Luminal starch substrate "brake" on maltase-glucoamylase activity is located within the glucoamylase subunit

The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.

Mucosal maltase-glucoamylase plays a crucial role in starch digestion and prandial glucose homeostasis of mice

Starch is the major source of food glucose and its digestion requires small intestinal alpha-glucosidic activities provided by the 2 soluble amylases and 4 enzymes bound to the mucosal surface of enterocytes. Two of these mucosal activities are associated with sucrase-isomaltase complex, while another 2 are named maltase-glucoamylase (Mgam) in mice. Because the role of Mgam in alpha-glucogenic digestion of starch is not well understood, the Mgam gene was ablated in mice to determine its role in the digestion of diets with a high content of normal corn starch (CS) and resulting glucose homeostasis. Four days of unrestricted ingestion of CS increased intestinal alpha-glucosidic activities in wild-type (WT) mice but did not affect the activities of Mgam-null mice. The blood glucose responses to CS ingestion did not differ between null and WT mice; however, insulinemic responses elicited in WT mice by CS consumption were undetectable in null mice. Studies of the metabolic route followed by glucose derived from intestinal digestion of (13)C-labeled and amylase-predigested algal starch performed by gastric infusion showed that, in null mice, the capacity for starch digestion and its contribution to blood glucose was reduced by 40% compared with WT mice. The reduced alpha-glucogenesis of null mice was most probably compensated for by increased hepatic gluconeogenesis, maintaining prandial glucose concentration and total flux at levels comparable to those of WT mice. In conclusion, mucosal alpha-glucogenic activity of Mgam plays a crucial role in the regulation of prandial glucose homeostasis.

Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus

Aspergillus niveus produced high levels of alpha-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35 degrees C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70 degrees C and 4.5, respectively. Amylase activity was stable for 1 h at 60 degrees C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (alpha and beta), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an alpha-amylase and a glucoamylase.

Heat-moisture treatment and enzymatic digestibility of Peruvian carrot, sweet potato and ginger starches

The aim of this work was to study the effects of heat-moisture treatment (27% moisture, 100 degrees C, 16 h) and of enzymatic digestion (alpha-amylase and glucoamylase) on the properties of sweet potato (SP), Peruvian carrot (PC) and ginger (G) starches. The structural modification with heat-moisture treatment (HMT) affected crystallinity, enzyme susceptibility and viscosity profile. The changes in PC starch were the most pronounced, with a strong decrease of relative crystallinity (from 0.31 to 0.21) and a shift of X-ray pattern from B- to A-type. HMTof SP and G starch did not change the Xray pattern (A-type). The relative crystallinity of these starches changed only slightly, from 0.32 to 0.29 (SP) and from 0.33 to 0.32 (G). The extent of these structural changes (PC > SP > G) altered the susceptibility of the starches to enzymatic attack, but not in same order (PC > G > SP). HMT increased the starches digestion, probably due to rearrangement of disrupted crystallites, increasing accessible areas to attack of enzymes. The viscosity profiles and values changed significantly with HMT, resulting in higher pasting temperatures, decrease of viscosity values and no breakdown, i.e., stability at high temperatures and shear rates. Changes in pasting properties appeared to be more significant for PC and SP starch, whereas the changes for G starch were small. Setback was minimized following HMT in SP and G starches.

Stabilization of an amylase from Neurospora crassa by immobilization on highly activated supports

The amylase from Neurospora crassa is an interesting enzyme, having higher stability than amylase from Aspergillus oryzea under a broad range of pH values. Moreover, the N. crassa enzyme may be immobilized on different supports with good retention of enzyme activity. The best stabilizations were achieved using Eupergit C 250 L or glyoxyl agarose, with which the enzyme remained fully active at 60C for 24 h while the soluble enzyme remained about 17%. The glyoxyl agarose immobilized enzyme had high thermostability, high optimal temperature (65C) and broad pH/activity profile, suggesting that this enzyme has potential for food and industrial applications for starch modification.

Evidence of thermostable amylolytic activity from Rhizopus microsporus var. rhizopodiformis using wheat bran and corncob as alternative carbon source

Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions-Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.

Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus

An extracellular alpha-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-d-glucopyranoside and presented an optimum temperature and pH of 65A degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60A degrees C, presenting t (50) of 90 min at 65A degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an alpha-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

Production of fungal protein by solid substrate fermentation of cactus Cereus peruvianus and Opuntia ficus indica

Agricultural wastes from cactus Cereus peruvianus and Opuntia ficus indica were investigated for protein production by solid substrate fermentation. Firstly, the polyelectrolytes were extracted and used in water cleaning as auxiliary of flocculation and coagulation. The remaining fibrous material and peels were used as substrate for fermentation with Aspergillus niger. Glucoamylase and cellulase were the main enzymes produced. Amino acids were determined by HPLC and protein by Lowry's method. After 120 hours of fermentation the protein increased by 12.8%. Aspartic acid (1.27%), threonine (0.97%), glutamic acid (0.88%), valine (0.70%), serine (0.68%), arginine (0.82%), and phenylalanine (0.51%) were the principal amino acids produced.

Production of glucose and fructose syrups from cassava (Manihot esculenta Crantz) starch using enzymes produced by microorganisms isolated from Brazilian Cerrado soil

The high demands for sugars and the development of enzymatic technology have increased the production of sweeteners, especially for glucose and fructose syrups. This work describe a technology for glucose and fructose syrups from Brazilian cassava starch using enzymes produced by soil microrganisms isolated from the Brazilian Cerrado soil. Firstly, Aspergillus niger and Streptomyces sp. were isolated from the soil and used as glucoamylase (GA) and glucose isomerase (GI) producer sources. After characterization, GA and GI exhibited optimum pH 4.5 and 8.0, respectively. GA showed maximum activity at 60 ºC and GI at 85 ºC. GA and GI retained 65 and 80%, respectively, of initial activity after 180 minutes of incubation at 60 ºC. The kinetic parameters Km and Vmáx were 0.476 (mg.mL-1) and 8.58 (µmol/minute) for GA and 0.082 (M) and 48.20 (µmol/minute) for GI. The maximum glucose syrups production occurred after 24 hours of reaction with a 98% yield. The production of fructose syrups with 42% (w/v) was reached after 96 hours of reaction.

Sugarcane starch: quantitative determination and characterization

Starch is found in sugarcane as a storage polysaccharide. Starch concentrations vary widely depending on the country, variety, developmental stage, and growth conditions. The purpose of this study was to determine the starch content in different varieties of sugarcane, between May and November 2007, and some characteristics of sugarcane starch such as structure and granules size; gelatinization temperature; starch solution filterability; and susceptibility to glucoamylase, pullulanase, and commercial bacterial and fungal α-amylase enzymes. Susceptibility to debranching amylolytic isoamylase enzyme from Flavobacterium sp. was also tested. Sugarcane starch had spherical shape with a diameter of 1-3 µm. Sugarcane starch formed complexes with iodine, which showed greater absorption in the range of 540 to 620 nm. Sugarcane starch showed higher susceptibility to glucoamylase compared to that of waxy maize, cassava, and potato starch. Sugarcane starch also showed susceptibility to debranching amylolytic pullulanases similar to that of waxy rice starch. It also showed susceptibility to α-amylase from Bacillus subtilis, Bacillus licheniformis, and Aspergillus oryzae similar to that of the other tested starches producing glucose, maltose, maltotriose, maltotetraose, maltopentose and limit α- dextrin.

Acid activated montmorillonite: an efficient immobilization support for improving reusability, storage stability and operational stability of enzymes

Three enzymes, α-amylase, glucoamylase and invertase, were immobilized on acid activated montmorillonite K 10 via two independent techniques, adsorption and covalent binding. The immobilized enzymes were characterized by XRD, N2 adsorption measurements and 27Al MAS-NMR spectroscopy. The XRD patterns showed that all enzymes were intercalated into the clay inter-layer space. The entire protein backbone was situated at the periphery of the clay matrix. Intercalation occurred through the side chains of the amino acid residues. A decrease in surface area and pore volume upon immobilization supported this observation. The extent of intercalation was greater for the covalently bound systems. NMR data showed that tetrahedral Al species were involved during enzyme adsorption whereas octahedral Al was involved during covalent binding. The immobilized enzymes demonstrated enhanced storage stability. While the free enzymes lost all activity within a period of 10 days, the immobilized forms retained appreciable activity even after 30 days of storage. Reusability also improved upon immobilization. Here again, covalently bound enzymes exhibited better characteristics than their adsorbed counterparts. The immobilized enzymes could be successfully used continuously in the packed bed reactor for about 96 hours without much loss in activity. Immobilized glucoamylase demonstrated the best results.

A study on the Immobilizaton of Enzymes on Polymeric Supports

This work was focused to study the immobilization of enzymes on polymers. A large range of polymer matrices have been employed as supports for enzyme immobilization. Here polyaniline (PAN!) and poly(0~toluidine) (POT) were used as supports. PANI and POT provides an excellent support for enzyme immobilization by virtue of its facile synthesis, superior chemical and physical stabilities, and large retention capacity. We selected industrially important starch hydrolyzing enzymes a-amylase and glucoamylase for the study. In this work the selected enzymes were immobilized via adsorption and covalent bonding methods.To optimize the catalytic efficiency and stability of the resulting biocatalysts, the attempt was made to understand the immobilization effects on enzymatic properties. The effect of pH of the immobilization medium, time of immobilization on the immobilization efficiency was observed. The starch hydrolyzing activity of free 0:-amylase and glucoamylase were compared with immobilized forms. Immobilization on solid supports changes the microenvironment of the enzyme there by influences the pH and temperature relationship on the enzymatic activity. Hence these parameters also optimized. The reusability and storage stability of immobilized enzymes an important aspect from an application standpoint, especially in industrial applications. Taking in to consideration of this, the reusability and the long tenn storage stability of the immobilized enzyme investigated.

Catalysis by Enzymes immobilized on Tuned Mesoporous Silica

Mesoporous silica nanoparticles provide a non-invasive and biocompatible delivery platform for a broad range of applications in therapeutics, pharmaceuticals and diagnosis. Additionally, mesoporous silica materials can be synthesized together with other nanomaterials to create new nanocomposites, opening up a wide variety of potential applications. The ready functionalization of silica materials makes them ideal candidates for bioapplications and catalysis. These properties of mesoporous silica like high surface areas, large pore volumes and ordered pore networks allow them for higher loading of drugs or biomolecules. Comparative studies have been made to evaluate the different procedures; much of the research to date has involved quick exploration of new methods and supports. Requirements for different enzymes may vary, and specific conditions may be needed for a particular application of an immobilized enzyme such as a highly rigid support. In this endeavor, mesoporous silica materials having different pore size were synthesized and easily modified with active functional groups and were evaluated for the immobilization of enzymes. In this work, Aspergillus niger glucoamylase, Bovine liver catalase, Candida rugosa lipase were immobilized onto support by adsorption and covalent binding. The structural properties of pure and immobilized supports are analyzed by various characterization techniques and are used for different reactions of industrial applications.