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Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. Key points: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch. [ABSTRACT FROM AUTHOR]

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR–Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. Key points: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated. [ABSTRACT FROM AUTHOR]

This article focuses on engineering Corynebacterium glutamicum to produce L-lysine efficiently from starch using combined method of "classical breeding" and "genome breeding." Firstly, a thermo-tolerable L-lysine-producing C. glutamicum strain KT45-6 was obtained after multi-round of acclimatization at high temperature. Then, amylolytic enzymes were introduced into strain KT45-6, and the resultant strains could use starch for cell growth and L-lysine production except the strain with expression of isoamylase. In addition, co-expression of amylolytic enzymes showed a good performance in starch degradation, cell growth and L-lysine production, especially co-expression of α-amylase (AA) and glucoamylase (GA). Moreover, L-lysine yield was increased by introducing AA-GA fusion protein (i.e., strain KT45-6S-5), and finally reached to 23.9 ± 2.3 g/L in CgXIIIPM-medium. It is the first report of an engineered L-lysine-producing strain with maximum starch utilization that may be used as workhorse for producing amino acid using starch as the main feedstock. Key points: • Thermo-tolerable C. glutamicum was obtained by temperature-induced adaptive evolution. • The fusion order between AA and GA affects the utilization efficiency of starch. • C. glutamicum with starch utilization was constructed by optimizing amylases expression. [ABSTRACT FROM AUTHOR]

Thermoplasma trehalase Tvn1315 is predicted to be composed of a β-sandwich domain (BD) and a catalytic domain (CD) based on the structure of the bacterial GH15 family glucoamylase (GA). Tvn1315 as well as Tvn1315 (Δ5), in which the 5 N-terminal amino acids are deleted, could be expressed in Escherichia coli as active enzymes, but deletion of 10 residues (Δ10) led to inclusion body formation. To further investigate the role of the N-terminal region of BD, we constructed five mutants of Δ5, in which each of the 5th to 10th residues of the N-terminus of Tvn1315 was mutated to Ala. Every mutant protein could be recovered in soluble form, but only a small fraction of the Y9A mutant was recovered in the soluble fraction. The Y9A mutant recovered in soluble form had similar specific activity to the other proteins. Subsequent mutation analysis at the 9th position of Tvn1315 in Δ5 revealed that aromatic as well as bulky hydrophobic residues could function properly, but residues with hydroxy groups impaired the solubility. Similar results were obtained with mutants based on untruncated Tvn1315. When the predicted BD, Δ5BD, Δ10BD, and BD mutants were expressed, the Δ10BD protein formed inclusion bodies, and the BD mutants behaved similarly to the Δ5 and full-length enzyme mutants. These results suggest that the hydrophobic region is involved in the solubilization of BD during the folding process. Taken together, these results indicate that the solubility of CD depends on BD folding. Key points: • N-terminal hydrophobic region of the BD is involved in the protein folding. • The N-terminal hydrophobic region of the BD is also involved in the BD folding. • BD is able to weakly interact with the insoluble β-glucan. [ABSTRACT FROM AUTHOR]

Diastatic strains of Saccharomyces cerevisiae possess the unique ability to hydrolyze and ferment long-chain oligosaccharides like dextrin and starch. They have long been regarded as important spoilage microbes in beer, but recent studies have inspired a re-evaluation of the significance of the group. Rather than being merely wild-yeast contaminants, they are highly specialized, domesticated yeasts belonging to a major brewing yeast lineage. In fact, many diastatic strains have unknowingly been used as production strains for decades. These yeasts are used in the production of traditional beer styles, like saison, but also show potential for creation of new beers with novel chemical and physical properties. Herein, we review results of the most recent studies and provide a detailed account of the structure, regulation, and functional role of the glucoamylase-encoding STA1 gene in relation to brewing and other fermentation industries. The state of the art in detecting diastatic yeast in the brewery is also summarized. In summary, these latest results highlight that having diastatic S. cerevisiae in your brewery is not necessarily a bad thing. Key Points: •Diastatic S. cerevisiae strains are important spoilage microbes in brewery fermentations. •These strains belong to the 'Beer 2' or 'Mosaic beer' brewing yeast lineage. •Diastatic strains have unknowingly been used as production strains in breweries. •The STA1-encoded glucoamylase enables efficient maltotriose use. [ABSTRACT FROM AUTHOR]

Diastatic strains of Saccharomyces cerevisiae are common contaminants in beer fermentations and are capable of producing an extracellular STA1-encoded glucoamylase. Recent studies have revealed variable diastatic ability in strains tested positive for STA1, and here, we elucidate genetic determinants behind this variation. We show that poorly diastatic strains have a 1162-bp deletion in the promoter of STA1. With CRISPR/Cas9-aided reverse engineering, we show that this deletion greatly decreases the ability to grow in beer and consume dextrin, and the expression of STA1. New PCR primers were designed for differentiation of highly and poorly diastatic strains based on the presence of the deletion in the STA1 promoter. In addition, using publically available whole genome sequence data, we show that the STA1 gene is prevalent among the 'Beer 2'/'Mosaic Beer' brewing strains. These strains utilize maltotriose efficiently, but the mechanisms for this have been unknown. By deleting STA1 from a number of highly diastatic strains, we show here that extracellular hydrolysis of maltotriose through STA1 appears to be the dominant mechanism enabling maltotriose use during wort fermentation in STA1+ strains. The formation and retention of STA1 seems to be an alternative evolutionary strategy for efficient utilization of sugars present in brewer's wort. The results of this study allow for the improved reliability of molecular detection methods for diastatic contaminants in beer and can be exploited for strain development where maltotriose use is desired. [ABSTRACT FROM AUTHOR]

High-throughput function-based screening techniques remain the major bottleneck in the novel biocatalysts development pipeline. In the present study, we customized protocols for amylolytic activity determination (Somogyi-Nelson and starch-iodine tests) to micro-volume thermalcycler-based assays (linearity range 60-600 μM of reducing sugar, R2 = 0.9855; 0-2 mg/mL of starch, R2 = 0.9921, respectively). Exploitation of a thermalcycler enabled rapid and accurate temperature control, further reduction of reagents and samples volumes, and limited evaporation of the reaction mixtures, meeting several crucial requirements of an adequate enzymatic assay. In the optimized micro-volume Somogyi-Nelson protocol, we were able to reduce the time required for high-temperature heating sixfold (down to 5 min) and further increase sensitivity of the assay (tenfold), when compared to the previous MTP-based protocol. The optimized microassays have complementary scope of specificities: micro-starch-iodine test for endoglucanases, micro-Somogyi-Nelson test for exoglucanases. Due to rapid, micro-volume and high-throughput character, the methods can complement toolbox assisting development of novel biocatalysts and analysis of saccharides-containing samples. [ABSTRACT FROM AUTHOR]

Raw starch-degrading enzymes (RSDEs) are capable of directly degrading raw starch granules below the gelatinization temperature of starch, which may significantly reduce the cost of starch-based biorefining. However, low yields of natural RSDEs from filamentous fungi limit their industrial application. In this study, transcriptomic and secretomic profiling was employed to screen strongest promoters and signal peptides for use in overexpression of a RSDE gene in Penicillium oxalicum. Top five strong promoters and three signal peptides were detected. Using a green fluorescent protein (GFP) as the reporter, the inducible promoter pPoxEgCel5B of an endoglucanase gene PoxEgCel5B and the signal peptide spPoxGA15A of a raw starch-degrading glucoamylase PoxGA15A were respectively identified as driving the highest GFP production in P. oxalicum. PoxGA15A-overexpressed P. oxalicum strain OXPoxGA15A, which was constructed based on both pPoxEgCel5B and spPoxGA15A, produced significantly higher amounts of recombinant PoxGA15A than the parental strain ∆PoxKu70. Furthermore, crude enzyme from the OXPoxGA15A strain exhibited high activities towards raw starch from cassava, potato, and uncooked soluble starch. Specifically, raw cassava starch-degrading enzyme activity reached 241.6 U/mL in the OXPoxGA15A, which was 3.4-fold higher than that of the ∆PoxKu70. This work provides a feasible method for hyperproduction of RSDEs in P. oxalicum. [ABSTRACT FROM AUTHOR]

