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PubMed2006

Expression of Vitreoscilla hemoglobin enhances growth and levels of alpha-amylase in Schwanniomyces occidentalis.

A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native alpha-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular alpha-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of alpha-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.

Antifungal Agents↗
PubMed1997

Effect of growth rate on alpha-amylase production by Streptomyces sp. IMD 2679.

The alpha-amylase of Streptomyces sp. IMD 2679 was subject to catabolite repression. Four different growth rates were achieved when the organism was grown at 40 degrees C and 55 degrees C in the presence and absence of cobalt, with an inverse relationship between alpha-amylase production and growth rate. Highest alpha-amylase yields (520 units/ml) were obtained at the lowest growth rate (0.062 h-1), at 40 degrees C in the absence of cobalt, while at the highest growth rate (0.35 h-1), at 55 degrees C in the presence of cobalt, alpha-amylase production was decreased to 150 units/ml. As growth rate increased, the rate of specific utilisation of the carbon source maltose also increased, from 46 to 123 micrograms maltose (mg biomass)-1 h-1. The pattern and levels of alpha-glucosidase (the enzyme degrading maltose) detected intracellularly in each case, indicate that growth rate effectively controls the rate of feeding of glucose to the cell, and thus catabolite repression.

Bacterial Proteins↗
PubMed1999

A variant of Saccharomyces cerevisiae pep4 strain with improved oligotrophic proliferation, cell survival and heterologous secretion of alpha-amylase.

A variant of Saccharomyces cerevisiae pep4 strain 20B12, with improved oligotrophic proliferation, cell survival and secretion of heterologous mouse alpha-amylase, is described. Previously we reported a procedure to enrich NI transformants that are not inhibited by cytotoxic expression of hepatitis B virus surface antigen in the secretion pathway of the protease-A-deficient (pep4) strain. To use the NI cells as a host for heterologous expression, we tried to amend the introduced pYAS/12S vector and obtain a host strain, NI-C, with stable NI phenotype and trp1 marker restored. Southern analysis of genomic DNA of NI-C suggested that the original pYAS/12S was abnormally rearranged and not completely corrected. Further assay showed that the viability and mitotic ability of the NI-C strain were increased. While using the NI-C strain as host for plasmid transformation and heterologous expression of mouse alpha-amylase, we observed that transformed colonies grew more quickly and secreted more alpha-amylase than general yeast strains. A further test showed that the NI-C strain was able to use mouse alpha-amylase as a positive selection marker to form transformed colonies on nitrogen-starved plates that contain starch as the sole carbon source. The results imply that the NI-C variant is an improved pep4 strain that can be used for heterologous expression and for the development of new selective markers in the yeast transformation system.

Animals↗
PubMed2001

Influence of carbon source on alpha-amylase production by Aspergillus oryzae.

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.

Aspergillus oryzae↗
PubMed2001

The N-terminal signal sequence and the last 98 amino acids are not essential for the secretion of Bacillus sp. TS-23 alpha-amylase in Escherichia coli.

A truncated Bacillus sp. TS-23 alpha-amylase gene lacking 96 and 294 bp at its 5' and 3' end respectively was prepared by polymerase chain reaction and cloned into Escherichia coli expression vector, pQE-30, under the control of T5 promoter. SDS-PAGE and activity staining analyses showed that the His6-tagged amylase had a molecular mass of approximately 54 kDa. Isopropyl-beta-d-thiogalactopyranoside (IPTG) induction of E. coli M15 cells bearing the recombinant plasmid resulted in the extracellular production of active amylase. Western blot analysis also revealed that the truncated amylase was present in the periplasmic space and culture medium.

Amino Acid Sequence↗
PubMed2001

Mutations of barley beta-amylase that improve substrate-binding affinity and thermostability.

Three allelic forms of barley beta-amylase (Sd1, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd1 and Sd2L beta-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis. The results showed that an R115C mutation is responsible for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the beta-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability index T50 by 1.9 degrees C and 2.1 degrees C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding of the protein during heating, while the replacement of V233 by alanine appears to cause an acceleration of the refolding after heating. Because the different beta-amylase properties determined by the three mutations (R115C, V233A and L347S) are associated with malting quality of barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering is discussed.

Alleles↗
PubMed2005

Expression and secretion of an acid-stable alpha-amylase gene in Bacillus Subtilis by SacB promoter and signal peptide.

Alpha amylase gene from Bacillus licheniformis was mutated by site-directed mutagenesis to improve its acid stability. The mutant gene was expression in Bacillus subtilis under the control of the promoter of sacB gene which was followed by either the alpha-amylase leader peptide of Bacillus licheniformis or the signal peptide sequence of sacB gene of Bacillus subtilis. Both peptides efficiently directed the secretion of alpha-amylase from the recombinant B. subtilis cells. The extracellular alpha-amylase activities in two recombinants were 1001 and 2012 U ml(-1), respectively. The purity of the recombinant product was confirmed by SDS-PAGE.

