Chemical Modification of Amino Groups of Acid-stable α-Amylase and Acid-unstable α-Amylase from Aspergillus niger

Monochlorotrifluoro-p-benzoquinone (CFQ) was used for investigating the state of the amino groups of acid-stable α-amylase and acid-unstable α-amylase. About half of the total amino groups in both enzyme molecules were reacted with the reagent. The unreactive amino groups seemed to exist in a different state from the reactive ones. Both enzymes whose amino groups were modified by CFQ still maintained the α-phenylmaltosidase activity in spite of losing or decreasing the amylase activity. These facts suggest that the amino groups of both enzymes were not in the active site but the modification of them caused steric hindrance.

The pH-stability of the acid-unstable α-amylase whose one or two amino groups were modified with succinic anhydride or 2,4,6-trinitrobenzene-l-sulfonate (TNBS) increased on the acidic side and decreased on the alkaline side, but further modification of them led to decrease the stability on both sides.     

Soluble, nuclear and mitochondrial forms of dehydrogenases, pentose-phosphate pathway transferases and nucleases in chicken liver]

The sub-cellular topography of oxidative and non-oxidative enzymes of the pentose phosphate pathway of carbohydrates metabolism and enzymes of the nucleic exchange (acid and alkaline deoxyribonucleases and ribonucleases) in chicken liver is studied. Nuclear and mitochondrial forms of the enzymes are discovered. The activity of the enzymes studied of carbohydrates metabolism is shown to correlate with that of the enzymes of nucleic metabolism in cytosol, nucleic and mitochondrial liver fractions.     

Oxidation of O-Alkyl-O-(subst. phenyl) phenylphosphonothioates

It has been found that exocellular α-amylase could be fixed to mycelia of Aspergillus oryzae at an acidic pH region and fixed α-amylase was released reversely at an alkaline pH region. The fixation has more remarkably been observed in mycelia obtained from a phosphate deficient medium where endocellular accumulation of a-amylase occurs more easily than in an ordinary mycelia which secrete a large amount of α-amylase into medium. Bound form of α-amylase was more resistant to low pH and less active than the free form. The results appear to support the previous suggestion that a large quantity of endocellular α-amylase might be located on mycelial surface of the mold.     

Structural elucidation and molecular characterization of Marinobacter sp. α-amylase

Halophiles have been perceived as potential source of novel enzymes in recent years. The interest emanates from their ability to catalyze efficiently under high salt and organic solvents. Marinobacter sp. EMB8 α-amylase was found to be active and stable in salt and organic solvents. A study was carried out using circular dichroism (CD), fluorescence spectroscopy, and bioinformatics analysis of similar protein sequence to ascertain molecular basis of salt and solvent adaptability of α-amylase. Structural changes recorded in the presence of varying amounts of NaCl exhibited an increase in negative ellipticity as a function of salt, confirming that salt stabilizes the protein and increases the secondary structure, making it catalytically functional. The data of intrinsic and extrinsic fluorescence (using 1-anilinonaphthalene 8-sulfonate [ANS] as probe) further confirmed the role of salt. The α-amylase was active in the presence of nonpolar solvents, namely, hexane and decane, but inactivated by ethanol. The decrease in the activity was correlated with the loss of tertiary structure in the presence of ethanol. Guanidine hydrochloride and pH denaturation indicated the molten globule state at pH 4.0. Partial N-terminal amino acid sequence of the purified α-amylase revealed the relatedness to Pseudoalteromonas sp. α-amylase. “FVHLFEW” was found as the N-terminal signature sequence. Bioinformatics analysis was done using M. algicola α-amylase protein having the same N-terminal signature sequence. The three-dimensional structure of Marinobacter α-amylase was deduced using the I-TASSER server, which reflected the enrichment of acidic amino acids on the surface, imparting the stability in the presence of salt. Our study clearly indicate that salt is necessary for maintaining the secondary and tertiary structure of halophilic protein, which is a necessary prerequisite for catalysis.     

