Zearalenone Removal from Corn Oil by an Enzymatic Strategy.

The estrogen-like mycotoxin zearalenone (ZEN) is one of the most widely distributed contaminants especially in maize and its commodities, such as corn oil. ZEN degrading enzymes possess the potential for counteracting the negative effect of ZEN and its associated high safety risk in corn oil. Herein, we targeted enhancing the secretion of ZEN degrading enzyme by Pichia pastoris through constructing an expression plasmid containing three optimized expression cassettes of zlhy-6 codon and signal peptides. Further, we explored various parameters of enzymatic detoxification in neutralized oil and analyzed tocopherols and sterols losses in the corn oil. In addition, the distribution of degraded products was demonstrated as well by Agilent 6510 Quadrupole Time-of-Flight mass spectrometry. P. pastoris GSZ with the glucoamylase signal was observed with the highest ZLHY-6 secretion yield of 0.39 mg/mL. During the refining of corn oil, ZEN in the crude oil was reduced from 1257.3 to 13 µg/kg (3.69% residual) after neutralization and enzymatic detoxification. Compared with the neutralized oil, no significant difference in the total tocopherols and sterols contents was detected after enzymatic detoxification. Finally, the degraded products were found to be entirely eliminated by washing. This study presents an enzymatic strategy for efficient and safe ZEN removal with relatively low nutrient loss, which provides an important basis for further application of enzymatic ZEN elimination in the industrial process of corn oil production.

Identification of patients with Pompé disease using routine pathology results: PATHFINDER (creatine kinase) study.

AIMS: Adult-onset inherited errors of metabolism can be difficult to diagnose. Some cases of potentially treatable myopathy are caused by autosomal recessive acid α-1,4 glucosidase (acid maltase) deficiency (Pompé disease). This study investigated whether screening of asymptomatic patients with elevated creatine kinase (CK) could improve detection of Pompé disease. METHODS: Pathology databases in six hospitals were used to identify patients with elevated CK results (>2× upper limit of normal). Patients were recalled for measurement of acid α-1,4 glucosidase activity in dried blood spot samples. RESULTS: Samples were obtained from 812 patients with elevated CK. Low α-glucosidase activity was found in 13 patients (1.6%). Patients with neutropaenia (n=4) or who declined further testing (n=1) were excluded. Confirmation plasma specimens were obtained from eight individuals (1%) for a white cell lysosomal enzyme panel, and three (0.4%) were confirmed to have low α-1,4-glucosidase activity. One patient was identified as a heterozygous carrier of an acid α-1,4 glucosidase c.-32-13 G>T mutation. Screening also identified one patient who was found to have undiagnosed Fabry disease and one patient with McArdle's disease. One patient later presented with Pompé's after an acute illness. Including the latent case, the frequency of cases at 0.12% was lower than the 2.5% found in studies of patients with raised CK from neurology clinics (p<0.001). CONCLUSIONS: Screening pathology databases for elevated CK may identify patients with inherited metabolic errors affecting muscle metabolism. However, the frequency of Pompé's disease identified from laboratory populations was less than that in patients referred for neurological investigation.

Reevaluating the pathogenicity of the mutation c.1194 +5 G>A in GAA gene by functional analysis of RNA in a 61-year-old woman diagnosed with Pompe disease by muscle biopsy.

Glycogen storage disease type II, or Pompe disease, is an autosomal recessive disorder caused by deficiency of lysosomal acid alpha-glucosidase (GAA). We performed genetic analysis to confirm the diagnosis of Pompe disease in a 61-year-old patient with progressive weakness in extremities, severe Sleep Apnea-Hypopnea Syndrome, a significant reduction of alpha-glucosidase in liquid sample of peripheral blood and muscular biopsy diagnosis. GAA gene sequencing showed the patient is homozygous for the splice-site mutation c.1194+5G>A, considered as nonpathogenic in Pompe Center mutation database. Further molecular RNA characterization of GAA transcripts allowed us to identify abnormal processing of pre-mRNA, leading to aberrant transcripts and a significant reduction of GAA mRNA levels. Our results indicate that c.1194+5G>A is a pathogenic splice-site mutation and should be considered as such for diagnostic purposes. This study emphasizes the potential role of functional studies to determine the consequences of mutations with no evident pathogenicity.

Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease.

Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa(-/-) mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders.

[Variability in the clinical presentation of Pompe disease: development following enzyme replacement therapy]. / Variabilidad en la presentación clínica en la enfermedad de Pompe: evolución tras terapia de reemplazo enzimático.

INTRODUCTION: Pompe disease is a generalized progressive disease caused by a deficiency of the lysosome enzyme acid alpha-glucosidase (GAA). We present three cases with different clinical symptomatology and treated with enzyme replacement therapy (ERT) with positive evolution. CASE REPORTS: Case 1: three-month old male, with weakness and rejecting meals; mild hepatomegaly, discrete macroglossia and muscular hypotony; and increased muscular enzymes. Case 2: five-month old male, with delayed motor development, severe neurosensory deafness, and respiratory disorder of difficult evolution; muscular hypotony; and mild increase in creatine kinase. Case 3: 22-year old male, with progressive dyspnea, with history of increased creatine kinase and transaminases, and hypercholesterolemia. He suffered from severe respiratory failure requiring endotraqueal intubation Muscular biopsy showed glycogen storage suggestive of Pompe disease. In the three cases, the EMG showed a characteristic pattern with pseudomyotonic discharges and the deficiency in GAA was confirmed in lymphocytes. One single mutation was observed in one case and two in the other two cases. Every patient received ERT showing a favorable evolution; with disappearance of cardiac disorders in case 1, improvement in motor development in both infants and no longer need for mechanical ventilation in case 3. CONCLUSION: Pompe disease has a wide variability in clinical expression. ERT achieves a good response, especially in infant forms of the disease. The survival of treated patients with these Pompe disease forms will allow knowing further the course of the disease.

TITLE: Variabilidad en la presentacion clinica en la enfermedad de Pompe: evolucion tras terapia de reemplazo enzimatico.Introduccion. La enfermedad de Pompe es un trastorno generalizado progresivo producido por el deficit de la enzima alfa-glucosidasa acida (AGA) de los lisosomas. Se presentan tres casos manifestados de forma muy diferente y tratados con terapia enzimatica sustitutiva (TES), con evolucion favorable. Casos clinicos. Caso 1: varon de 3 meses, con debilidad y rechazo de la alimentacion, hepatomegalia leve, ligera macroglosia e hipotonia, y aumento de las enzimas musculares. Caso 2: varon de 5 meses, con retraso del desarrollo motor, sordera neurosensorial grave, trastornos respiratorios de repeticion de evolucion torpida, hipotonia y leve elevacion de la creatincinasa. Caso 3: varon de 22 años con disnea progresiva, con antecedentes de elevacion de la creatincinasa y las transaminasas, e hipercolesterolemia. Sufrio insuficiencia respiratoria grave que preciso intubacion endotraqueal. La biopsia muscular presento depositos de glucogeno sugestivos de la enfermedad. En los tres casos, el estudio electromiografico dio un patron caracteristico, con descargas pseudomiotonicas, y se confirmo el deficit de AGA en los linfocitos. Se encontro una mutacion en un caso y dos mutaciones en los otros dos. Todos recibieron TES con evolucion favorable: desaparicion de las alteraciones cardiacas en el caso 1, mejoria en los hitos motores en los dos casos infantiles y retirada del respirador en el caso 3. Conclusion. La enfermedad de Pompe tiene una amplia variabilidad en la expresion clinica. La TES consigue una buena respuesta, especialmente en las formas infantiles. La supervivencia a largo plazo de las formas infantiles tratadas permitira conocer mas aspectos del curso de la enfermedad.

Evaluation of Readministration of a Recombinant Adeno-Associated Virus Vector Expressing Acid Alpha-Glucosidase in Pompe Disease: Preclinical to Clinical Planning.