Bacillus subtilis↗
PubMed2006

A mutant alpha-amylase with only part of the catalytic domain and its structural implication.

A truncated mutant alpha-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type alpha-amylases (Xa-WT) through random mutagenesis that contained 167 amino acid residues (approx 65% shorter than that of Xa-WT). Secondary structure prediction implied that Xa-S2, would be unable to form the whole (beta/alpha)(8)-barrel catalytic domain and did not have the three conserved catalytic residues of wild type alpha-amylase, but it still displays the starch-hydrolyzing activity. Xa-S2 was prepared, characterized and compared to the recombinant wild-type enzymes. The K (m) for starch was 32 mg/ml; activity was optimal at pH 6.2 and 30 degrees C. In contrast, the K (m) for starch of Xa-WT was 8 mg/ml and optimal enzyme activity was at pH 6.0-6.2 and 45-50 degrees C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of alpha-1,4-glucosidic bonds.

Amino Acid Sequence↗
PubMed2005

Flat-chip microanalytical enzyme sensor for salivary amylase activity.

It is considered that measurement of salivary alpha-amylase activity is a useful tool for evaluating the sympathetic nervous system. The purpose of this research is to demonstrate a new design of a flat-chip microanalytical enzyme sensor (flat-chip sensor) for salivary amylase activity as a Micro-Electro-Mechanical Systems (MEMS), which may be used for wearable analytical systems. To meet this purpose, the biosensor needs to be miniaturized and to possess high-sensitivity. A pre-column and a flat-enzyme electrode were incorporated in a flow cell of volume 25.7 ml. In order to miniaturize the flow cell, two enzymatic membranes containing maltose phosphorylase obtained from Enterococcus hirae (MP membrane) and glucose oxidase and peroxidase (GOD-POD membrane) were immobilised on the same planar surface. As a result, a flat-chip sensor incorporating a flow cell as small as a C battery was produced. The optimum conditions of three parameters of the fabricated flat-chip sensor, the immobilising method of the enzymatic membrane, dropping volume of the mixed enzymatic solution and flow rate of the sample solution, were examined. An analytical system for 0-190 kU/l amylase activity with R(2) of 0.97 was fabricated with a sample volume of 50 microl. This research indicates the possibility of a wearable biosensor for continuous monitoring of salivary amylase activity.

Biosensing Techniques↗
PubMed2005

Molecular cloning and expression of an alpha-amylase inhibitor from rye with potential for controlling insect pests.

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new alpha-amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of alpha-amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic alpha-amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.

Amino Acid Sequence↗
PubMed1975

Serum amylase changes during pregnancy.

Study of the behavior of serum amylase activity in 200 preganant women in various stages of pregnancy indicated that: (1) serum amylase rises gradually during pregnancy until the twenty-fifth week and thereafter falls slightly; (2) serum amylase values may be found in normal pregnant women during the second and third trimesters that exceed those in normal men and nonpregnant women; (3) during the second trimester of pregnancy there may be an alteration in the relative distribution of the pancreatic and salivary-type isoamylases with the salivary type tending to dominate. Knowledge of these changes is of importance in the clinical assessment of serum amylase values in pregnant women complaining of abdominal pain and other symptoms suggestive of complicating acute pancreatitis. An explanation for the observed changes is not readily available and further study is required.

Adolescent↗
PubMed1975

Significance of serum amylase level in evaluating pancreatic trauma.

A series of fifty-one patients who underwent exploratory laparotomy for blunt abdominal trauma is reviewed. Attempts to correlate preoperative serum amylase levels with pancreatic injury were unsuccessful. We could find no statistically significant correlation between elevated serum amylase and pancreatic injury. The accuracy of the test improved somewhat with markedly abnormal serum amylase levels (greater than 300 Somogyi units/100 ml), but even at these levels the correlation was not significant. One must conclude that there is no correlation between elevated serum amylase and pancreatic injury in blunt abdominal trauma. Decisions as to the advisability of operation in patients with such trauma should therefore be based on other parameters.

Abdominal Injuries↗
PubMed1987

The amylase profile: a discriminant in biliary and pancreatic disease.

Serial serum amylase determinations were made in 85 consecutive patients who presented with an initially elevated value. In 35 patients (Group A), the clinical findings were consistent with biliary tract disease. All underwent cholecystectomy for documented cholelithiasis within a week of diagnosis, and 23 percent had choledocholithiasis as well. Fifty patients (Group B) presented with a first episode of acute alcoholic pancreatitis. An amylase profile, consisting of initial and final values and the daily rate of change, was significantly different between the two groups. The initial serum amylase value was higher in Group A patients and decreased more rapidly to a lower value than in Group B patients. We conclude that both the initial value and pattern of serum amylase decay distinguish the hyperamylasemia of biliary tract disease from that of alcoholic pancreatitis.

Acute Disease↗
PubMed1988

Selective inhibition of histidine-modified pancreatic alpha-amylase by proteinaceous inhibitor from Phaseolus vulgaris.

Chemical modification of two histidine residues of porcine pancreatic alpha-amylase (EC 3.2.1.1) by diethyl pyrocarbonate in the presence of a high concentration of maltotriose caused a decrease of amylase activity and an increase of maltosidase activity (hydrolysis of p-nitrophenyl-alpha-maltoside). By binding a proteinaceous inhibitor from Phaseolus vulgaris (white kidney bean) with the modified enzyme, the amylase activity was further decreased but the maltosidase activity was retained to about 100% that of the native enzyme. Both amylase and maltosidase activities of the native enzyme were almost completely inhibited by the proteinaceous inhibitor. The increase of maltosidase activity by histidine modification was due to an increase of kcat, whereas the Km value was not changed; but binding of the proteinous inhibitor affected mainly the Km value of the modified enzyme.

Animals↗
PubMed1989

New substrate specificity of modified porcine pancreatic alpha-amylase.

Conversion of the substrate specificity of porcine pancreatic alpha-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the alpha-D-(1,4)glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous alpha-amylase inhibitor (Mr 60,000) purified from white kidney bean (Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the alpha-D-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the alpha-D-(1,4)glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.

Amylases↗
PubMed1983

The binding of human salivary alpha-amylase by oral strains of streptococcal bacteria.

The ability of various oral streptococci to bind salivary alpha-amylase to their cell surfaces was investigated. Samples of cells were mixed with whole cleared saliva and the alpha-amylase remaining after removal of the cells was assayed by radial diffusion in starch-containing agarose. Seventy-five per cent of Streptococcus sanguis strains bound the enzyme but strains of Streptococcus mutans and Streptococcus mitior did not. SDS-polyacrylamide gel electrophoretic analysis of Strep. sanguis cells which had been mixed with saliva showed that alpha-amylase is bound to the surface of the cells and can be recovered from them intact. The ability of Strep. sanguis strains to bind the enzyme did not correlate with biotype, IPS production or ability to adhere to saliva-coated hydroxyapatite. The cell-surface receptor responsible for the binding of alpha-amylase to Strep. sanguis (NCTC 7865) is a heat-stable component, possibly protein in nature, which is closely associated with the cell wall.

Humans↗
PubMed1987

Amylase activity, protein and urea in saliva of the red kangaroo (Macropus rufus).

Parotid and mandibular salivas, produced at flow rates of 0.055 +/- 0.0038 to 4.45 +/- 0.101 and 0.052 +/- 0.0059 to 4.30 +/- 0.072 ml min-1 respectively by intracarotid infusion of acetylcholine or methacholine, were analysed. During acetylcholine stimulation, the protein concentration ranged from 86.4 +/- 10.17 to 235.0 +/- 23.5 mg l-1 in parotid and from 102.9 +/- 11.54 to 379.6 +/- 38.01 mg l-1 in mandibular saliva. The minimum protein concentrations in parotid and mandibular salivas occurred at 0.5 and 1.0 ml min-1 respectively with the concentration rising progressively at lower and higher flow rates. Both glands maintained constant levels of protein during 90 min of sustained stimulation at constant flow rates of 0.5 and 2.0 ml min-1. The optimum pH for salivary amylase activity lay between 7.4 and 7.9 (mean 7.5-7.6). The amylase activity of parotid saliva ranged between 32.2 +/- 3.81 and 94.5 +/- 24.05 mukat l-1 and the activity/protein ratio was similar at all flow rates (mean, 0.39 +/- 0.049 mukat amylase/mg protein). Amylase levels in arterial plasma were constant within experiments but varied from 8.0 +/- 0.07 to 26.4 +/- 0.24 mukat l-1 between experiments, and were always less than corresponding parotid values. Mandibular amylase activities were low, highly variable or often absent. Methacholine-evoked parotid saliva had lower protein concentrations but similar amylase/protein ratios to acetylcholine-stimulated saliva. Urea concentrations in arterial plasma were constant within experiments but varied from 6.71 +/- 0.092 to 14.2 +/- 0.14 mmol l-1 between experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗
PubMed1988

Relationship between serum-amylase activity and intraductal pressures in the rat parotid and submandibular salivary glands after administration of pilocarpine or isoprenaline.

Ligation of the parotid ducts induced marked elevation of serum-amylase activity after administration of pilocarpine or isoprenaline. Secretion of saliva and serum-amylase activity increased most rapidly in the first 15 min after injection of pilocarpine, whereas isoprenaline induced a constant rate of secretion and serum-amylase activity over 120 min. Amylase activity in the serum was closely connected with intraductal pressure due to saliva secretion.

Amylases↗