The glucoamylase from Aspergillus wentii: Purification and characterization.

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.

Application and Analysis of Rhizopus oryzae Mycelia Extending Characteristic in Solid-state Fermentation for Producing Glucoamylase.

Enhanced application of solid-state fermentation (SSF) in industrial production and the influence of SSF of Rhizopus K1 on glucoamylase productivity were analyzed using the flat band method. A growth model was implemented through SSF of Rhizopus K1 in this experiment, and spectrophotometric method was used to determine glucoamylase activity. Results showed that in bran and potato culture medium with 70% moisture in a loose state, µ of mycelium reached to 0.15 h-1 after 45 h of culture in a thermostatic water bath incubator at 30°C. Under a low-magnification microscope, mycelial cells appeared uniform, bulky with numerous branches, and were not easily ruptured. The generated glucoamylase activity reached to 55 U/g (dry basis). This study has good utilization value for glucoamylase production by Rhizopus in SSF.

Amylolytic fungi in starter cakes for rice beer production.

Two types of starter cakes, viz. amou and perok-kushi, used in the production of rice beer in Assam, India, by the Bodo and Deori communities, respectively, were used for the isolation of amylolytic fungi. Based on the sequencing of their internal transcribed spacer (ITS) regions the fungi were identified as Amylomyces rouxii and Rhizopus oryzae, and given the strain names TU460 and TU465, respectively. Both the strains showed the ability to degrade and saccharify starch. The glucoamylase activity was considerably high in A. rouxii TU460 (14.92 µmol/min) as compared to R. oryzae TU465 (1.41 µmol/min), whereas α-amylase activity was found to be closely related, i.e. 7.02 and 6.09 unit mL-1, respectively. SDS PAGE for the determination of the molecular size of the glucoamylase enzymes revealed the production of two distinct units of 59 kDa and 31 kDa by A. rouxii TU460, and one unit of 72 kDa by R. oryzae TU465. LC MS/MS analysis revealed that no mycotoxins were produced by either of the strains. The overall study indicated a good amylolytic property of both strains and a potential for application in the starch processing industries.

Development of a method for efficient cost-effective screening of Aspergillus niger mutants having increased production of glucoamylase.

OBJECTIVES: To develop an efficient cost-effective screening process to improve production of glucoamylase in Aspergillus niger. RESULTS: The cultivation of A. niger was achieved with well-dispersed morphology in 48-deep-well microtiter plates, which increased the throughput of the samples compared to traditional flask cultivation. There was a close negative correlation between glucoamylase and its pH of the fermentation broth. A novel high-throughput analysis method using Methyl Orange was developed. When compared to the conventional analysis method using 4-nitrophenyl α-D-glucopyranoside as substrate, a correlation coefficient of 0.96 by statistical analysis was obtained. CONCLUSION: Using this novel screening method, we acquired a strain with an activity of 2.2 × 103 U ml-1, a 70% higher yield of glucoamylase than its parent strain.

Inaccuracy of AOAC method 2009.01 with amyloglucosidase for measuring non-digestible oligosaccharides and proposal for an improvement of the method.

We wished to clarify the inaccuracy of AOAC method 2009.01 for the measurement of non-digestible oligosaccharides and to propose an improved method using porcine intestinal enzymes. Amyloglucosidase used in AOAC method 2009.01 scarcely hydrolyses sucrose, palatinose and panose (which are readily digested by intestinal enzymes). Hence, oligosaccharides could not be measured accurately by AOAC method 2009.01. To confirm the inaccuracy of the method, we used porcine intestinal enzymes instead of amyloglucosidase. Using the improved method, fructooligosaccharide and galactooligosaccharide were measured accurately as non-digestible oligosaccharides, but sucrose, palatinose, panose and isomaltooligosaccharide were not. The improved method hydrolysed digestible oligosaccharides into monosaccharides. These results demonstrate that the inaccuracy of AOAC method 2009.01 for oligosaccharide measurement is due to incomplete hydrolysis by amyloglucosidase. We propose that amyloglucosidase should be replaced with porcine intestinal enzymes for such measurements.

