas, así: procesado de maíz, de papa y de yuca. Los criterios utilizados para el aislamiento de las cepas amilolíticas, son el crecimiento sobre los medios modificados con almidón y la positividad frente al Test de Lugol. Los medios empleados fueron CZAPEK, PCA y MRS modificados. Las cepas obtenidas son debidamente purificadas, crío conservadas e identificadas bioquímicamente, obteniendo un total de 103 cepas nativas. De éste total de cepas, se seleccionan debido a la similitud fisiológica entre microorganismos aislados, 9 cepas. Estas últimas son identificadas bioquímicamente y se obtienen los géneros Bacillus sp ,Clostridium sp y Kurthia sp. Las enzimas amilolíticas obtenidas a partir de las cepas nativas presentan pesos moleculares que oscilan entre 30 y 150 kDa aproximadamente

Effects of malnutrition on the digestive enzymes of the large bowel of young rhesus monkeys.

The aim of the present study was to investigate the effect of mild-to-moderate protein-energy malnutrition (PEM) and rehabilitation on the digestive enzymes of the large bowel in young rhesus monkeys. The presence of these enzymes has already been reported in the large bowel by many authors. The activities of the digestive enzymes, i.e. lactase, sucrase, maltase, trehalase, glucoamylase, leucine aminopeptidase, alkaline phosphatase and gamma-glutamyl transpeptidase, from different parts of the large bowel were determined in 6 controls, 6 PEM and 6 rehabilitated young rhesus monkeys. These monkeys had been used to study the effect of malnutrition on the small intestine and the results have already been published. There was a significant decrease in the sucrase in the ascending colon (p < 0.05); maltase in all the parts of the large bowel (p < 0.05); and glucoamylase activities (p < 0.05) in the caecum segment of the large bowel in the PEM group. The activity of other enzymes, i.e. lactase, trehalase, alkaline phosphatase, gamma-glutamyl transpeptidase and leucine aminopeptidase, was unaffected in the PEM group. The changes in the enzyme activities recovered on rehabilitation of 21 weeks. The result of this study suggest that even mild-to-moderate malnutrition affects the enzyme activity of the large bowel, which recovers on rehabilitation.

Fermentation of starch to ethanol by an amylolytic yeast Saccharomyces diastaticus SM-10.

A total of fifteen yeast strains were isolated from natural sources including fruits, soil, molasses, honey and a variety of indigeneous fermented foods. Screening of these strains for growth, ethanol production and glucoamylase activity led to selection of a yeast strain SM-10 identified as S. diastaticus having maximum glucoamylase activity (80 units ml(-1)) and ethanol production from starch (3.5%). Ethanol production from wheat flour was found to be 1.75% which could be increased to 5.2% after treatment of wheat flour with pepsin, diastase and glucoamylase.

Serum enzyme pattern and local enzyme gradients in chronic chagasic patients

Histochemical studies of myocardial biopsies from chronic chagasic patients at different evolutive stages showed a pattern primarily characterized by a marked increment in tissue enzymes such as mono-amine oxidase and lysosomal acid phosphatase. This cellular damage can be reflected by changes in certain serum enzymes associated with myocardial metabolism, specially in the coronary sinus, where the blood metabolized by the heart is drained. However, little is known about the possible changes in blood enzyme activity during chronic Chagas disease. In this investigation, the activity of the following enzymes glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), alkaline phosphatase (ALP), acid maltase (AM), lactate dehydrogenase (LDH), alpha-hydroxybutyric dehydrogenase (alpha-HBDH or LDH1) and creatine phosphokinase (CPK) was measured in blood serum of the superior cava vein (SCV), coronary sinus (CS) and pulmonary (PA) and femoral (FA) arteries of 45 chronic chagasic patients, ages between 20 and 55 yr, at different evolutive stages (groups IA, IB, II and III). The results demonstrate that the average activity of the enzymes studied in chagasic patients, except LDH and CPK, are significantly altered (p < 0.05) in the majority of the arterial and venous blood samples. The finding of released GOT, GPT, ALP, acid maltase and alpha-HBDH in groups IA and IB is an indication of early myocardial damage in chronic chagasic patients without clinical evidence of cardiac disease. In conclusion, it is suggested that the possible evolutive pattern for myocardial damage could be established by the increment in coronary sinus blood of the enzymes GOT, acid maltase and alpha-HBDH

Construction of recipient strain of expression-secretion system in filamentous fungi / 生物工程学报

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.

Cardioprotective Effect of Ischemic Preconditioning: Relationship between Intracellular Glycogen and Protein Kinase C

BACKGROUND: Recent studies suggest that the cardioprotective effect of ischemic preconditioning (IPC) is related to intracellular glycogen content in rat hearts, however, controversies still remain. METHODS: To test this hypothesis, isolated Langendorff-perfused rabbit hearts were subjected to 45 min global ischemia followed by 120 min reperfusion with IPC (n=0) or without IPC (ischemic control, n=). IPC was induced by one cycle of 5 min global ischemia and 10 min reperfusion. In the glucose (G)-free preconditioned group (n=0), G depletion-repletion was induced by perfusion with G-free Tyrode solution for 5 min and then G-containing Tyrode solution for 10 min followed by 45 min ischemia and 120 min reperfusion. For glycogen depletion or loading, hearts were treated with sodium acetate (NA, 5 mM, n=) or insulin (Ins, 1 unit/L, n=) for 15 min before 45 min ischemia. Left ventricular function and coronary flow (CF) were continuously recorded during experiments. Myocardial cytosolic and membrane protein kinase C (PKC) activities were measured by 32P-gamma-ATP incorporation into PKC-specific pepetide; glycogen content in the cardiac myocytes was determined by spectrophotometry with amyloglucosidase; expression of PKC isozymes was determined by Western blot with monoclonal antibodies. Infarct size was determined by staining with tetrazolium salt and planimetry. Data were analyzed by ANOVA and Tukey's post-hoc test. RESULTS: IPC or G-free preconditioning enhanced LV functional recovery; NA did not influence on functional recovery but Ins depressed it. Infarct size was significantly reduced by IPC, G-free preconditioning, and NA treatment (35.3+/-2.1% in the ischemic control, 18.7+/-1.2% in the IPC, 22.1+/-1.2% in the G-free preconditioned, 16.3+/-1.2% in the NA-treated group, and 32.8+/-1.6% in the Ins-treated group, p<0.05). Membrane PKC activities significantly increased by IPC, IPC and 45 min ischemia, G-free preconditioning, and G-free preconditioning and 45 min ischemia; especially, expression of membrane PKC-epsilon increased by IPC and G-free preconditioning. Glycogen content decreased by 45 min ischemia, IPC, G-free preconditioning, and by NA treatment, but increased by Ins treatment. CONCLUSION: These results suggest that in rabbit heart, intracellular glycogen may not significantly be related with the cardioprotective effect of IPC; G-free preconditioning could not improve post-ischemic contractile dysfunction but it has an infarct size-limiting effect; this cardioprotective effect may be related in part to activation of PKC, especially epsilon isozyme.