Production of acid and alkaline phosphatases byMyxococcus coralloides

Acid and alkaline phosphatase ofMyxococcus coralloides were examined during vegetative growth in a liquid medium. Two extracellular phosphatases and two cell-bound phosphatases, acid and alkaline in both cases, were produced. The phosphatase production was unaltered by the presence of high concentrations of inorganic phosphate. Both enzymes were produced constitutively. These two hydrolases were released into the growth medium during the exponential growth phase (approximately 10% of total activity). The production of these enzymes was modified by the presence of organic acids and metal ions in the medium.     

Metabolic Changes in Developing Rice Seeds

Metabolic changes in developing rice seeds were studied with respect to respiration, carbohydrate and nitrogen fractions, nucleic acids and hydrolytic enzymes, viz. α-amylase, adenosine triphosphatase (ATPase) and phytase. — Respiration rate was maximum after 12 days from the date of pollination and became feeble afterwards with the fall in the moisture content of the maturing seeds. In the early stage, there was a preponderance of reducing sugars which were replaced later by nonreducing forms. Dry matter accumulation was mainly due to the steady rise in starch content. There was a gradual accumulation of protein nitrogen throughout the experiment, the rate being highest between 12–16 days. RNA content increased steadily till the seeds became mature, while DNA formed rapidly during the first 20 days and was maintained at the same level thereafter. —α-Amylase activity increased up to 20 days and declined sharply afterwards. The peak activities of ATPase and phytase were recorded at 32 and 24 days after pollination, respectively.     

Pentose phosphate pathway and nucleic acid metabolism in red and white muscles of fish

The activities of the pentose phosphate cycle enzymes, the content of nucleic acids and the activities of acid and alkaline RNAses in the heart and red and white muscles of cartilaginous and teleost fish were determined. It was found that the rates of the dehydrogenase and transferase reactions of the pentose phosphate pathway and the nucleic acid metabolism in the red muscles and heart are much higher than those in the white muscles of the species under study.     

Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Effects of poly(ethylene glycol) and salt on the binding of α-amylase from the fermentation broth of Bacillus amyloliquefaciens by Cu2+-β-CD affinity adsorbent

α-Amylase from Bacillus amyloliquefaciens was purified by the immobilized metal ion affinity adsorbent, β-CD_cl-IDA-Cu²⁺. The adsorbent was prepared by reacting the cross-linked β-cyclodextrin (β-CD) with the ligand, iminodiacetic acid (IDA). The copper ion was further linked to the adsorbent. Poly(ethylene glycol) (PEG) was added to the fermentation broth to improve the adsorption efficiency of the adsorbent toward α-amylase. The effort was to provide hydrophobic interactions with the impurities which might interfere with the adsorption of α-amylase. It also provided a polymer shielding effect to prevent non-specific interactions. With the addition of PEG, the adsorption efficiency could be increased to 98%. Imidazole containing a phosphate buffer and NaCl was used to elute the bound α-amylase. By consecutive adsorption/desorption steps, up to 81% of the α-amylase activity could be recovered. Regarding the reutilization of the affinity adsorbents, α-amylase could be adsorbed and desorbed six times consecutively without a significant loss of α-amylase activity.     

Cloning of the α-Amylase cDNA of Aspergillus shirousamii and Its Expression in Saccharomyces cerevisiae

α-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3? non-coding regions, respectively, so far determined. The α-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active α-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Biochemical characterisation of digestive α-amylase of Red Palm Weevil,Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae)

The Red Palm Weevil, Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae), is a serious pest of a wide range of plant species including coconut, sago, date and oil palms. The α-amylases are the hydrolytic enzymes that are involved in carbohydrate metabolism in insects. So far nothing is done to demonstrate α-amylase activity of R. ferrugineus. Thus, the aim of the current study was to identify and characterise the α-amylase activity to gain a better understanding of digestive physiology of the insect. Thus, the α-amylase in the gut of red palm weevil was isolated and characterised using starch as a substrate. The study showed that the α-amylase is present in the gut of the insect for carbohydrate digestion. The α-amylase has an optimum pH and temperature of 5 and 40°C. The activity of α-amylase was increased by NaCl and KCl and inhibited by other compounds such as MgCl₂, CaCl₂, urea, ethylenediaminetetraacetic acid and sodium dodecylsulfate. Native-PAGE electrophoresis of α-amylase showed two isoenzymes, one major and one minor band showing α-amylase importance in the carbohydrate metabolism of the insect. Understanding of the digestive physiology and α-amylase activity of Red Palm Weevil is important when new management strategies for this economically important pest are devised.     

Hyperproduction of Proteinase and Some Hydrolytic Enzymes by Mutants of Aspergillus sojae

Mutant strains of Aspergillus sojae exhibited coordinate increases of acid proteinase, α-amylase, and cellulase and a decrease of pectin trans-eliminase accompanied with the hyperproduction of alkaline proteinase in wheat bran koji culture. The production of these enzymes in the wheat bran solid medium, liquid wheat bran-defatted soybean medium, and liquid glucose-peptone medium were surveyed. The analyses on the production patterns of these enzymes under the different cultural conditions suggest that mutation in these mutants producing elevated levels of the above enzymes is due to a more complex alteration than a single gene mutation.     

EFFECT OF PHOSPHORUS DEFICIENCY ON TWO ALGAE GROWING IN CHEMOSTATS1

phosphorus-limited chemostats were used to study the effect of degree of phosphorus deficiency on several aspects of the composition and metabolism of Anabaena variabilis Kütz. and Scenedesmus quadricauda (Turp.) Bréb. The changes as a function of the dilution rate could be placed into 3 patterns. Most aspects of the composition showed it progressive change with dilution rate. The carbohydrate content generally increased while cellular P and nitrogen, protein, nucleic acid and chlorophyll contents generally decreased over the entire range of increasing deficiency studied. The changes in metabolism fell into a second pattern, showing great sensitivity to the onset of P deficiency. The ability to take up phosphate and the alkaline phosphatase activity increased most markedly with increasing deficiency at the higher dilution rates. The third pattern was confined to the, lipid content of S. quadricauda, which increased with deficiency only at the lowest dilution rates.     

Overexpression of a wheat α-amylase type 2 impact on starch metabolism and abscisic acid sensitivity during grain germination

Despite being of vital importance for seed establishment and grain quality, starch degradation remains poorly understood in organs such as cereal or legume seeds. In cereals, starch degradation requires the synergetic action of different isoforms of α-amylases. Ubiquitous overexpression of TaAmy2 resulted in a 2.0–437.6-fold increase of total α-amylase activity in developing leaf and harvested grains. These increases led to dramatic alterations of starch visco-properties and augmentation of soluble carbohydrate levels (mainly sucrose and α-gluco-oligosaccharide) in grain. Interestingly, the overexpression of TaAMY2 led to an absence of dormancy in ripened grain due to abscisic acid (ABA) insensitivity. Using an allosteric α-amylase inhibitor (acarbose), we demonstrated that ABA insensitivity was due to the increased soluble carbohydrate generated by the α-amylase excess. Independent from the TaAMY2 overexpression, inhibition of α-amylase during germination led to the accumulation of soluble α-gluco-oligosaccharides without affecting the first stage of germination. These findings support the hypotheses that (i) endosperm sugar may overcome ABA signalling and promote sprouting, and (ii) α-amylase may not be required for the initial stage of grain germination, an observation that questions the function of the amylolytic enzyme in the starch degradation process during germination.     

Differential rates of α-amylase and acid phosphatase induction by Ga3 in embryoless half-seeds and isolated aleurones of wheat

The induction of α-amylase and acid phosphatase by gibberellic acid (GA₃) was significantly higher (2–4-fold) in embryoless half-seeds of wheat than that observed in the excised aleurones. Addition of endosperm extract to excised aleurones enhanced the stimulatory effect of GA₃ on amylase activity by approximately 2-fold. Substitution of endosperm extract by 19 amino acids in GA₃-treated aleurones also brought about a 2–2.5-fold stimulation of α-amylase activity. Subsequent studies revealed that the addition of seven non-polar amino acids (0.5 mM each) was sufficient for the enhanced induction of α-amylase (1.8–2.5-fold) in GA₃-treated aleurones. A similatory effect of endosperm extract and amino acids on acid phosphatase activity was observed in GA₃-treated wheat aleurones. These observations are of physiological significance since an increased pool of free amino acids (5-fold) was also witnessed in the incubation medium of GA₃-treated half-seeds in comparison to the hormone-treated aleurones. The relative abundance of free amino acids in half-seed seems vital for the maximal induction of α-amylase and acid phosphatase. Thus, the presence of endosperm tissue associated with the aleurone layers is crucial for enhanced rate of production of GA₃-induced α-amylase and acid phosphatase in the wheat system.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Medium pH, carbon and nitrogen concentrations modulate the phosphate solubilization efficiency of Penicillium purpurogenum through organic acid production