A recombinant serotype 9 adeno-associated virus (rAAV9) vector carrying a transgene that expresses codon-optimized human acid alpha-glucosidase (hGAA, or GAA) driven by a human desmin (DES) promoter (i.e., rAAV9-DES-hGAA) has been generated as a clinical candidate vector for Pompe disease. The rAAV9-DES-hGAA vector is being developed as a treatment for both early- and late-onset Pompe disease, in which patients lack sufficient lysosomal alpha-glucosidase leading to glycogen accumulation. In young patients, the therapy may need to be readministered after a period of time to maintain therapeutic levels of GAA. Administration of AAV-based gene therapies is commonly associated with the production of neutralizing antibodies that may reduce the effectiveness of the vector, especially if readministration is required. Previous studies have demonstrated the ability of rAAV9-DES-hGAA to correct cardiac and skeletal muscle pathology in Gaa(-/-) mice, an animal model of Pompe disease. This article describes the IND-enabling preclinical studies supporting the program for a phase I/II clinical trial in adult patients with Pompe. These studies were designed to evaluate the toxicology, biodistribution, and potential for readministration of rAAV9-DES-hGAA injected intramuscularly into the tibialis anterior muscle using an immune modulation strategy developed for this study. In the proposed clinical study, six adult participants with late-onset Pompe disease will be enrolled. The goal of the immune modulation strategy is to ablate B-cells before the initial exposure of the study agent in one leg and the subsequent exposure of the same vector to the contralateral leg four months after initial dosing. The dosing of the active agent is accompanied by a control injection of excipient dosing in the contralateral leg to allow for blinding and randomization of dosing, which may also strengthen the evidence generated from gene therapy studies in the future. Patients will act as their own controls. Repeated measures, at baseline and during the three months following each dosing will assess the safety, biochemical, and functional impact of the vector.

A floppy baby.

Pompe disease is a rare inherited disorder of glycogen metabolism. We present a case of a 9-month-old infant who presented to the emergency department with generalized hypotonia and respiratory distress and was found to have Pompe disease. In this article, we will review the differential diagnosis of hypotonia in the infant, presentations of hypotonia that are relevant to the emergency department physician, as well as the diagnosis, management, and prognosis of Pompe disease.

BAFF blockade prevents anti-drug antibody formation in a mouse model of Pompe disease.

Antibodies formed against the therapeutic protein are a life-threatening complication that arises during enzyme replacement therapy for Pompe disease (acid α-glucosidase deficiency; GAA). To provide an effective alternative to current practices, we investigated the capacity of anti-B-cell activating factor (BAFF) as a novel drug candidate to prevent antibody formation in a Pompe disease mouse model. A BAFF-neutralizing antibody was administered prophylactically and with maintenance doses in association with enzyme replacement therapy using recombinant human GAA in Gaa(-/-) mice. BAFF blockade delayed antibody production and increased GAA activity within tissues with protection from anaphylaxis. Anti-BAFF also resolved antibody formation during an immune response and precluded the maturation of antibody secreting cells from entering the bone marrow compartment. This treatment modality may therefore be a viable alternative for the clinical management of antibody formation for Pompe disease and has potential use against antibody formation in other protein replacement therapies.

Direct utilization of purple sweet potato by sake yeasts to produce an anthocyanin-rich alcoholic beverage.

OBJECTIVE: To produce an alcoholic beverage containing anthocyanins that can act as antioxidants and have anticarcinogenic activities and antihypertensive effects. RESULTS: High starch-assimilating sake yeast strain of Saccharomyces cerevisiae co-expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis using the double rDNA-integration system was developed. The new strain grew substantially using 5 % (w/v) purple sweet potato flour as the sole carbon source. Its cell yield reached 14.5 mg ml(-1) after 3 days. This value was 2.4-fold higher than that of the parental wild-type strain. It produced 12 % (v/v) ethanol from 20 % (w/v) purple sweet potato flour and consumed 98 % of the starch content in purple sweet potato flour after 5 days of fermentation. CONCLUSION: We have produced a health-promoting alcoholic beverage abundant in anthocyanins from purple sweet potato.

[Variability in the clinical presentation of Pompe disease in infancy: two case reports and response to treatment with human recombinant enzyme]. / Variabilidad en la presentacion clinica de la enfermedad de Pompe infantil: presentacion de dos casos y respuesta al tratamiento con enzima recombinante humana.

