Separation and purification of useful proteins using hydrogel ultrafiltration

The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a data base obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53–65% separation efficiency and 78–83% enzyme recovery. Wild and recombinantAutographa californica nuclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.     

Dubiumin, a chymotrypsin-like serine protease from the seeds of Solanum dubium Fresen

A serine protease was purified 6.7-fold and with 35% recovery from the seeds Solanum dubium Fresen by a simple purification procedure that combined ammonium sulfate fractionation, cation exchange and gel filtration chromatographies. The enzyme, named dubiumin, has a molecular mass of 66 kDa as estimated by gel filtration and SDS-PAGE. Carbohydrate staining established the existence of a carbohydrate moiety attached to the enzyme. Inhibition of enzyme activity by serine protease inhibitors such as PMSF and chymostatin indicated that the enzyme belongs to the chymotrypsin-like serine protease class. Dubiumin is a basic protein with pI value of 9.3, acts optimally at pH 11.0, and is stable over a wide range of pH (3.0-12.0). The enzyme is also thermostable retaining complete activity at 60 °C after 1 h and acts optimally at 70 °C for 30 min. Furthermore, it is highly stable in the presence of various denaturants (2.0% SDS, 7.0 M urea and 3.0 M guanidine hydrochloride) and organic solvents [CH₃CN-H₂O (1:1, v/v) and MeOH-H₂O (1:1, v/v)] when incubated for 1 h. The enzyme showed a high resistance to autodigestion even at low concentrations.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Properties of laccase purified from nitrogen limited culture of white-rot fungus Coriolus hirsutus

Laccase produced by nitrogen-limited culture of Coriolus hirsutus was purified to electrophoretic homogeneity (133-fold) with an overall yield of 40%. The molecular mass of the enzyme was determined as 82 kDa by SDS-PAGE and 80 kDa using gel filtration. It had a pI of 3.50. With ferulic acid and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as the substrate, the enzyme had optimal activity at pH 4.0 and 2.5, respectively. The enzyme was stable in the range pH 5.5 to 7.0 at 30 °C for 1 h. The enzyme was optimally active at 70 °C and it lost all activity within 15 min at 80 °C. The apparent Km value of enzyme toward ABTS was 67 °M and had highest affinity toward sinapinic acid. The enzyme was totally inhibited by 0.01 mM cysteine.     

Immobilization of endo-polygalacturonase from Aspergillus ustus on silica gel

Endo-polygalacturonase from Aspergillus ustus when immobilized on to modified silica gel retained 28% of its original activity. The immobilized enzyme could be re-used through 10 cycles of reaction with almost 90% retention of its original activity. It had increased thermostability over its soluble form: the half-life of the soluble enzyme at 40 °C was less than 10 h whereas the immobilized enzyme retained 82% of its activity after 10 h at 40 °C. Similarly, at 50 °C the half-life of the soluble enzyme was 30 min whereas that of the immobilized enzyme was 5 h.     

Spray drying of the dehalogenating bacterium Rhodococcus sp.

The bacteria Rhodococcus sp. and Xanthobacter autotrophicus have the ability to dehalogenate a broad range of halogenated hydrocarbons. The applicability of spray drying to the preservation of the microorganisms and the intracellular enzyme halidohydrolase (E.C.3.8.1.1) was examined. K₂SO₄, MgSO₄, glutamate and sucrose were added as stabilizers and carriers. Spray drying was carried out at inlet air temperatures of 100–120 °C and outlet air temperatures of 65–72 °C. Best results were obtained by the addition of 5% K₂SO₄ and at 107 °C air inlet temperature. Dried preparations of Rhodococcus sp. exhibited a crystalline consistency and a 95% recovery of cellular activity. After storage at 4 °C for six months the enzyme preparation showed no loss in activity. Spray dried preparations of Xanthobacter autotrophicus showed only a 4% recovery of cellular activity.List of Symbols MSG Monosodiumglutamate - RC % Recovery of stabilizer and biomass - RCA % Recovery of cellular activity ([U/g biomass after the spraydrying]/[U/g biomass of untreated cells]) 100 - RCB % Recovery of biomass - SR % Survival rate - T ₁ °C Inlet air temperature - T ₂ °C Exit air temperature - W % Water content - Y.Akt % Yield in enzyme activity This work was supported by the Jubiläumsfond der Oesterreichischen Nationalbank, Projekt No. 4499.     

Pullulan 4-glucanohydrolase from Aspergillus niger

Pullulan 4-glucanohydrolase, a novel pullulan-hydrolyzing enzyme from Aspergillus niger, was highly purified by means of acetone precipitation, chromatography on P-cellulose and DEAE-cellulose, and gel filtration on Sephadex G-150. More than 430-fold purification was achieved through these procedures from crude extract of wheat bran culture. The enzyme can liberate a large amount of isopanose and a small amount of tetrasaccharide from pullulan. The optimum pH of the enzyme action on pullulan was 3.0–3.5 and the optimum temperature was 40 °C at pH 3.5. The enzyme activity remained intact after heating at 50 °C for 30 min at pH 3.7–4.5. The enzyme was stable at pH 2.0–8.0 on storage at 5 °C for 24 hr. The purified enzyme attacked reducing end α-1,4-glucosidic linkages adjacent to α-1,6-glucosidic linkages in pullulan, 6³-α-glucosylmaltotriose, 6²-α-maltosylmaltose and panose, to liberate isopanose, isomaltose and maltose, isopanose and glucose, and isomaltose and glucose, respectively. The molecular weight of the enzyme determined by gel filtration on Bio-Gel P-150 was about 74,000.     

