Evaluation of extruded starch foam for glucose-supplying biomaterials.

The survival rate of mesenchymal stem cells (MSC), a crucial factor in tissue engineering, is highly dependent on glucose supply. The purpose of this paper is to study the potential of starch foams as glucose suppliers. It is investigated through in vitro hydrolysis by amyloglucosidase in conditions that respect physiological constraints (37 °C and pH 7.4), including a duration of 21 days, and no stirring. Nine extruded starch foams with amylose contents ranging from 0 to 74 %, with various cell wall thicknesses (50 to 300 µm), and different crystallinities (0-30 %) were hydrolysed. These kinetics were fitted by a model which shows that the maximum rate of hydrolysis varies from 7 to 100 %, and which allows the rate of hydrolysis at 21 days to be calculated precisely. The results reveal the major role of amylose in glucose delivery kinetics, and the secondary roles of crystallinity and cell wall thickness of the foams. Additional hydrolysis of starch films revealed that thickness positively influences the amylose chain reorganisation during hydrolysis, which, in slows down and limits glucose delivery. A simple glucose delivery kinetics analysis procedure is proposed to select samples for testing as MSC glucose suppliers.

Immobilized glucoamylase based on ZIF-8: Preparation, response surface optimization, characterization.

The glucoamylase@ZIF-8 was prepared using ZIF-8 material as the carrier in this study. The preparation process was optimized by response surface methodology, and the stability of glucoamylase@ZIF-8 was determined. The material was characterized by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The results showed that the optimum preparation process of glucoamylase@ZIF-8 was 1.65 mol 2-methylimidazole, 5.85 mL glucoamylase, 33°C stirring temperature, 90 min stirring time, and 84.0230% ± 0.6006% embedding rate. At 100°C, the free glucoamylase completely lost its activity, whereas the glucoamylase@ZIF-8 still had a retained enzyme activity of 12.0123% ± 0.86158%; at pH 3-6, the highest activity of glucoamylase@ZIF-8 was 95.9531% ± 0.96181%, and about 80% of glucoamylase activity could be retained under alkaline conditions. When the ethanol concentration was 13%, the retained enzyme activity was 7.9316% ± 0.19805%, significantly higher than free enzymes. The Km of glucoamylase@ZIF-8 and free enzyme were 1235.6825 and 80.317 mg/mL, respectively. Vmax was 0.2453 and 0.149 mg/(mL min), respectively. The appearance, crystal strength, and thermal stability of glucoamylase@ZIF-8 were improved after optimization, and they had high reusability.

Enzymatic deposition of gold nanoparticles at vertically aligned carbon nanotubes for electrochemical stripping analysis and ultrasensitive immunosensing of carcinoembryonic antigen.

Herein we combine the sandwich immunoreaction at a vertically aligned single-walled carbon nanotube (SWCNT)-based immunosensor and the enzymatically catalytic deposition of gold nanoparticles (Au NPs) by a gold nanoprobe to develop a novel electrochemical immunosensing method. The vertically arranged nanostructure was prepared through the covalent linking of terminally carboxylated SWCNTs at an aryldiazonium-modified electrode. It not only provides an excellent platform for the high density immobilization of antibodies to obtain the immunosensor but also serves as useful molecular wires to accelerate electron transfer during the electrochemical immunosensing process. Meanwhile, the enzymatic reaction of the nanoprobe prepared by surface functionalization of the nanocarrier of Au NPs by high-content glucoamylases can catalyze the deposition of a large number of Au NPs at the immunosensor. The electrochemical stripping analysis of these nanoparticles enabled the convenient signal transduction of the method. Due to the sensitive gold stripping analysis at the vertically aligned SWCNTs and the multi-enzyme signal amplification of the nanoprobe, the electrochemical signal response was greatly enhanced. Thus, the method can be used for the ultrasensitive detection of the tumor biomarker of carcinoembryonic antigen in a wide linear range of 5 orders of magnitude with a low detection limit of 0.48 pg mL-1. Considering its obvious performance superiorities, this immunosensing method exhibits an extensive prospect for practical applications.

A dandelion-like liposomes-encoded magnetic bead probe-based toehold-mediated DNA circuit for the amplification detection of MiRNA.

