Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Hydrolysis and transformation of cellulose with Aspergillus niger IBT-90 enzymes

Hydrolysis and transformation of Fibrenier cellulose (USA) with enzymes from Aspergillus niger IBT-90 was studied. The process was performed at 50°C and pH 4.8 for 24 h using an enzyme complex either as a properly diluted culture filtrate or as a mixture of isolated and purified enzymes from A.niger IBT-90. In the latter experiments, enzyme-substrate ratios expressed as units of activity per 1 g of cellulose were as follows: endoglucanase E₁ and E₂, 40; β-glucosidase, 40 and cellobio-hydrolase, 2. Cellulose concentration was 5%. It was proved that the crude celluloytic complex from A. niger IBT-90 exhibits higher efficiency in the decomposition of cellulose in comparison to the mixture of enzymes isolated from this complex, as was revealed in assays of reducing sugars and determinations of light transmission throughout cellulose fibres using a computer analysis of the microscopic image. Comparison of both the endoglucanases E₁ and E₂ showed that the first enzyme is more active against cellulose. It liberated more reducing sugars and caused more significant decomposition of fibres. The predominant effect of the endoglucanase E₂ was a smoothing of the fibre surface. The cellobiohydrolase split a cellulose fibre into many short fibres.     

The oxidation state of active site thiols determines activity of saccharopine dehydrogenase at low pH

Saccharopine dehydrogenase catalyzes the NAD-dependent conversion of saccharopine to generate l-lysine and α-ketoglutarate. A disulfide bond between cysteine 205 and cysteine 249, in the vicinity of the dinucleotide-binding site, is observed in structures of the apoenzyme, while a dithiol is observed in a structure with AMP bound, suggesting preferential binding of the dinucleotide to reduced enzyme. Mutation of C205 to S gave increased values of V/E_t and V/KE_t at pH 7 compared to wild type. Primary deuterium and solvent deuterium kinetic isotope effects suggest the catalytic pathway, which includes the hydride transfer and hydrolysis steps, contributes more to rate limitation in C205S, but the rates of the two steps relative to one another remain the same. There is a large increase in the rate constants V₁/E_t and V₁/K_NADEt at pH values below 7 compared to WT. Data indicate the low pH increase in activity results from a decreased sensitivity of the C205S mutant enzyme to the protonation state of an enzyme group with a pK_a of about 7, likely responsible for a pH-dependent conformational change. Reduction of WT and C205S mutant enzymes with TCEP gives equal activities at pH 6, consistent with the increased activity observed for the C205S mutant enzyme.     

Micronization of lysozyme by supercritical assisted atomization

Supercritical Assisted Atomization (SAA) has been used to produce lysozyme microparticles. Lysozyme has been micronized using water, buffered water at pH 6.2 and water–ethanol mixtures at different volume percentages. Precipitated lysozyme particles were spherical, with a narrow particle size distribution (PSD) ranging from 0.1 to 4 µm. The concentration of lysozyme in the liquid solvent mixture had a nonlinear effect on the particle distribution, with an increase of the X_0.9 from about 1 to 3 µm varying the enzyme concentration from 5 to 20 mg/mL. Precipitation temperature was set as low as possible to avoid enzyme degradation. High‐performance liquid chromatography analysis showed no degradation of lysozyme and the enzyme activity, measured by turbidimetric enzymatic assay, only slightly decreased after SAA processing. Depending on the process conditions lysozyme retained from 95% to 100% of the biological activity compared to the untreated enzyme. Biotechnol. Bioeng. 2009; 104: 1162–1170. © 2009 Wiley Periodicals, Inc.     

Extracellular Isoamylase Produced by the Yeast Lipomyces kononenkoae

A strain of the starch-converting yeast Lipomyces kononenkoae produced, when grown on starch, a debranching enzyme that proved to be an isoamylase (glycogen 6-glucanohydrolase; E.C. 3.2.1.68). So far, only bacteria have been found to produce extracellular isoamylases. The yeast isoamylase enhanced β-amylolysis of amylopectin and glycogen and completely hydrolyzed these substrates into maltose when combined with a β-amylase but had no action on dextran or pullulan. By isopropanol precipitation and carboxymethyl cellulose chromatography, L. kononenkoae isoamylase was partially purified from the supernatant of cultures grown on a mineral medium with soluble starch. Optimum temperature and pH for activity of the isoamylase were 30°C and 5.6. The molecular weight was around 65,000, and the pI was at pH 4.7 to 4.8. The K_m (30°C, pH 5.5) for soluble starch was 9 g liter⁻¹.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

Mechanism of the aromatic aminotransferase encoded by the Aro8 gene from Saccharomyces cerevisiae