Thermostable liquefying α-amylase fromBacillus amyloliquefaciens

Summary An extracellular -amylase is purified to homogeneity with 62 % recovery of the enzyme activity using heat treatment, ion-exchange and gel filtration chromatographies. The purified enzyme has a molecular weight of 68, 000, isoelectric point 6.25, optimal activity at pH 6 and temperature 65 °C, k_est 8.8×10⁸ s¹ liquefying amylase units, and K_m for starch 2.9 mg ml^–1. The enzyme is further characterized for its endo action on the starch and related polymers. Calcium stabilizes the active conformation of the enzyme during prolonged exposure to the extremes of pH and temperature. The enzyme retains 100 % activity for 24 h at 65°C and exhibits a half-life of 9, 3, and 0.5 h at 80°, 85° and 90°C, respectively.     

A thermostable manganese-containing superoxide dismutase from the thermophilic fungus Thermomyces lanuginosus

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN₃, but not by KCN or H₂O₂, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55°C. It was thermostable at 50 and 60°C and retained 55% activity after 60 min at 70°C. The half-life of the SOD at 80°C was approximately 28 min and even retained 20% activity after 20 min at 90°C.     

Isolation,partial purification,and some properties of protease I from a thermophilic mold Thermoascus aurantiacus var. levisporus

When the thermophilic mold Thermoascus aurantiacus var. levisporus was grown in a modified Czapek Dox medium containing casein the filtrate was found to contain proteolytic activity. The maximum production of activity occurred at 50 ° C in a medium containing 8% casein. The filtrate was subjected to ammonium sulfate fractionation and chromatography on DEAE-cellulose. Two proteases were separated. No further work was done on protease II. Protease I was further purified by gel filtration on Sephadex G 100–200. It showed a 40-fold purification with a final recovery of approximately 25%. It is a neutral protease with a pH optimum at 7.0. The optimal activity of the enzyme occurred in 0.02 M phosphate buffer but was completely inhibited at a concentration of 0.1 M. The optimum temperature for casein hydrolysis was found to be 55 ° C. The enzyme was inhibited by Hg⁺⁺ but was greatly stimulated by Cu⁺⁺ and mercaptoethanol. Metallo and sulfhydryl agents had no significant effect on enzyme activity.     

Production and properties of fibrinolytic enzyme in solid state cultures of Fusarium pallidoroseum

Summary Ten local fungal isolates were screened for their ability to produce extracellular fibrinolytic enzyme activity in solid state and skaken cultures. Fusarium pallidoroseum was the most active in wheat bran solid cultures. Maximum activity was obtained at 25 °C and 50% moisture content. Addition of 2% casein to the culture increased the activity by 1.7-folds. The 65% ammonium sulphate fraction showed the highest fibrinolytic activity it was further purified by gel filtration on Sephadex G-100 followed by rechromatography of the most active peak on DEAE-cellulose. The pure enzyme was highly active on human fibrin and showed an optimum reaction temperature of 40 °C and pH 7. The enzyme was relatively sensitive to heat treatment at 55 °C and strongly inhibited by EDTA, it restored its activity by adding cobalt ions to the reaction.     

Extracellular glucose-producing exodextranase of the yeast Lipomyces lipofer

A dextran-hydrolysing enzyme from Lipomyces lipofer IGC 4042 was purified from the supernatant of cultures grown on a mineral medium with dextran, by ultrafiltration and gel filtration on Bio Gel A-0.5 m. This preparation gave only one band by disc gel electrophoresis. Glucose was the only product of dextran hydrolysis. Optimum pH and temperature for the activity of the enzyme were pH 4.5–5.0 and 45°C, respectively. The enzyme was most stable over a pH range of 4.5–6.0, and after 2 hours at 50°C maintained over 60% of its original activity. The molecular weight was 29,000 daltons and the isoelectric point was at pH 7. K_m (45°C, pH 5) for dextran T-40 was 1.2×10^–5 M. Glucose inhibited the enzyme competitively with a K_i (45°C, pH 5) of 0.5 mM.     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

A kinetic evaluation of anaerobic treatment of swine wastewater at two temperatures in a temperate climate zone

A static granular bed reactor (SGBR) was used to treat swine wastewater at 24 and 16 °C. At 24 °C, the organic loading rate (OLR) was 0.7-5.4 kg COD/m³ day and the average chemical oxygen demand (COD) removal efficiency was 88.5%, respectively. Meanwhile, at 16 °C, the OLR was 1.6-4.0 kg COD/m³ day and the average COD removal efficiency was 68.0%, respectively. The SGBR acted as a bioreactor as well as a biofilter. After backwashing, the recovery of COD removal was not a function of an OLR but recovery time, while that of TSS removal was not a function of either recovery time or the OLR. The maximum substrate utilization rate (k_max) ratio was 1.89 between 24 and 16 °C, and the half velocity constant (K_s) ratio was 1.22, and the maximum specific growth rate (μ_max) ratio was 4.71. In addition, the temperature-activity coefficient in this study was determined to be 1.09.     