The development of facile and sensitive miRNA quantitative detection methods is a central challenge for the early diagnosis of miRNA-related diseases. Herein, we propose a strategy for a liposome-encoded magnetic bead-based DNA toehold-mediated DNA circuit for the simple and sensitive detection of miRNA based on a toehold-mediated circular strand displacement reaction (TCSDR) coupled with a personal glucometer (PGM ). In this strategy, a glucoamylase-encapsulated liposomes (GELs)-encoded magnetic bead (GELs-MB) probe is designed to integrate target binding, magnetic separation, and signal response. Upon sensing the target miRNA-21, a GELs-MB probe-based toehold-mediated circular strand displacement reaction (TCSDR) was initiated with the help of fuel-DNA, constructing a DNA circuit system, and realizing target recycling amplification and the disassembly of the liposomes. The disassembled liposomes were finally removed via magnetic separation, and the encapsulated glucoamylase was liberated to catalyze amylose hydrolysis with multiple turnovers to glucose for a PGM readout. Benefiting from target recycling amplification initiated by the toehold-mediated DNA circuit and the liposome multiple-label amplification, a small quantity of target miRNA-21 can be transformed into a large glucose signal. The strategy realized the quantification of miRNA-21 down to a level of 0.7 fM without enzymatic amplification or precise instrumentation. Moreover, the high-density GELs-MB probe allows the sensitive detection of miRNA-21 to be accomplished within 1.5 h. Furthermore, this strategy exhibits the advantages of specificity and simplicity, since a toehold-mediated strand displacement reaction, magnetic separation and portable PGM were used. Importantly, this strategy has been demonstrated to allow the high-confidence quantification of miRNA. Therefore, with the advantages of low cost, ease of use, portability, and sensitivity, the reported method holds great potential for the early diagnosis of miRNA-related diseases.

Enzyme-assisted development of biofunctional polyphenol-enriched buckwheat protein: physicochemical properties, in vitro digestibility, and antioxidant activity.

BACKGROUND: During the last decade buckwheat was reported to have positive health effects. The present study investigated a high-polyphenol buckwheat protein (Fagopyrum esculentum Moench) prepared by enzyme-assisted processing, together with its physicochemical properties, in vitro digestibility, and antioxidant activity. RESULTS: Buckwheat protein prepared from the synergistic enzymatic action of α-amylase and amyloglucosidase (E-BWP) had much higher polyphenol content than buckwheat protein prepared by isoelectric precipitation (I-BWP) or salt extraction (S-BWP). Rutin degraded during the process, giving quercetin. The protein constituents and amino acid composition of E-BWP were very similar to those of native buckwheat and were able to meet the WHO/FAO requirements for both children and adults. During in vitro digestion, E-BWP showed anti-digestive behavior with a nitrogen release that was lower than that of I-BWP or S-BWP. The positive effect of the polyphenol content of E-BWP resulted in a higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) content and greater reducing activity. CONCLUSION: Buckwheat protein with high polyphenol content was successfully developed by enzyme-assisted processing. It had a well-balanced amino acid profile, antidigestive behavior, and high antioxidant activities. The results suggest that enzyme-assisted processing is promising in the production of polyphenol-enriched cereal protein, contributing higher functionality with good nutritional and antioxidant properties. © 2018 Society of Chemical Industry.

Spectroscopic techniques investigation on the interaction of glucoamylase with 1-deoxynojirimycin: Mechanistic and conformational study.

1-Deoxynojirimycin (DNJ), a representative polyhydroxylated alkaloids, is widely used in the field of antidiabetic, antitumor, and anti-HIV. The present study tried to clarify the interaction mechanism of DNJ with glucoamylase by multi-spectroscopic techniques, dynamic light scattering in combination with molecular modeling strategies from biophysics point of view. Fluorescence and UV-vis data indicated that fluorescence quenching mechanism of glucoamylase and DNJ was a dynamic manner. The association constant, binding site and thermodynamic parameters were also obtained from fluorescence spectrum at different temperatures. Synchronous fluorescence, circular dichroism and dynamic light scattering methods demonstrated that their interaction induced microenvironment changes around tryptophan residue and protein conformational alteration. The main driving force was hydrophobic interaction and hydrogen bonding. In addition, molecular docking study indicated that 1-deoxynojirimycin could bind in the catalytic domain of glucoamylase and interact with amino acid residues Arg78, Asp79, Glu203 and Glu424 by forming hydrogen bonds. Molecular dynamics simulation demonstrated that profiles of atomic fluctuation remained the rigidity of ligand binding site. This study elucidated the detailed interaction mechanism of DNJ with glucoamylase, which will be helpful for pharmaceutical companies to design new α-glucosidase inhibitor drugs based on polyhydroxylated alkaloids compound like DNJ.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification for portable and accurate quantification of microRNAs.

Development of reliable and affordable quantitative methods for miRNAs with high specificity and sensitivity is a central challenge to make miRNA testing a routine part of medical care with respect to cancer. Herein, we propose a strategy for glucoamylase-encapsulated liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification (P-ERCA) for portable and accurate quantification of miRNA by using a glucometer readout.

Preparation and characterization of a green nano-support for the covalent immobilization of glucoamylase from Neurospora sitophila.