A novel diazoresin/poly(N-vinyl aminobutyric acid) covalent capillary coating for the analysis of proteins by capillary electrophoresis.

A novel method for the preparation of covalently linked capillary coatings of poly(N-vinyl aminobutyric acid) (PVAA) obtained from hydrolyzed polyvinylpyrrolidone was demonstrated using photosensitive diazoresin (DR) as a coupling agent. A layer-by-layer self-assembled film of DR and PVAA based on ionic bonding was first fabricated on the inner wall of capillary, then ionic bonding was converted into covalent bonding after treatment with UV light through a unique photochemical reaction of DR. The covalently bonded coatings suppressed protein adsorption on the inner surface of the capillary, and thus a baseline separation of lysozyme, cytochrome c, BSA, amyloglucosidase, and myoglobin was achieved using CE. Compared with bare capillary or noncovalently bonded DR/PVAA coatings, the covalently linked DR/PVAA capillary coatings not only improved the CE separation performance for proteins, but also exhibited good stability and repeatability. Due to the replacement of the highly toxic and moisture-sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide a green and easy way to make covalently coated capillaries for CE.

[Determination of exogenous gamma-amylase residue in honey].

A novel method for the determination of exogenous gamma-amylase residue in honey using liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) was established. After pre-separation by gel column chromatography, the gamma-amylase in honey samples was separated from the sugars. The gamma-amylase was then used to catalyze maltose into glucose. This enzymatic reaction was under the conditions of 55 degrees C and 0.03 mol/L phosphate buffer solution (pH 4.5) for 48 h. The maltose and glucose in the above enzymatic reaction solution were separated using liquid chromatography. By measuring the content of glucose with isotope ratio mass spectrometry, the gamma-amylase in honey can be determined. The linear range of gamma-amylase was 5 - 200 U/kg with the quantification limit of 5 U/kg. The recoveries were between 89.6% and 108.2% with the relative standard deviations from 3.3% to 4.9%. This method was used to analyze 38 honey and rice syrup samples, and the detection rate of gamma-amylase was 76.3%. To further verify the detection capability of this method, an authentic honey was adulterated with 15% (mass fraction) rice syrup. The gamma-amylase content in this sample was 10.2 U/kg. This method can effectively identify honey adulteration with rice syrups from the perspective of enzymology.

Fast and reliable method for simultaneous zymographic detection of glucoamylase and α-amylase in fungal fermentation.

Detection of α-amylase and glucoamylase in crude fermentation extracts using a single native electrophoresis gel and zymogram is described in this article. Proteins were printed on substrate gel and simultaneously onto a membrane in a three-sandwich gel. α-Amylase was detected on the substrate gel with copolymerized ß-limit dextrins and iodine reagent. Glucoamylases were detected on the membrane using a coupled assay for glucose detection. Both amylases were detected in native gel using starch and iodine reagent. The described technique can be a helpful tool for monitoring and control of fermentation processes because fungal amylase producers almost always synthesize both amylases.

Improved enzyme production by bio-pellets of Aspergillus niger: targeted morphology engineering using titanate microparticles.

The present study describes the design of bio-pellet morphologies of the industrial working horse Aspergillus niger strains in submerged culture. The novel approach recruits the intended addition of titanate microparticles (TiSiO(4), 8 µm) to the growth medium. As tested for two recombinant strains producing fructofuranosidase and glucoamylase, the enzyme titer by the titanate-enhanced cultures in shake flasks was increased 3.7-fold to 150 U/mL (for fructofuranosidase) and 9.5-fold to 190 U/mL (for glucoamylase) as compared to the control. This could be successfully utilized for improved enzyme production in stirred tank reactors. Stimulated by the particles, the achieved final glucoamylase activity of 1,080 U/mL (fed-batch) and 320 U/mL (batch) was sevenfold higher as compared to the conventional processes. The major reason for the enhanced production was the close association between the titanate particles and the fungal cells. Already below 2.5 g/L the micromaterial was found inside the pellets, including single particles embedded as 50-150 µm particle aggregates in the center resulting in core shell pellets. With increasing titanate levels the pellet size decreased from 1,700 µm (control) to 300 µm. Fluorescence based resolution of GFP expression revealed that the large pellets of the control were only active in a 200 µm surface layer. This matches with the critical penetration depth for nutrients and oxygen typically observed for fungal pellets. The biomass within the titanate derived fungal pellets, however, was completely active. This was due a reduced thickness of the biomass layer via smaller pellets as well as the core shell structure. Moreover, also the created loose inner pellet structure enabled a higher mass transfer and penetration depths for up to 500 µm. The creation of core-shell pellets has not been achieved previously by the addition of microparticles, for example, made of talc or alumina. Due to this, the present work opens further possibilities to use microparticles for tailor-made morphology design of filamentous fungi, especially for pellet based processes which have a long and strong industrial relevance for industrial production.

Enzyme analysis for Pompe disease in leukocytes; superior results with natural substrate compared with artificial substrates.

Enzyme analysis for Pompe disease in leukocytes has been greatly improved by the introduction of acarbose, a powerful inhibitor of interfering alpha-glucosidases, which are present in granulocytes but not in lymphocytes. Here we show that the application of acarbose in the enzymatic assay employing the artificial substrate 4-methylumbelliferyl-alpha-D: -glucoside (MU-alphaGlc) is insufficient to clearly distinguish patients from healthy individuals in all cases. Also, the ratios of the activities without/with acarbose only marginally discriminated Pompe patients and healthy individuals. By contrast, when the natural substrate glycogen is used, the activity in leukocytes from patients (n = 82) with Pompe disease is at most 17% of the lowest control value. The use of artificial substrate in an assay with isolated lymphocytes instead of total leukocytes is a poor alternative as blood samples older than one day invariably yield lymphocyte preparations that are contaminated with granulocytes. To diagnose Pompe disease in leukocytes we recommend the use of glycogen as substrate in the presence of acarbose. This assay unequivocally excludes Pompe disease. To also exclude pseudo-deficiency of acid alpha-glucosidase caused by the sequence change c.271G>A (p.D91N or GAA2; homozygosity in approximately 1:1000 caucasians), a second assay employing MU-alphaGlc substrate plus acarbose or DNA analysis is required.

Glycogen catabolism enzymes and protein fractions in the third and fourth larval stages of Anisakis simplex.

Extracts of Anisakis simplex third (L3) and fourth (L4) larval stages were assayed for protein content and activity and properties of alpha-amylase, glucoamylase and glycogen phosphorylase. Protein content in L4 was twice that in L3. SDS-PAGE applied to both larval stages revealed 22 protein fractions in each, including five stage-specific fractions in each larval stage. The L3 extracts contained three amylase isoenzymes: alpha 1, alpha 2 and alpha 3; their molecular weights were 64, 29 and 21 kDa, respectively. Only one amylase isoenzyme (64 kDa) was found in the L4 extracts. Glycogen in L3 was found to be broken down mostly by hydrolysis because of low glycogen phosphorylase activity. The alpha-amylase activity in L4 was higher than that in L3 by half and the glycogen phosphorylase activity was ten times higher. In addition, the same enzymes isolated from L3 and L4 were found to differ in their properties. These differences could be manifestations of metabolic adaptations of A. simplex larvae to host switch from fish (L3) to mammals (L4), i.e. adaptations to a new habitat.

Cloning of a gene encoding thermostable glucoamylase from Chaetomium thermophilum and its expression in Pichia pastoris.