The amino acid l-lysine is synthesized in Saccharomyces cerevisiae via the α-aminoadipate pathway. An as yet unidentified PLP-containing aminotransferase is thought to catalyze the formation of α-aminoadipate from α-ketoadipate in the l-lysine biosynthetic pathway that could be the yeast Aro8 gene product. A screen of several different amino acids and keto-acids showed that the enzyme uses l-tyrosine, l-phenylalanine, α-ketoadipate, and l-α-aminoadipate as substrates. The UV–visible spectrum of the aminotransferase exhibits maxima at 280 and 343 nm at pH 7.5. As the pH is decreased the peak at 343 nm (the unprotonated internal aldimine) disappears and two new peaks at 328 and 400 nm are observed representing the enolimine and ketoenamine tautomers of the protonated aldimine, respectively. Addition, at pH 7.1, of α-ketoadipate to free enzyme leads to disappearance of the absorbance at 343 nm and appearance of peaks at 328 and 424 nm. The V/E_t and V/K_{α-ketoadipate}E_t pH profiles are pH independent from pH 6.5 to 9.6, while the V/K_l-tyrosine pH-rate profile decreases below a single pK_a of 7.0 ± 0.1. Data suggest the active enzyme form is with the internal aldimine unprotonated. We conclude the enzyme should be categorized as a α-aminoadipate aminotransferase.     

Bacterial dextranase immobilised on zirconia coated alkylamine glass using glutaraldehyde

Bacterial dextranase has been immobilised on zirconia coated alkylamine glass through the process of glutaraldehyde coupling. The immobilised enzyme preparation exhibited 62% of the initial enzyme activity with a conjugation yield of 18 mg/g support. K_m of the immobilised enzyme exhibited a decline in its value as compared to the soluble enzyme while V_max remained unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme had its optimal pH at 5.4 while the alkylamine conjugated dextranase was optimally active in the pH range 5.2–6.2. The immobilised enzyme has also been characterised through its pI by a new method. The industrial importance of this work is discussed.     

Pyruvate Kinase Activity in Crude Extracts of Leaves of Cynodon dactylonand Other C4Plants

A new extraction procedure and an LDH-coupled assay method are presented for the study of pyruvate kinase (PK) in leaf crude extracts from Cynodon dactylon(L.) Pers and other C₄plants. Extraction at pH 6.8 and assay at pH 6.2 facilitated the measuring of PK activity by eliminating phosphoenolpyruvate carboxylase interference more effectively than the thermal inactivation or chemical inhibition previously used. The method suggested did not affect the kinetic properties of PK as compared to the purified enzyme from C. dactylon.     

Purification and Characterization of a Thermo- and Organic Solvent-Tolerant Alkaline Protease from Bacillus sp. JER02

Bacillus sp. JER02 is a bacterial strain that can be grown in a medium containing organic solvents and produce a protease enzyme. JER02 protease was purified with a yield of 31.9% of total protein and 328.83-fold purification. K _m and V_max of this protease were established as 0.826 µM and 7.18 µmol/min, respectively. JER02 protease stability was stimulated about 80% by cyclohexane. It exhibited optimum temperature activity at 70°C. Furthermore, this enzyme was active in a wide range of pH (4-12) and showed maximum activity at pH 9.0. The nonionic detergents Tween-20 and Triton X-100 improved the protease activity by 30 and 20%, respectively. In addition, this enzyme was shown to be very stable in the presence of strong anionic surfactants and oxidizing agents, since it retained 77%, 93%, and 98% of its initial activity, after 1 hr of incubation at room temperature with sodium dodecyl sulfate (SDS), sodium perborate (1%, v/v) and H₂O₂ (1%, v/v), respectively. Overall, the unique properties of the Bacillus sp. JER02 protease suggested that this thermo- and detergent-stable, solvent-tolerant protease has great potential for industrial applications.     

Immobilization of Bacillus licheniformis 5A1 milk-clotting enzyme and characterization of its enzyme properties

Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated 5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting enzyme. The activation energy (E _A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E _A = 1.59 and 1.99 Kcal mol⁻¹ respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0 for both free and immobilized enzyme. The Michaelis constant K _m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Limited Hydrolysis of Ricin D with Alkaline Protease from Bacillus subtilis

Streptomyces naraensis was inoculated into 100 ml of culture broth, containing 50 µCi of ⁶⁵Zn, diluted with ZnCl₂ solution to make 10^-4 m Zn²⁺ ion, at 27°C for 5 days with shaking. ⁶⁵Zn-labeled neutral proteinase from Streptomyces naraensis was prepared by the method described previously. The preparation was homogeneous by disc electrophoresis and contained 1 g-atom of zinc per mole of enzyme in calculation by radioactivity.

It was suggested that the protein-bound zinc of neutral proteinase was not essential for enzymatic activity. Thus, this zinc was an essential component for the higher order structure of the protein, and the removal of zinc treated with EDTA* inactivated the enzyme. The enzymatic activity was maintained in the presence of calcium ion.     