The preparation of green nano supports for the covalent immobilization of enzymes is of special interest both from the economic and environmental point of view. In this contribution, we report on the synthesis of phytochemicals coated silver nanoparticles, which were used as a novel green support for the covalent immobilization of glucoamylase isolated from Neurospora sitophila. The aqueous extract of Fagonia indica was used as a source of reducing and capping agents for the reduction of silver ions into silver nanoparticles. The prepared nanoparticles were characterized by various analytical techniques. UV-visible spectroscopy was used to detect the characteristic surface plasmon resonance bands (426, 438nm) of the silver nanoparticles. The biosynthesized silver nanoparticles were mostly spherical in shapes with an average particle size of 30-40nm (TEM and DLS measurements). X-ray diffraction and energy dispersive X-ray studies confirmed the face centered cubic crystalline form and elemental composition of the biogenic silver nanoparticles respectively. FTIR study revealed that plant polyphenolics and protein were mainly involved in the reduction and capping of silver ions. Glucoamylase from Neurospora sitophila was covalently immobilized to these nanoparticles via EDC (1-(3-(dimethylamino) propyl) 3-ethylcarbodiimidehydrochloride) coupling reaction. The immobilized enzyme exhibited higher pH and thermal stabilities as compared to the free enzyme. The kinetic constant (KM) value for the immobilized glucoamylase was higher (0.73mg/mL) than its free counterpart (0.44mg/mL), whereas the Vmax value was slightly higher for the immobilized glucoamylase. The findings of this study conclude that the newly developed green method for the synthesis of green nano-support is simple, cost effective and could be successfully used for the immobilization of various enzymes and other macromolecules.

BcGs1, a glycoprotein from Botrytis cinerea, elicits defence response and improves disease resistance in host plants.

In this study, a necrosis-inducing protein was purified from the culture filtrate of the necrotrophic fungus Botrytis cinerea BC-98 strain. Secreted proteins were collected and fractionated by liquid chromatography. The fraction with the highest necrosis-inducing activity was further purified. A glycoprotein named BcGs1 was identified by 2D electrophoresis and mass spectrometry. The BcGs1 protein consisted of 672 amino acids with a theoretical molecular weight of 70.487 kDa. Functional domain analysis indicated that BcGs1 was a glucan 1,4-alpha-glucosidase, a cell wall-degrading enzyme, with a Glyco_hydro_15 domain and a CBM20_glucoamylase domain. The BcGs1 protein caused necrotic lesions that mimicked a typical hypersensitive response and H2O2 production in tomato and tobacco leaves. BcGs1-treated plants exhibited resistance to B. cinerea, Pseudomonas syringae pv. tomato DC3000 and tobacco mosaic virus in systemic leaves. In addition, BcGs1 triggered elevation of the transcript levels of the defence-related genes PR-1a, TPK1b and Prosystemin. This is the first report of a Botrytis glucan 1,4-alpha-glucosidase triggering host plant immunity as an elicitor. These results lay a foundation for further study of the comprehensive interaction between plants and necrotrophic fungi.

Preparation and characterization of starch crosslinked with sodium trimetaphosphate and hydrolyzed by enzymes.

Crosslinked porous starch samples were produced by first crosslinking corn starch with sodium trimetaphosphate (STMP) and then partially hydrolyzing it with a mixture of α-amylase and glucoamylase. The granule morphology, porosity, swelling power, adsorption capacity, crystalline nature, molecular structure, melting and viscometric properties of these starch samples were measured and analyzed. The results showed that the porous starch which was crosslinked with 6% (w/w) STMP (ScPS-6) possessed remarkable superiority in terms of thermal and shear resistance among all the starch samples tested. The ScPS-6 also had the highest porosity and largest average pore diameter values. The swelling power of crosslinked porous starch was 56.3% lower than that of uncrosslinked porous starch. First order reaction kinetics equation was found to excellently (R(2) ≥ 0.99, average error = 6.03%) predict the experimental adsorption kinetics data of methylene blue for the crosslinked porous starch samples.

Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.

Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies.

[Influence of UV-radiation on physical-chemical and functional properties of free immobilized glucoamylase from Aspergillus awamori].

The effect of UV light (240-390 nm) at the doses of 151-1510 J/m2 on the catalytic activity of free and im- mobilized glucoamylase from Aspergillus awamori was investigated. It was established that the loss of catalytic activity of the enzyme was associated with photochemical transformations of Trp-120, which is part of the active site of glucoamylase. It was shown that the use of collagen as a carrier for immobilization of glucoamylase reduces the constant photoinactivation of the enzyme. It was suggested that the protective effect of collagen in relation to the glucoamylase subjected to UV irradiation may be related to the acceptance of reactive oxygen species by collagen and the formation of the complex carrier-enzyme which is more photostable than the native enzyme.