AIMS: Chaetomium thermophilum is a soil-borne thermophilic fungus whose molecular biology is poorly understood. Only a few genes have been cloned from the Chaetomium genus. This study attempted to clone, to sequence and to express a thermostable glucoamylase gene of C. thermophilum. METHODS AND RESULTS: First strand cDNA was prepared from total RNA isolated from C. thermophilum and the glucoamylase gene amplified by using PCR. Degenerate primers based on the N-terminal sequences of the purified glucoamylase according to our previous works and a cDNA fragment encoding the glucoamylase gene was obtained through RT-PCR. Using RACE-PCR, full-length cDNA of glucoamylase gene was cloned from C. thermophilum. The full-length cDNA of the glucoamylase was 2016 bp and contained a 1797-bp open reading frame encoding a protein glucoamylase precursor of 599 amino acid residues. The amino-acid sequence from 31 to 45 corresponded to the N-terminal sequence of the purified protein. The first 30 amino acids were presumed to be a signal peptide. The alignment results of the putative amino acid sequence showed the catalytic domain of the glucoamylase was high homology with the catalytic domains of the other glucoamylases. The C. thermophilum glucoamylase gene was expressed in Pichia pastoris, and the glucoamylase was secreted into the culture medium by the yeast in a functionally active form. The recombinant glucoamylase purified was a glycoprotein with a size of about 66 kDa, and exhibited optimum catalytic activity at pH 4.5-5.0 and 65 degrees C. The enzyme was stable at 60 degrees C, the enzyme activity kept 80% after 60 min incubation at 70 degrees C. The half-life was 40 and 10 min under incubation at 80 and 90 degrees C respectively. CONCLUSIONS: A new thermostable glucoamylase gene of C. thermophilum was cloned, sequenced, overexpressed successfully in P. pastoris. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its thermostability and overexpression, this glucoamylase enzyme offers an interesting potential in saccharification steps in both starch enzymatic conversion and in alcohol production.

Purification and characterization of heterogeneous glucoamylases from Monascus purpureus.

Two forms of an extracellular glucoamylase, MpuGA-I and MpuGA-II, were purified to homogeneity from Monascus purpureus RY3410. The molecular weights of these enzymes were estimated to be 60,000 (MpuGA-I) and 89,000 (MpuGA-II). These enzymes were glycoproteins with a carbohydrate content of 15.0% (MpuGA-I) and 16.2% (MpuGA-II) respectively. The pH optima were 5.0 for both enzymes, and the optimal temperatures were 50 degrees C (MpuGA-I) and 65 degrees C (MpuGA-II). The Km values for soluble starch were calculated to be 4.0+/-0.8 mg/ml (MpuGA-I) and 1.1+/-0.2 mg/ml (MpuGA-II) respectively.

Dynamic coating using methylcellulose and polysorbate 20 for nondenaturing electrophoresis of proteins on plastic microchips.

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%).

Effect of bacterial monoassociation on brush-border enzyme activities in ex-germ-free piglets: comparison of commensal and pathogenic Escherichia coli strains.

This study was designed to investigate the effect of monoassociation of germ-free piglets with Escherichia coli strains on the development of intestinal brush-border enzyme activities. Piglets were delivered by hysterectomy, reared for seven days under germ-free conditions and fed milk formula diet. One group was maintained germ-free, the other four groups were monoassociated on day eight with one of four E. coli strains: non-pathogenic O86 or O83 and G58-1, or pathogenic 933D. The development of brush-border digestive enzyme functions in the small intestine was evaluated after 15 days. Germ-free controls exhibited slower developmental declines of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and delayed increases of sucrase and glucoamylase compared to conventionally grown animals. Association of germ-free piglets with the non-pathogenic E. coli strains O86 and O83 resulted in increased enterocyte differentiation along the length of the small intestine, accompanied by declining activities of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and elevated activities of maturational markers such as sucrase and glucoamylase. Maturational changes also occurred along the villus-crypt axis, as revealed by histochemical localization of aminopeptidase N on the villi tips in piglets colonized with E. coli O83. Interestingly, colonization with the pathogenic E. coli strain 933D stimulated changes in the main differentiation enzyme markers lactase, sucrase and glucoamylase to an extent comparable with those produced by the non-pathogenic and probiotic E. coli strains. In conclusion, germ-free piglets represent a valuable tool to study the consequences of colonization of the immature sterile gut with defined strains of bacteria.

Branching mutants of Aspergillus oryzae with improved amylase and protease production on solid substrates.