INTRODUCTION: Pompe disease/glycogen storage disease type II is a congenital metabolic disorder. It is an autosomal recessive disease where there is a deficiency of acid alpha-glucosidase (GAA), an enzyme required for lysosomal glycogen degradation. We describe two infantile onset cases with heterogeneous presentations. CASE REPORTS: The first case is a newborn with maintained bradycardia, a cardiologic study revealed severe biventricular hypertrophy. The second case is a 14 months old patient with motor delay and arreflexic hypotonia. Creatine kinase, lactate dehydrogenase, glutamic-oxaloacetic transaminase and glutamic-pyruvic trans aminase were high. The electromyogram showed myopathic alteration and the muscle biopsy vacuolar myopathy with glycogen accumulation. In both of them, GAA activity was decreased and the genetic exam of the GAA gene revealed heterozygous mutations already described in Pompe disease. They started enzyme replacement therapy at 1 month old and 18 months old respectively, improving the cardiomyopathy hypertrophy, motor wise however less advance was seen. At the present time, the patients are 9 months and 5 years old respectively, the last one has a Gross Motor Function Measure (GMFM) III level. CONCLUSION: GAA is the only authorized option for Pompe disease treatment; the effects observed are similar to the ones described in the literature, with excellent outcome in the hypertrophic cardiomyopathy but less effective in the skeletal muscle.

TITLE: Variabilidad en la presentacion clinica de la enfermedad de Pompe infantil: presentacion de dos casos y respuesta al tratamiento con enzima recombinante humana.Introduccion. La enfermedad de Pompe o glucogenosis tipo II es un error innato del metabolismo, de herencia autosomica recesiva, causado por un deficit de la enzima lisosomal alfa-glucosidasa acida (GAA), que ocasiona un acumulo multisistemico de glucogeno. Describimos dos casos de inicio temprano (infantil) con diferentes formas de presentacion. Casos clinicos. El primer caso se trata de un neonato que presento una bradicardia mantenida desde el nacimiento, lo que motivo el estudio cardiologico, en el que se evidencio una hipertrofia biventricular grave. El segundo caso se trata de un niño de 14 meses con retraso motor e hipotonia con arreflexia. En la analitica destacaban una creatincinasa, lactato deshidrogenasa, transaminasa glutamico oxalacetica y transaminasa glutamico piruvica elevadas. El electromiograma mostro signos de afectacion miopatica, y la biopsia muscular, una miopatia vacuolar con deposito de glucogeno. En ambos, la actividad de la GAA estaba disminuida y el analisis genetico del gen GAA evidencio que eran portadores heterocigotos de mutaciones descritas en la enfermedad de Pompe. Iniciaron tratamiento con enzima recombinante humana al mes y a los 18 meses de vida, respectivamente, con mejoria evidente de la miocardiopatia hipertrofica, aunque mas limitada a nivel motor. En la actualidad tienen, respectivamente, 9 meses y 5 años, y este ultimo presenta un nivel III de la escala GMFM (Gross Motor Function Measure). Conclusion. La GAA recombinante humana es la unica opcion terapeutica autorizada para estos pacientes. Los efectos que hemos observado son similares a los descritos en otros casos, con una excelente evolucion de la miocardiopatia hipertrofica y un efecto variable sobre la musculatura esqueletica.

Novel GAA sequence variant c.1211 A>G reduces enzyme activity but not protein expression in infantile and adult onset Pompe disease.