Clostridium sp. G0005 glucoamylase (CGA) is composed of a β-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae produces a large amount of secreted proteins in solid-state culture, and some proteins such as glucoamylase (GlaB) and acid protease (PepA) are specifically produced in solid-state culture, but rarely in submerged culture. From the disruption mutant library of A. oryzae transcriptional regulators, we successfully identified a disruption mutant showing an extremely low production level of GlaB but a normal level of α-amylase production. This strain was a disruption mutant of the CH-type transcription factor, FlbC, which is reported to be involved in the regulation of conidiospore development. Disruption mutants of other upstream regulators comprising a conidiation regulatory network had no apparent effect on GlaB production in solid-state culture. In addition to GlaB, the production of acid protease in solid-state culture was also markedly decreased by flbC disruption. Northern blot analyses revealed that transcripts of glaB and pepA were significantly decreased in the flbC disruption strain. These results suggested that FlbC is involved in the transcriptional regulation of genes specifically expressed under solid-state cultivation conditions, possibly independent of the conidiation regulatory network. [ABSTRACT FROM AUTHOR]

Recombinant yeast strains that display heterologous amylolytic enzymes on their cell surface via the glycosylphosphatidylinositol (GPI)-anchoring system are considered as promising biocatalysts for direct ethanol production from starchy materials. For the effective hydrolysis of these materials, the ratio optimization of multienzyme activity displayed on the cell surface is important. In this study, we have presented a ratio control system of multienzymes displayed on the yeast cell surface by using different GPI-anchoring domains. The novel gene cassettes for the cell-surface display of Streptococcus bovis α-amylase and Rhizopus oryzae glucoamylase were constructed using the Saccharomyces cerevisiae SED1 promoter and two different GPI-anchoring regions derived from Saccharomyces cerevisiae SED1 or SAG1. These gene cassettes were integrated into the Saccharomyces cerevisiae genome in different combinations. Then, the cell-surface α-amylase and glucoamylase activities and ethanol productivity of these recombinant strains were evaluated. The combinations of the gene cassettes of these enzymes affected the ratio of cell-surface α-amylase and glucoamylase activities and ethanol productivity of the recombinant strains. The highest ethanol productivity from raw starch was achieved by the strain harboring one α-amylase gene cassette carrying the SED1-anchoring region and two glucoamylase gene cassettes carrying the SED1-anchoring region (BY-AASS/GASS/GASS). This strain yielded 22.5 ± 0.6 g/L of ethanol from 100 g/L of raw starch in 120 h of fermentation. [ABSTRACT FROM AUTHOR]

Thermoanaerobacter tengcongensis MB4 glucoamylase (TteGA) contains a catalytic domain (CD), which is structurally similar to eukaryotic GA, and a β domain (BD) with ambiguous function. Firstly, BD is found to be essential to TteGA activity because CD alone could not hydrolyze soluble starch. However, starch hydrolysis activity, similar to that of intact TteGA, was restored to CD in the presence of BD. Secondly, BD is found to be an important helper in the correct folding of CD because CD was mainly expressed in the inclusion bodies on its own in Escherichia coli. By contrast, intact TteGA, BD, and CD combined with BD could be expressed as soluble proteins. Additionally, BD is essential to the thermostability of TteGA because CD displayed lower thermostability compared with the intact TteGA and exhibited enhanced thermostability in the presence of BD in vitro. Truncation of TteGA or mutagenesis of the residues that participate in the interdomain interaction at its BD also led to the reduced thermostability of TteGA. [ABSTRACT FROM AUTHOR]

The Aspergillus oryzae glucoamylase-encoding gene glaB is expressed specifically and strongly only during solid-state cultivation (SSC). To elucidate the basis for the specificity, the glaB promoter was analyzed by electrophoretic gel mobility shift assay (EMSA) which indicated two protein-binding elements from −382 to −353 and from −332 to −313. To confirm that these regions contained cis-elements, deletion analysis of the promoter was undertaken using β-glucuronidase as a reporter. The results of the deletion analysis were consistent with the EMSA results. The promoter missing the −332 to −313 element was not induced by low water activity stress during SSC. [ABSTRACT FROM AUTHOR]

Glucoamylase (GA) was immobilized onto polyaniline (PANI)-grafted magnetic poly(2-hydroxyethylmethacrylate-co-glycidylmethacrylate) hydrogel (m-p(HEMA-GMA)-PANI) with two different methods (i.e., adsorption and adsorption/cross-linking). The immobilized enzyme preparations were used for the hydrolysis of 'starch' dextrin. The amount of enzyme loading on the ferrogel was affected by the medium pH and the initial concentration of enzyme. The maximum loading capacity of the enzyme on the ferrogel was found to be 36.7 mg/g from 2.0 mg/mL enzyme solution at pH 4.0. The adsorbed GA demonstrated higher activity (59%) compared to adsorbed/cross-linked GA (43%). Finally, the immobilized GA preparations exhibited greater stability against heat at 55 °C and pH 4.5 compared to free enzyme (50 °C and pH 5.5), suggesting that the ferrogel was suitable support for immobilization of glucoamylase. [ABSTRACT FROM AUTHOR]

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of FeO/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic FeO/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization. [ABSTRACT FROM AUTHOR]

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3′,5,5′-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days. [ABSTRACT FROM AUTHOR]

This study demonstrates the potential of Aspergillus niger as a candidate expression system for virus-like particle production using gene fusion. Hepatitis B surface antigen (HBsAg) production, targeted through the secretory pathway in A. niger, resulted in completely assembled and properly folded HBsAg. This was achieved by implementing a gene fusion strategy using the highly expressed catalytic domain of the native glucoamylase gene ( GlaA) fused to the HBsAg S gene. The inducible glucoamylase promoter ( GlaA) was used to control transcription in the A. niger D15 host. The gene fusion strategy was designed for cleavage of the fused product by the KEX2-like protease, resulting in intracellular accumulation of HBsAg and extracellular secretion of glucoamylase. Immunodetection using a monoclonal HBsAg antibody could not detect the fused GlaA ::S product in intracellular and extracellular fractions, indicating that full assembly and maturation of HBsAg occurred after cleavage of the fused product in the Golgi complex. Several breakdown products showing an immunoreactive response to the glucoamylase polyclonal antibody indicated a level of intracellular degradation. The choice of carbon source used in cultivation significantly affected HBsAg production levels through induction of the glucoamylase promoter. The highest specific HBsAg production was observed during growth on inducing substrates of starch and its degradation products (maltodextrin and maltose), although residual glucose accumulation in the mid-exponential phase reduced HBsAg production. HBsAg production in starch-based cultures may be improved further by optimization of the rates of starch hydrolysis by glucoamylase and subsequent glucose consumption by the host. [ABSTRACT FROM AUTHOR]

Next to cellulose, starch is the most abundant hexose polymer in plants, an import food and feed source and a preferred substrate for the production of many industrial products. Efficient starch hydrolysis requires the activities of both α-1,4 and α-1,6-debranching hydrolases, such as endo-amylases, exo-amylases, debranching enzymes, and transferases. Although amylases are widely distributed in nature, only about 10 % of amylolytic enzymes are able to hydrolyse raw or unmodified starch, with a combination of α-amylases and glucoamylases as minimum requirement for the complete hydrolysis of raw starch. The cost-effective conversion of raw starch for the production of biofuels and other important by-products requires the expression of starch-hydrolysing enzymes in a fermenting yeast strain to achieve liquefaction, hydrolysis, and fermentation (Consolidated Bioprocessing, CBP) by a single organism. The status of engineering amylolytic activities into Saccharomyces cerevisiae as fermentative host is highlighted and progress as well as challenges towards a true CBP organism for raw starch is discussed. Conversion of raw starch by yeast secreting or displaying α-amylases and glucoamylases on their surface has been demonstrated, although not at high starch loading or conversion rates that will be economically viable on industrial scale. Once efficient conversion of raw starch can be demonstrated at commercial level, engineering of yeast to utilize alternative substrates and produce alternative chemicals as part of a sustainable biorefinery can be pursued to ensure the rightful place of starch converting yeasts in the envisaged bio-economy of the future. [ABSTRACT FROM AUTHOR]

Magnetic carbon nanotubes (MCNTs) with necklace-like nanostructures was prepared via hydrothermal method, and hyperbranched poly(amidoamine) (PAMAM) was grafted on the surface of MCNTs on the basis of the Michael addition of methyl acrylate and the amidation of the resulting ester with a large excess of ethylenediamine (EDA), which could achieve generational growth under such uniform stepwise reactions. The terminal -NH groups from the dendritic PAMAM were reacted with differently functionalized groups to form functionalized MCNTs. Subsequently, enzyme was immobilized on the functionalized MCNTs through adsorption, covalent bond, and metal-ion affinity interactions. The immobilization of glucoamylase, hereby chosen as model enzyme, onto the differently functionalized MCNTs is further demonstrated and assessed based on its activity, thermal stability, as well as reusability. Besides ease in recovery by magnetic separation, the immobilized glucoamylase on functionalized MCNTs offers superior stability and reusability, without compromising the substrate specificity of free glucoamylase. Furthermore, the results indicate that the metal-chelate dendrimer offers an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase. [ABSTRACT FROM AUTHOR]

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 °C and pH 5.0. It is the most thermostable bacterial glucoamylase described to date with nearly no activity loss after incubation at 75 °C for 6 h. TtcGA can hydrolyze both α-1, 4- and α-1, 6-glycosidic linkages in various α-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. [ABSTRACT FROM AUTHOR]

A gene encoding an intracellular glucoamylase was identified in the genome of the extreme thermoacidophilic Archaeon Thermoplasma acidophilum. The gene taGA, consisting of 1,911 bp, was cloned and successfully expressed in Escherichia coli. The recombinant protein was purified 22-fold to homogeneity using heat treatment, anion-exchange chromatography, and gel filtration. Detailed analysis shows that the glucoamylase, with a molecular weight of 66 kDa per subunit, is a homodimer in its active state. Amylolytic activity was measured over a wide range of temperature (40–90°C) and pH (pH 3.5–7) and was maximal at 75°C and at acidic condition (pH 5). The recombinant archaeal glucoamylase uses a variety of polysaccharides as substrate, including glycogen and amylose. Maximal activity was measured towards amylopectin with a specific activity of 4.2 U/mg and increased almost threefold in the presence of manganese. Calcium ions have a pronounced effect on enzyme stability; in the presence of 5 mM CaCl2, the half-life increased from 15 min to 2 h at 80°C. [ABSTRACT FROM AUTHOR]

It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF™ proteomics Analyzer indicated the enzyme to be 1,4-alpha- D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50°C, pH 4, and specific activity of 12.34 U mg of total protein−1 under the conditions, using diosgenin-3- O-α- L-rhamnopyranosyl(1→4)-[α-L-rhamnopyranosyl (1→2)]-β- D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin. [ABSTRACT FROM AUTHOR]

Purified glucoamylase (GA) from Fusarium solani was chemically modified by cross-linking with aniline hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 [aniline-coupled glucoamylase-1 (ACG-1)], 7 (ACG-7), and 13 min (ACG-13). The aniline coupling of GA had a profound enhancing effect on temperature, pH optima, and p K a’s of active site residues. The specificity constants ( K cat/ K m) of native, ACG-1, ACG-7, and ACG-13 were 136, 244, 262, and 208 at 55°C for starch, respectively. The enthalpy of activation (Δ H*) and free energy of activation (Δ G*) for soluble starch hydrolysis were lower for the chemically modified forms compared to native GA. Proteolysis of ACGs by α-chymotrypsin and subtilisin resulted in activation. [ABSTRACT FROM AUTHOR]