To study the relation between the number of hyphal tips and protein secretion during growth on a solid substrate, we have constructed two mutant strains of Aspergillus oryzae with increased hyphal branching. We have analysed hydrolytic enzyme activities during growth on wheat kernels (WK) of A. oryzae strains carrying the disrupted allele of the pclA gene encoding a secretion pathway specific (KEX2-like) endo-protease and the disrupted allele of the pg/pi-tp gene encoding a phosphatidylglycerol/phosphatidylinositol transfer protein. The biomass levels produced by the pclA and pg/pi-tp disrupted strains on wheat-based solid media were similar as found for the wild-type strain. However, the pclA disrupted strain showed much more compact colony morphology than the other two strains. Sporulation of the pclA and pg/pi-tp disrupted strains occurred, respectively, 2 days and 1 day later, compared to the wild type during fermentation on ground WK. During surface growth, microscopic analysis revealed that the hyphal growth unit length (L (hgu)) of the pclA and pg/pi-tp disrupted strains was, on average, 50 and 74% of that of the wild-type strain. This implies that in both mutant strains, a higher branching frequency occurs than in the wild-type strain. Compared to the wild-type strain, the pclA and pg/pi-tp disrupted strains produced at least 50% more amylase, at least 100% more glucoamylase and at least 90% more protease activity levels after growth on WK. These results support the hypothesis that branching mutants with an increased branching frequency can improve the solid state fermentation process.

[Effect of maternal toxic hepatitis on the functional characteristics of the lactation process]. / Vliianie toksicheskogo gepatita u materi na funktsional'nye osobennosti protsessa laktatsii.

The authors have studied the influence of chronic heliotrine intoxication on female rats subjected to it before their pregnancy and on the quantity of proteins, carbohydrates, activity of enzymes and immunocompetent cells (ICC) of rats milk during the period of breast feeding. Decrease in an amount of proteins and carbohydrates since the third day of lactation, lowering in dipeptidhydrolase, r-amilase and maltase activity were observed in the study. It has been also seen the decrease in an amount of ICC (monocytes, macrophages, small lymphocytes) just after parturitions. This deacrease was mostly expressed on the 14th day of lactation. IC cells were not determined in milk on the 21st day of breast feeding.

Analysis of secreted proteins during conidial germination of Aspergillus oryzae RIB40.

To broaden our understanding of extracellular proteins of Aspergillus oryzae at the conidial germination stage, analyses of the secreted proteins during germination were carried out. Taka-amylase A (TAA), glucoamylase (GLAA), and aspergillopepsin A (PEPA) were identified as the main products by peptide mass fingerprinting. TAA and PEPA were detected simultaneously with the formation of germ tubes. With the development of germination, the pH of the medium fell from 5.5 to 3.5. The secreted PEPA had a pro-sequence and likely shifted from 42 kDa to 41 kDa below pH 4.6, indicating that the precursor of PEPA was secreted and underwent pH-dependent processing. Furthermore, the 41 kDa protein was trapped by the addition of pepstatin A, the specific inhibitor of PEPA, suggesting that the maturation of pro-PEPA was a stepwise autoprocessing upon acidification of the medium and itself was an intermediate of the processing. It was implied that PEPA plays an important role at the early germination stage.

Rhizopus microsporus var. rhizopodiformis: a thermotolerant funguswith potential for production of thermostable amylases

The effect of several nutritional and environmental parameters on growth and amylase production from Rhizopus microsporus var. rhizopodiformis was analysed. This fungus was isolated from soil of the Brazilian «cerrado» and produced high levels of amylolytic activity at 45 degrees C in liquid medium supplemented with starch, sugar cane bagasse, oat meal or cassava flour. Glucose in the culture medium drastically repressed the amylolytic activity. The products of hydrolysis were analysed by thin layer chromatography, and glucose was detected as the main component. The amylolytic activity hydrolysed several substrates, such as amylopectin, amylase, glycogen, pullulan, starch, and maltose. Glucose was always the main end product detected by high-pressure liquid chromatography analysis. These results indicated that the amylolytic activity studied is a glucoamylase, but there were also low levels of alpha-amylase. As compared to other fungi, R. microsporus var. rhizopodiformis can be considered an efficient producer of thermostable amylases, using raw residues of low cost as substrates. This information is of technological value, considering the importance of amylases for industrial hydrolysis (AU)

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