Pompe disease is a clinically and genetically heterogeneous autosomal recessive disorder caused by lysosomal acid α-glucosidase (GAA) deficiency. We report on two affected members of a non-consanguineous Caucasian family, including a classical infantile-onset patient with severe cardiomyopathy (IO) and his paternal grandmother with the adult-onset (AO) form. Two compound heterozygous sequence variants of the GAA gene were identified in each patient by mutation analyses (IO=c.1211A>G and c.1798C>T; AO=c.1211A>G and c.692+5G>T). For this study, the biochemical phenotype resulting from the missense mutation c.1211A>G in exon 8, which converts a highly conserved aspartate to glycine (p.Asp404Gly), was of specific interest because it had not been reported previously. Western blotting revealed a robust expression of all GAA isoforms in quadriceps muscle of both patients (fully CRIM positive), while enzymatic activity was 3.6% (IO) and 6.6% (AO) of normal controls. To further validate these findings, the c.1211A>G sequence variant was introduced in wild type GAA cDNA and over-expressed in HEK293T cells. Site-directed mutagenesis analyses confirmed that the mutation does not affect processing or expression of GAA protein, but rather impairs enzyme function. Similar results were reported for c.1798C>T (p.Arg600Cys), which further supports the biochemical phenotype observed in IO. The third mutation (c.692+5G>T, in intron 3) was predicted to affect normal splicing of the GAA mRNA, and qPCR indeed verified a 4-fold lower mRNA expression in AO. It is concluded that the novel sequence variant c.1211A>G results in full CRIM but significantly lower GAA activity, which in combination with c.1798C>T leads to infantile-onset Pompe disease. We surmise that the difference in disease severity between the two family members in this study is due to a milder effect of the intronic mutation c.692+5G>T (vs. c.1798C>T) on phenotype, partially preserving GAA activity and delaying onset in the proband (paternal grandmother).

Production of a functional human acid maltase in tobacco seeds: biochemical analysis, uptake by human GSDII cells, and in vivo studies in GAA knockout mice.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II (GSDII) or Pompe's disease. To investigate whether we could generate a functional recombinant human GAA enzyme (tobrhGAA) in tobacco seeds for future enzyme replacement therapy, we subcloned the human GAA cDNA into the plant expression plasmid-pBI101 under the control of the soybean ß-conglycinin seed-specific promoter and biochemically analyzed the tobrhGAA. Tobacco seeds contain the metabolic machinery that is more compatible with mammalian glycosylation-phosphorylation and processing. We found the tobrhGAA to be enzymatically active was readily taken up by GSDII fibroblasts and in white blood cells from whole blood to reverse the defect. The tobrhGAA corrected the enzyme defect in tissues at 7 days after a single dose following intraperitoneal (IP) administration in GAA knockout (GAA(-/-)) mice. Additionally, we could purify the tobrhGAA since it bound tightly to the matrix of Sephadex G100 and can be eluted by competition with maltose. These data demonstrate indirectly that the tobrhGAA is fully functional, predominantly proteolytically cleaved and contains the minimal phosphorylation and mannose-6-phosphate residues essential for biological activity.

[Clinical sequelae of 17 cases with glycogen storage disease type II/Pompe disease].

OBJECTIVE: To analyze and summarize the characteristics of glycogen storage disease type II (Pompe disease) patients according to the clinical description and prognosis. METHOD: Seventeen Chinese patients diagnosed by acid alpha-glucosidase (GAA) enzyme activity test were reviewed. Clinical data tables were designed. Interviews were made via phone calls. Information was collected to reach the objective. RESULT: Four of 17 patients diagnosed by acid alpha-glucosidase are infantile-onset, symptoms started between 2 to 6 months after birth with increased serum creatine kinase and cardiac problems, with or without respiratory concerns. Other 13 patients were later-onset cases, and their symptoms started between 2 to 22 years of age with increased serum creatine kinase. Eleven later-onset patients started with muscle weakness, 2 patients developed respiratory insufficiency, 2 patients showed scoliosis, and 1 patient expressed increased serum creatine kinase with abnormal liver function. Just 3 of the later-onset patients were treated with mechanical ventilator and adjuvant therapy, others were not. All patients' acid alpha-glucosidase (GAA) enzyme activity analysis showed lower than 10% of normal. Fourteen patients were tested by muscle biopsy pathology, and 9 of them progressed to glycogen storage disease type II; 10 patients received genetic analysis, and 6 of them had two mutations which cause the disorder. Twelve of the 17 patients were interviewed successfully. In 3 of the infant-onset patients the disease resulted in death from respiratory failure, and 1 is still alive at the age of 1 year and 7 months. In 4 of 8 later-onset patients the disease resulted in death from respiratory failure between 3 to 5 years after onset of symptoms. Three of 4 survivors had increased muscle weakness, and 1 patient kept alive with ventilator without any changes. Seven of 12 interviewed patients died, the mortality rate was 58.3%. CONCLUSION: Glycogen storage disease type II (Pompe disease) present differently in the clinic. Infant-onset Pompe disease is mainly characterized by generalized muscle weakness and obvious cardiac involvement. It's a dangerous disease, with high mortality rate. Later-onset Pompe disease is characterized by chronic proximal muscle weakness and respiratory insufficiency. GAA enzyme activity analysis, muscle biopsy and genetic analysis used to support the diagnosis of Pompe disease. Prognosis of the disease depends on age of onset and respiratory muscle involvement.

Novel mutations in the gene encoding acid α-1,4-glucosidase in a patient with late-onset glycogen storage disease type II (Pompe disease) with impaired intelligence.

A 17-year-old Japanese man was referred to our hospital because of highly elevated serum levels of creatine kinase (CK) and transaminases. On admission, the proximal muscles of the lower extremities were found to be predominantly affected, and a score of 3/5 was obtained on Medical Research Council (MRC) scale. Muscular atrophy was evident and Gowers' sign was positive. His functional vital capacity (FVC) was markedly reduced. The results of the third edition of the Wechsler Adult Intelligence Scale (WAIS-III) indicated impairment of the patient's intelligence. Muscle biopsy showed scattered intracytoplasmic vacuoles with basophilic amorphous materials inside which were strongly stained by both periodic acid Schiff (PAS) and acid phosphatase. Biochemical analysis of the muscle tissue confirmed the diagnosis of GSDII because the glucosidase activity was 1.0 nmol/4 MU/mg/30 min (control range, 7.3 ± 2.2). Genetic analysis revealed a novel compound heterozygous missense mutation in GAA--c.1814 G >A (p.Gly605Asp) and c.1846 G >A (p.Asp616Asn) both in exon 13.

Raw starch fermentation to ethanol by an industrial distiller's yeast strain of Saccharomyces cerevisiae expressing glucoamylase and α-amylase genes.

Industrial strains of a polyploid, distiller's Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l(-1)) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.

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.

Novel strategy for yeast construction using delta-integration and cell fusion to efficiently produce ethanol from raw starch.

We developed a novel strategy for constructing yeast to improve levels of amylase gene expression and the practical potential of yeast by combining delta-integration and polyploidization through cell fusion. Streptococcus bovis alpha-amylase and Rhizopus oryzae glucoamylase/alpha-agglutinin fusion protein genes were integrated into haploid yeast strains. Diploid strains were constructed from these haploid strains by mating, and then a tetraploid strain was constructed by cell fusion. The alpha-amylase and glucoamylase activities of the tetraploid strain were increased up to 1.5- and tenfold, respectively, compared with the parental strain. The diploid and tetraploid strains proliferated faster, yielded more cells, and fermented glucose more effectively than the haploid strain. Ethanol productivity from raw starch was improved with increased ploidy; the tetraploid strain consumed 150 g/l of raw starch and produced 70 g/l of ethanol after 72 h of fermentation. Our strategy for constructing yeasts resulted in the simultaneous overexpression of genes integrated into the genome and improvements in the practical potential of yeasts.

A CBM20 low-affinity starch-binding domain from glucan, water dikinase.

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

Identification and characterization of a novel fibril forming peptide in fungal starch binding domain.

Scanty information is available regarding the chemical basis for structural alterations of the carbohydrate-binding modules (CBMs). The N-terminal starch binding domain (SBD) of Rhizopus oryzae glucoamylase (GA) forms fibrils under thermal stress, presenting an unusual conformational change from immunoglobulin-like to beta-sheet-rich structure. Site-directed mutagenesis revealed that the C-terminal Lys of SBD played a crucial role in the fibril formation. The synthetic peptide (DNNNSANYQVSTSK) representing the C-terminal 14 amino acid residues of SBD was further demonstrated to act as a fibril-forming segment, in which terminal charges and an internal NNNxxNYQ motif were key fibril-forming determinants. The formation of fibril structure in a fungal SBD, caused by its chemical and biophysical requirements, was demonstrated for the first time.

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.