Production of cellulases by Thermomucor indicae-seudaticae: characterization of a thermophilic β-glucosidase

Abstract

The current study evaluated the production and characterization of β-glucosidase by the thermophilic fungus Thermomucor indicae-seudaticae in solid-state fermentation of wheat bran. Isolated fungi have significant amounts of β-glucosidase, an enzyme that may be applied to different industrial processes, such as the production of fuels, food, and other chemical compounds. Maximal enzyme activity occurred in pH 3.5–4.5 and at 70?°C. The enzyme exhibited high thermostability, for 1?h, up to 60?°C, and good tolerance to glucose (10?mM) and ethanol (10%). The optimization of fermentative parameters on the production of β-glucosidase was carried out by evaluating the best supplementary nutrient source, pH of nutrient solution, initial substrate moisture and fermentation temperature. The optimization of the above fermentation parameters increased enzyme activity by 120.0%. The highest enzymatic activity (164.0?U/g) occurred with wheat bran containing 70% initial moisture, supplemented with 1.0% (NH₄)₂SO₄ solution at pH 5.5–6.0 and fungus incubated at 40?°C. A more detailed study of β-glucosidase suggested that Sulfur is an important component of the main amino acid present in this enzyme. The enhancer of the enzyme activity occurred when the fungus was grown on wheat bran supplemented with a sulfur-containing solution. In fact, increasing the concentration of sulfur in the solution increased its activity.     

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

A truncated mutant α-amylase, Xa-S2, was obtained from Xanthomonas campestris wild type α-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 (β/α)₈-barrel catalytic domain and did not have the three conserved catalytic residues of wild type α-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°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°C. Our results suggested that Xa-S2 is a new amylase with a minimal catalytic domain for hydrolyzing substrates with of α-1,4-glucosidic bonds. T. Ke and X. D. Ma contributed equally to this work     

Production,purification and characterisation of a novel halostable xylanase from Bacillus sp. NTU-06

Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0–20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had K_m and V_max values of 3.45 mg mL⁻¹ and 387.3 µmol min⁻¹mg⁻¹, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed.     

Isolation of Allo-isoleucic Acid (α-Hydroxy-β-methylvaleric Acid) and β-Hydroxy-β-methylvaleric Acid from Turkish Tobacco Leaves

A new method determining the activity of tannin acyl hydrolase (tannase) was made. This method was based on the change in optical density of substrate tannic acid at 310 mμ. In this method, the error of measurement was about 1~3%, and many samples could be tested at one time because of its simplicity.

The procedure was as follows; To four parts of substrate (0.350 w/v% of tannic acid dissolved in 0.05m citrate buffer, pH 5.5), one part of the enzyme solution was added.

After t minutes reaction at 30°C, 0.1 part of the mixture was added to ten parts of 90% ethanol.

The optical density of the ethanol solution at 310 mμ was measured. Tannase activity (unit/ml) was given by following equation. $u=114\times \frac{E_{t1}?E_{t2}}{t_2?t_1}$

Where E_t1 and E_t2 mean the optical density of the ethanol solution at 310 mμ prepared after t₁ and t₂ minutes reaction, and one unit of the enzyme means the amount of the enzyme which is able to hydrolyze one μ mole of the ester bond in tannic acid in one minute.

The substrate tannic acid used in this determining method was purified. It was composed of one mole of glucose and nine moles of gallic acid, and eight moles of which formed four moles of m-digallic acid.     

Serratia Protease

A strain of Serratia, isolated from an intestinal canal of a silkworm, produced a large quantity of protease. The enzyme was extracellular and was named Serratiopeptidase, tentatively. Protease production of this strain was over 3 times as much as that of Serratia marcescens which was known as a protease-producing organism. The highly purified enzyme was prepared from the culture supernatant through ammonium sulfate precipitation, acetone fractionation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-75.

The purified enzyme moved homogeneously with a sedimentation constant, s_20,w of 3.8 S in ultracentrifugation and the molecular weight was determined to be 6.0 × 10₄ by the Archibald method. Determination of the ultraviolet absorption spectrum indicated the E^1%_{280 mμ,1 cm} was 13.0. Neither carbohydrate nor sulfur-containing amino acid was detected in the purified enzyme preparation. The enzyme showed maximal activity at pH 9.0 and at 40°C, and was stable under lower temperatures over the pH range from 5 to 10, whereas it was unstable at 37°C in alkaline conditions. The enzyme was completely inactivated by heating at 55°C for 15 min.     

Recombinant pp60 c-src from Baculovirus-Infected Insect Cells: Purification and Characterization

A simple and effective method has been developed to purify the recombinant protein tyrosine kinase pp60^c-^src from a baculovirus-insect cell expression system. The procedure includes affinity chromatography and HPLC. Milligram quantities of protein have been isolated with an activity of 3.9 μmol/min/mg protein using the substrate poly E₄Y. This specific activity is many times higher than any published protocol. The enzyme is stable for months when stored in buffered 10% glycerol at ?70°C. This purification technique is compared to the immuno-affinity technique which is widely used for this enzyme. Enzyme kinetics were characterized with respect to substrate specificity, the effect of temperature, ionic strength, pH, and Mg⁺² versus Mn⁺² ions. Similar to the enzyme expressed in human cells, the recombinant enzyme demonstrated a higher Vmax and substrate specificity for poly E₄Y over 5V-Agt-II. An activation energy of 14.2 kcal/mol was determined. Inhibition by increasing ionic strength is mostly due to an increase in Km for the poly E₄Y substrate and hence was substrate dependent. The Km(ATP) was pH dependent while the Km(poly E₄Y) was pH independent.