Iron oxide filled magnetic carbon nanotube-enzyme conjugates for recycling of amyloglucosidase: toward useful applications in biofuel production process.

Biofuels are fast advancing as a new research area to provide alternative sources of sustainable and clean energy. Recent advances in nanotechnology have sought to improve the efficiency of biofuel production, enhancing energy security. In this study, we have incorporated iron oxide nanoparticles into single-walled carbon nanotubes (SWCNTs) to produce magnetic single-walled carbon nanotubes (mSWCNTs). Our objective is to bridge both nanotechnology and biofuel production by immobilizing the enzyme, Amyloglucosidase (AMG), onto mSWCNTs using physical adsorption and covalent immobilization, with the aim of recycling the immobilized enzyme, toward useful applications in biofuel production processes. We have demonstrated that the enzyme retains a certain percentage of its catalytic efficiency (up to 40%) in starch prototype biomass hydrolysis when used repeatedly (up to ten cycles) after immobilization on mSWCNTs, since the nanotubes can be easily separated from the reaction mixture using a simple magnet. The enzyme loading, activity, and structural changes after immobilization onto mSWCNTs were also studied. In addition, we have demonstrated that the immobilized enzyme retains its activity when stored at 4 °C for at least one month. These results, combined with the unique intrinsic properties of the nanotubes, pave the way for greater efficiency in carbon nanotube-enzyme bioreactors and reduced capital costs in industrial enzyme systems.

Species-specific differences in the processing of acid α-glucosidase are due to the amino acid identity at position 201.

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R(200) and A(204) inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA(H201L) but not rhGAA(WT) to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA(H201L) but not rhGAA(WT) was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.

Structure of the catalytic domain of glucoamylase from Aspergillus niger.

Glucoamylase from Aspergillus niger is an industrially important biocatalyst that is utilized in the mass production of glucose from raw starch or soluble oligosaccharides. The G1 isoform consists of a catalytic domain and a starch-binding domain connected by a heavily glycosylated linker region. The amino-terminal catalytic domain of the G1 isoform generated by subtilisin cleavage has been crystallized at pH 8.5, which is a significantly higher pH condition than used for previously characterized glucoamylase crystals. The refined structure at 1.9 Å resolution reveals the active site of the enzyme in complex with both Tris and glycerol molecules. The ligands display both unique and analogous interactions with the substrate-binding site when compared with previous structures of homologous enzymes bound to inhibitors.

Cloning, expression, and characterization of a thermostable glucoamylase from Thermoanaerobacter tengcongensis MB4.

A thermostable glucoamylase (TtcGA) from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli. The full-length gene (2112 bp) encodes a 703-amino acid polypeptide including a predicted signal peptide of 21 residues. The recombinant mature protein was partially purified to 30-fold homogeneity by heat treatment and gel filtration chromatography. The mature protein is a monomer with the molecular weight of 77 kD. The recombinant enzyme showed maximum activity at 75 degrees C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 degrees C for 6 h. TtcGA can hydrolyze both alpha-1, 4- and alpha-1, 6-glycosidic linkages in various alpha-glucans. It showed preference for maltooligosaccharides over polysaccharides with specific activity of 80 U/mg towards maltose. Kinetic studies revealed that TtcGA had the highest activity on maltooligosaccharide with four monosaccharide units. The cations Ca2+, Mn2+, Co2+, Mg2+, and reducing agent DTT showed no obvious effects on the action of TtcGA. In contrast, the enzyme was inactivated by Zn2+, Pb2+, Cu2+, and EDTA.

Small angle X-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution.

The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.

Dynamic cross-linking effect of Mg2+ to enhance sieving properties of low-viscosity poly(vinylpyrrolidone) solutions for microchip electrophoresis of proteins.

We report a dynamic cross-linking effect of Mg2+ that enhances the sieving properties of low-viscosity poly(vinylpyrrolidone) (PVP) solutions. A low-viscosity PVP solution was applied to nondenaturing microchip electrophoresis of protein samples using microchips made of poly(methyl methacrylate). The separation resolution of nondenatured protein markers in 1.8% PVP solution was improved by adding 1-20 mM MgCl2. We studied the effect of the ratio of cross-linking agent on mobility of protein samples and showed that protein retardation (ln micro/micro0) is correlated with the ratio of cross-linking agent to PVP ([cMg2+/cPVP]) as ln micro/micro0=A'[cMg2+/cPVP]b'. A' was related to the protein radius (R), and b' was found to be 0.72 for proteins with R=2.4 nm and 0.82 for proteins with R=1.85 nm. A structural study of PVP in semidilute solutions using dynamic light scattering showed that incremental increases of Mg2+ ion concentration from 5 to 20 mM in 1.8% PVP solution increased the hydrodynamic radius of PVP polymers by 20%.

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor.

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.