Aims: To study phosphate solubilization in Penicillium purpurogenum as function of medium pH, and carbon and nitrogen concentrations. Methods and Results: Tricalcium phosphate (CP) solubilization efficiency of P. purpurogenum was evaluated at acid or alkaline pH using different C and N sources. Glucose‐ and (NH₄)₂SO₄‐based media showed the highest P solubilization values followed by fructose. P. purpurogenum solubilizing ability was higher in cultures grown at pH 6·5 than cultures at pH 8·5. Organic acids were detected in both alkaline and neutral media, but the relative percentages of each organic acid differed. Highest P release coincided with the highest organic acids production peak, especially gluconic acid. When P. purpurogenum grew in alkaline media, the nature and concentration of organic acids changed at different N and C concentrations. A factorial categorical experimental design showed that the highest P‐solubilizing activity, coinciding with the highest organic acid production, corresponded to the highest C concentration and lowest N concentration. Conclusions: The results described in the present study show that medium pH and carbon and nitrogen concentrations modulate the P solubilization efficiency of P. purpurogenum through the production of organic acids and particularly that of gluconic acid. In the P solubilization optimization studies, glucose and (NH₄)₂SO₄ as C and N sources allowed a higher solubilization efficiency at high pH. Significance and Impact of the Study: This organism is a potentially proficient soil inoculant, especially in P‐poor alkaline soils where other P solubilizers fail to release soluble P. Further work is necessary to elucidate whether these results can be extrapolated to natural soil ecosystems, where different pH values are present. Penicillium purpurogenum could be used to develop a bioprocess for the manufacture of phosphatic fertilizer with phosphate calcium minerals.     

Immobilization of enzymes by radiopolymerization of acrylamide

A new and simple method for immobilization of enzymes by the aerobic radio-polymerization of acrylamide was developed. Irradiation treatment of acrylamide in the frozen state produces a spongy immobilized enzyme membrane without the addition of carriers. Aerobic polymerization yields of acrylamide in the frozen state were increased by the addition of starch and also by lyophilization. Glucose oxidase (activity recovery was 12.3–33.7%), invertase (69.2%), D -amono acid oxidase (25.0–70.5%), aminoacylase (39.2–43.7%), mold α-amylase (18.0%), malt β-amylase (4.1%), glucoamylase (6.5%), alkaline protease (5.3%), and neutral protease (10.5%) were immobilized by this method. Invertase entrapped by this method had a wider optium pH range and was active at higher temperatures.     

Ultrastructure of acid- and enzyme-modified cross-linked potato starch

Purified potato starch, cross-linked with epichlorohydrin (65–5.4 anhydroglucose units per cross-link) by an iterative method that preserved the native crystalline structure of the starch granule, was exposed to enzymic (B. subtilis α-amylase at 50°C) and acidic (3 N HCl at 40°C) hydrolysis. The stability of the granules to digestion by enzymes, as measured by their weight loss after incubation with the enzyme solution, increased with the degree of cross-linking; by contrast, the stability to acid attack decreased as a function of the degree of cross-linking. The morphology of these samples was studied by scanning electron microscopy. A lamellar structure, which was most evident after enzyme treatment, was observed in samples that had a weight loss of greater than 50%. The results reflect differences in accessibility, which are interpreted in terms of a statistical model of the ultrastructure with correlated fluctuations in density in the radial direction.     

The Amino Acid Content in Gamma-irradiated Rice†

High phosphate accumulating bacteria were isolated by autoradiography. One isoate, Arthrobacter globiformis PAB-6 accumulated phosphate intracellularly at 20% of dry cell mass in a simple synthetic medium. This amount was 3~7 times higher than type cultures examined. Almost no phosphate was released into the medium after cessation of growth. Fifty percent of total intracellular phosphate was fractionated as nucleic acids, while 20% each was recovered from cold PCA soluble fractions and polyphosphate fractions. The large content of nucleic acids in this bacterium appeared due to increased RNA content, specifically 4 S RNA fraction.