Purification and characterization of a heat-stable alkaline protease from Bacillus stearothermophilus F1

A thermophilic Bacillus stearothermophilus F1 that produced an extremely thermostable alkaline protease was isolated from decomposed oil palm branches. The isolated protease was purified to homogeneity by heat treatment, ultrafiltration and gel filtration chromatography with a 128-fold increase in specific activity and 75% recovery. The protease, which is a serine-type enzyme, has a relative molecular mass of 33 500 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis but only 20 000 by gel-filtration chromatography. The enzyme was optimally active at pH 9.0 and was stable for 24 h at 70° C and in the pH range from 8.0 to 10.0. It was capable of hydrolysing many soluble and insoluble protein substrates but no esterase activity was detected. The enzyme activity was markedly inhibited by Co²⁺ and Hg²⁺, whereas Mg²⁺, Fe²⁺, Cu²⁺, Zn²⁺ and Sr²⁺ had little or no inhibitory effect. However, Mn²⁺ strongly activated the protease activity. The protease exhibited a high degree of thermostability [t _1/2 (85° C) = 4 h, (90° C) = 25 min]. The stability at higher temperatures (85° C and above) was shown to be dependent on the presence of Ca²⁺. Correspondence to: A. B. Salleh     

Purification and partial characterisation of a broad-range L-amino acid oxidase from Bacillus carotarum 2Pfa isolated from soil

The L-amino acid oxidase (L-aao) from Bacillus carotarum 2Pfa was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from crude sonicated cell extract by a combination of anion exchange chromatography and gel filtration. The purified enzyme was a dimer with a native relative molecular mass of approximately 102,000 to 115,000 and comprised two identical subunits of 54,000. The isoelectric point of the L-aao was at pH 4.8 the ph optimum was at 8.0–8.5 and the temperature optimum was at approximately 50° C. It was stable for several months at + 4° C and at –20° C. The enzyme contained 2 mol flavin adenine dinucleotide (FAD)/mol enzyme and exhibited relatively broad range substrate specificity, oxidising a total of ten L-amino acids and , albeit to a much lesser extent, seven D-amino acids. Kinetic studies revealed that the three aromatic L-amino acids were the preferred substrates.     

Biochemical characterization of a 27 kDa 1,3-β-d-glucanase from Trichoderma asperellum induced by cell wall of Rhizoctonia solani

Trichoderma asperellum produces two extracellular 1,3-β-d-glucanase upon induction with cell walls from Rhizoctonia solani. A minor 1,3-β-d-glucanase was purified to homogeneity by ion exchange chromatography on Q-Sepharose and gel filtration on Sephacryl S-100. A typical procedure provided 13.8-fold purification with 70% yield. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 27 kDa. The enzyme exhibited optimum catalytic activity at pH 3.6 and 45 °C. It was thermostable at 40 °C, and retained 75% activity after 60 min at 45 °C. The K_m and V_max values for 1,3-β-d-glucanase, using laminarin as substrate, were 0.323 mg ml⁻¹ and 0.315 U min⁻¹, respectively. The enzyme was strongly inhibited by Hg²⁺ and SDS. The enzyme was only active toward glucans containing β-1,3-linkages. Peptide sequences showed similarity with two endo-1,3(4)-β-d-glucanases from Aspergillus fumigatus Af293when compared against GenBank non-redundant database.     

A thermostable alkaline β-1,3-glucanase produced by alkalophilic Bacillus sp. AG-430

Summary Alkalophilic Bacillus sp. AG-430 was isolated from soil and found to produce an extracellular -1,3-glucanase. This enzyme was purified by chromatography on DEAE-sepharose CL-6B, Sephadex G-75 and hydroxyapatite. The enzyme was extremely thermostable and lost only 10% of the original activity after incubation at 100°C for 10 min. The optimum temperature and pH for activity were 60°–65°C and 9–10, respectively. The molecular weight was estimated at about 35 000 on sodium dodecyl sulphate-polyacrylamide gel eletrophoresis and the pI was about 3.8. The enzyme hydrolysed laminaritetraose, but not laminaritriose, and the end-products detected in the hydrolysate were identified as glucose, laminaribiose and laminaritriose. The enzyme split laminarin at random and yielded glucose, laminaribiose, laminaritriose and higher oligosaccharides. The enzyme is a type of endo--1,3-glucanase.Offprint requests to: Y. Nogi     

Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells

The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography. The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0–6.5. The enzyme was stable against heat treatments between 4 and 50°C and works optimally at 52°C. The activity remained constant at 4°C for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis