Production of saccharifying enzyme using the wastewater of a Schochu distillery

A saccharifying enzyme was produced by Aspergillus awamori var. kawachi using wastewater generated by a Shochu distillery. The production of the saccharifying enzyme was of the non-growth associated type, and 80 U of activity per ml of broth was obtained in about 6 d of flask cultivation. Since the Shochu distillery wastewater contained high concentrations of volatile fatty acids that were converted to their free forms and severely inhibited cell growth at low pH, the optimum initial pH ranged from 4.5 to 6.0. It is suggested that cell autolysis facilitated the release of the saccharifying enzyme, but a released protease digested the saccharifying enzyme with a subsequent decrease in activity. The saccharifying enzyme was easy to purify, and the purified enzyme was homogeneous when analyzed by disc electrophoresis. The molecular weight was estimated to be 54,000 Da by SDS-PAGE, and the isoelectric point was found to be pH 3.6 by isoelectric focusing. The optimum temperature and pH for the reaction ranged from 50 to 55°C and 4.5 to 5.5, respectively. The saccharifying enzyme could not digest raw starch. The hydrolyzate of soluble starch hydrolyzed by the saccharifying enzyme was composed of two to four oligosaccharides. From these results and the amino acid sequence in the N-terminal, the enzyme produced was concluded to be α-amylase.     

Production of alkaline protease in a low-cost medium by alkalophilic Bacillus sp. and properties of the enzyme

The main object of this research was to obtain a large amount of alkaline protease in a low-cost medium, for which purpose Bacillus sp. B21-2 was isolated from soil. The enzyme production by this strain reached the maximum level of 15,000 u/ml in a short cultivation time of 24–28 h in alkaline medium consisting of cheap materials such as soybean meal and bonito extract. The optimum pH and temperature for activity of the purified enzyme were 11.5 and 60°C respectively. The enzyme was stable up to 50°C but inactivated after 10 min at 60°C. The enzyme activity was completely inhibited by diisopropylfluorophosphate.     

Purification and some properties of cellulose 1,4-β-cellobiosidase from a strain of Penicillium sp.

A cellulase was purified from the culture supernatant of a strain of Penicillium sp. The purified enzyme was homogenous on polyacrylamide disc gel electrophoresis. It was a glycoprotein with a molecular weight of 52,000 estimated by gel filtration. The optimum pH was about 4.0 and the optimum temperature was 60°C. The enzyme was stable in the pH range of 3.0–10.0 at 6°C for 48 h and on heating at 60°C for 10 min. The activity of the enzyme toward Avicel was about 3 times higher than toward carboxymethyl cellulose. The enzyme showed a low activity for cotton, newspaper, filter paper and cellulose powder. The main product from Avicel was cellobiose, with a trace of glucose.     

Purification and characterization of thermostable organic solvent-stable protease from Aeromonas veronii PG01

A mesophilic bacterium, Aeromonas veronii PG01, isolated from industrial wastes produced an extracellular thermostable organic solvent tolerant protease. The optimum condition for cell growth and protease production was pH 7.0 and 30 °C. The protease produced was purified 53-fold to homogeneity with overall yield of 32%, through ammonium sulphate precipitation, ion-exchange and gel permeation chromatography (GPC). The molecular weight, as determined by GPC–HPLC, was found to be about 67 kDa. SDS-PAGE revealed that the enzyme consisted of two subunits, with molecular weight of 33 kDa. The protease was active in broad range of pH from 6.0 to 10.0 with optimum activity at pH 7.5. The optimum temperature for this protease was 60 °C. The enzyme remained active after incubation at 50–60 °C for 1 h. This enzyme was stable and active after incubation with benzene and it was activated 1.3- and 1.5-fold by n-hexane and n-dodecane, respectively. This protease was inhibited completely by the classic metalloprotease inhibitor 1,10-phenanthroline and partially by the metal chelator EDTA but not by the serine protease inhibitor PMSF. The PG01 protease was found to contain 1.901 mol of zinc per mole of enzyme upon analysis by Inductively coupled plasma-optical emission spectroscopy. The thermostable and solvent tolerance property make it an attractive and promising biocatalyst for enzyme mediated synthesis.     

Studies on Protein Metabolism in Higher Plants

A leaf protease of tobacco whose activity was enhanced during curing was purified about 60 times with ammonium sulfate fractionation, ethanol precipitation, calcium phosphate gel treatment and Sephadex G-200 column chromatography, and some properties of the protease were examined. The purified enzyme showed the optimum pH at 5.5 and the optimum temperature at 60°C. The protease activity was stable between pH 4.5 and 5.5 at 50°G or at pH 5.5 below 40°C for 1 hr, but completely destroyed at 70°C during 1 hr. The protease activity was greatly activated by reducing agents such as cysteine, glutathione or mercaptoethanol and inhibited by p-chloromercuribenzoate, phenyl- mercuric acetate or silver ions. Metal ions except for silver ion and ethylenediamine tetraacetic acid did not affect the protease activity so far examined.     

Optimization of culture conditions for the production of halothermophilic protease from halophilic bacterium <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TVSP101

An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease. Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl₂ concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease.     

Purification and Characterization of Polygalacturonase-3 from Jamaica cherry (Muntingia calabura Linn)

Endo-polygalacturonase-3 (PG-3), the key enzyme of fruit ripening was purified to near homogeneity as judged by native PAGE from the fruit tissues of Jamaica cherry (Muntingia calabura) using ammonium sulphate fractionation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the PG-3 enzyme was determined as 85 kD, by size exclusion chromatography. SDS-PAGE of PG-3 revealed two dissimilar bands of 62 and 21 kD as heterogenous subunits. The optimum pH of PG-3 was found to be 4.0. The enzyme had an optimum temperature of 40°C and was relatively stable at 50°C and 60°C. K_m for the substrate polygalacturonic acid was found to be 0.27%. The purified enzyme was a glycoprotein with 6.6 % carbohydrate content.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Thermostable alkaline lipase from a newly isolated thermophilic Bacillus,strain A30-1 (ATCC 53841)

A thermophilic bacterium was isolated from a hot spring area of Yellowstone National Park. The organism grew optimally at 60–65°C and in the pH range of 6–9. It was characterized as Bacillus sp. In the presence of corn or olive oil (1.0%) as the growth substrate, this Bacillus produced an extracellular lipolytic activity (EC 3.1.1.3). The enzyme activity could be efficiently recovered by ultrafiltration of cell-free culture supernatant. The partially purified lipase preparation had an optimum temperature of 60°C, at an optimum pH of 9.5. It retained 100% of the original activity after being heated at 75°C for half an hour. The half life of the enzyme was 8 h at 75°C. The enzyme retained at least 90% of the original activity after it was incubated at 60°C for 15 h at pH's in the range of 5 to 10.5. The enzyme was active on triglycerides containing fatty acids having a carbon chain length of C16 : 0 to C22 : 0 as well as on natural fats and oils. The enzyme activity was stable to both hydrogen peroxide and alkaline protease which are detergent ingredients. The purified enzyme had an isoelectric point of 5.15 and an approximate molecular weight of 65,000.     

Starch-degrading enzymes of two species ofFusarium

Starch supported production of maximum α-amylases (dextrinizing and saccharifying) byFusarium oxysporum andF. scirpi. Addition of gibberellic acid resulted in an increased production of α-amylase. Presence of glucose depressed the enzyme production. pH 4.5 and 4.0 was found to be optimum for the dextrinizing enzyme secreted by both species. The temperature of 25 and 40 °C was optimum for the dextrinizing enzyme secreted byF. oxysporum andF. scirpi, respectively. Saccharifying enzymes of both species showed their optimum at pH 6.9. The optimum temperature for the activity of the saccharifying enzyme was 30 and 40 °C, respectively.     

Purification and characterization of a cold active alkaline protease from <Emphasis Type="Italic">Stenotrophomonas</Emphasis> sp., isolated from Kashmir,India

A Psychrotolerant alkaline protease producing bacterium IIIM-ST045 was isolated from a soil sample collected from the Thajiwas glacier of Kashmir, India and identified as Stenotrophomonas sp. on the basis of its biochemical properties and 16S ribosomal gene sequencing. The strain could grow well within a temperature range of 4–37°C however, showed optimum growth at 15°C. The strain was found to over-produce proteases when it was grown in media containing lactose as carbon source (157.50 U mg⁻¹). The maximum specific enzyme activity (398 U mg⁻¹) was obtained using soya oil as nitrogen source, however, the inorganic nitrogen sources urea, ammonium chloride and ammonium sulphate showed the lowest production of 38.9, 62.2 and 57.9 U mg⁻¹. The enzyme was purified to 18.45 folds and the molecular weight of the partially purified protease was estimated to be ~55 kDa by SDS-PAGE analysis. The protease activity increased as the increase in enzyme concentration while as the optimum enzyme activity was found when casein (1% w/v) was used as substrate. The enzyme was highly active over a wide range of pH from 6.5 to 12.0 showing optimum activity at pH 10.0. The optimum temperature for the enzyme was 15°C. Proteolytic activity reduced gradually with higher temperatures with a decrease of 56% at 40°C. The purified enzyme was checked for the removal of protein containing tea stains using a silk cloth within a temperature range of 10–60°C. The best washing efficiency results obtained at low temperatures indicate that the enzyme may be used for cold washing purposes of delicate fabrics that otherwise are vulnerable to high temperatures.     

Accumulation of propionic acid during anaerobic treatment of distillery wastewater from barley-Shochu making

Synthetic wastewater consisting aliphatic acids contained in distillery wastewater from barley-shochu making was treated anaerobically. It was suggested that propionic acid was produced from lactic acid and citric acid via succinic acid. Since it appears to be difficult to treat anaerobically wastewater in which propionic acid is accumulated, we attempted to repress the production of propionic acid during acidification. The amount of propionic acid produced increased with an increase in the hydraulic retention time (HRT) at pH 7. Although the treatment was examined using different pHs at a shorter HRT of 10 h, it was difficult to repress the production of propionic acid.     

Purification and characterization of a prolyl endopeptidase isolated from Aspergillus oryzae

A new fungal strain that was isolated from our library was identified as an Aspergillus oryzae and noted to produce a novel proly endopeptidase. The enzyme was isolated, purified, and characterized. The molecular mass of the prolyl endopeptidase was estimated to be 60 kDa by using SDS-PAGE. Further biochemical characterization assays revealed that the enzyme attained optimal activity at pH 4.0 with acid pH stability from 3.0 to 5.0. Its optimum temperature was 30 °C and residual activity after 30 min incubation at 55 °C was higher than 80 %. The enzyme was activated and stabilized by Ca²⁺ but inhibited by EDTA (10 mM) and Cu²⁺. The K _m and k _cat values of the purified enzyme for different length substrates were also evaluated, and the results imply that the enzyme from A. oryzae possesses higher affinity for the larger substrates. Furthermore, this paper demonstrates for the first time that a prolyl endopeptidase purified from A. oryzae is able to hydrolyze intact casein.     

Identification of Protease from Euphorbia amygdaloides Latex and its Use in Cheese Production

Abstract

In this research, protease enzyme was purified and characterized from milk of Euphorbia amygdaloides. (NH₄)₂SO₄ fractionation and CM‐cellulose ion exchange chromatography methods were used for purification of the enzyme. The optimum pH value was determined to be 5, and the optimum temperature was determined to be 60°C. The V_max and K_M values at optimum pH and 25°C were calculated by means of Linewearver‐Burk graphs as 0.27 mg/L min^?1 and 16 mM, respectively. The purification degree was controlled by using SDS‐PAGE and molecular weight was found to be 26 kD. The molecular weight of the enzyme was determined as 54 kD by gel filtration chromatography. These results show that the enzyme has two subunits.

In the study, it was also researched whether purified and characterized protease can be collapsed to milk. It was determined that protease enzyme can collapse milk and it can be used to produce cheese.     

Studies on Lotus Seed Protease

A protease from the lotus seed (Nelumbo nucifera Gaertn) was purified by acid-treatment, ammonium sulfate-fractionation, ethylalcohol-fractionation, TEAE-cellulose-treatment and Sephadex G-100 gel-filtration.

The enzyme was purified about 870-fold and was homogeneous in electrophoretic and ultracentrifugal analyses.

Purified lotus seed protease is an acid protease with a pH optimum at 3.8 toward urea-denatured casein. It is active for casein and hemoglobin. But other proteins such as edestin, zein, lotus seed globulin and soybean casein are slightly hydrolyzed and egg albumin is hardly hydrolyzed. This enzyme is most stable at pH 4.0 below 40°C. The enzyme is not a thiol protease, and its activity was completely inhibited by potassium permanganate, remarkably inhibited by sodium dodecylsulfate and accelerated by hydrogen peroxide.     

Highly thermostable neutral protease from Bacillus stearothermophilus

Bacillus stearothermophilus MK232, which produced a highly thermostable neutral protease, was isolated from a natural environment. By several steps of mutagenesis, a hyper-producing mutant strain, YG185, was obtained. The enzyme productivity was twice as much as that of the original strain. This extracellular neutral protease was purified and crystallized. The molecular weight of the enzyme was 34,000 by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.5 and 70°C, respectively, and the enzyme was stable at pH 5–10 and below 70°C. The thermostability and specific activity of the new protease are around 10% and 40% higher than those of thermolysin (the neutral protease from Bacillus thermoproteolyticus), respectively. The enzyme was inactivated by EDTA, but not by phenylmethylsulfonyl fluoride. These results indicate that the enzyme is a highly thermostable neutral-(metallo)protease.     

Purification of a novel cysteine protease,procerain B,from Calotropis procera with distinct characteristics compared to procerain

Proteases have applications in food, detergent and pharmaceutical industries. A novel protease has been purified from the latex of Calotropis procera and characterized. As another cysteine protease, procerain, is reported from the same source, the newly purified enzyme was named as procerain B. The enzyme shows distinct properties compared to procerain, in terms of cleavage recognition site, immunological properties and other physical properties like molecular weight, isoelectric point, etc. The newly purified enzyme shows a broad optimum pH (6.5–8.5) as well as broad optimum temperature (40–60 °C). Additionally, the enzyme retains its activity where most of other proteases are not active. Moreover, the enzyme appeared to be very efficient in hydrolysis of blood stain and may have potential application in detergent industries. Simple and economic purification of procerain B, together with easy availability of latex, makes the large-scale production of procerain B possible, thus enables to explore various industrial as well as biotechnological applications.     

Purification and characterization of an alkaline serine-protease produced by a new isolated Aspergillus clavatus ES1

An extracellular bleach stable protease from the fungus Aspergillus clavatus ES1, isolated from wastewater, was purified and characterized. The protease of ES1 strain was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and CM-Sepharose ion exchange chromatography, with a 7.5-fold increase in specific activity and 29% recovery. The molecular mass was estimated to be 32 kDa on SDS-PAGE. The optimum pH and temperature for the proteolytic activity were pH 8.5 and 50 °C, respectively. The enzyme was stable in the pH range of 7.0–9.0. The protease was activated by divalent cations such as Ca²⁺ and Mg²⁺.The alkaline protease showed extreme stability towards non-ionic surfactants (5% Tween 80 and 5% Triton X-100). In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 71 and 53% of its initial activity after 1 h incubation in the presence of 1 and 2% (w/v) sodium perborate, respectively.The N-terminal sequence of the first 15 amino acids of the purified alkaline protease of A. clavatus ES1 showed high similarity with other fungal alkaline proteases. The activity was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine-protease.     

A novel organic solvent-stable alkaline protease from organic solvent–tolerant Bacillus licheniformis YP1A

An organic solvent-stable alkaline protease producing bacterium was isolated from the crude oil contaminant soil and identified as Bacillus licheniformis. The enzyme retained more than 95% of its initial activity after pre-incubation at 40 °C for 1 h in the presence of 50% (v/v) organic solvents such as DMSO, DMF, and cyclohexane. The protease was active in a broad range of pH from 8.0 to 12.0 with the optimum pH 9.5. The optimum temperature for this protease activity was 60 °C, and the enzyme remained active after incubation at 50–60 °C for 1 h. This organic solvent-stable protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.     

UFEIII, a fibrinolytic protease from the marine invertebrate, Urechis unicinctus

A new serine protease with fibrinolytic activity from a marine invertebrate, Urechis unicinctus, was purified to electrophoretic homogeneity using column chromatography. SDS-PAGE of the purified enzyme showed a single polypeptide chain with MW ~20.8 kDa. Its N-terminal sequence was IIGGSQAAITSY. The purified enzyme, UFEIII, was stable at pH 6–10 below 60 °C with an optimum pH of 8.5 at approx. 55 °C. The enzyme activity was significantly inhibited by PMSF and SBTI suggesting that it was a serine protease. In fibrin plate assays, UFEIII was contained 1.46 × 10³ U (urokinase units) mg^?1 total fibrinolytic activity, which consisted of 692 U mg^?1 direct fibrinolytic activity and 769 U mg^?1 plasminogen-activator activity. K_m and V_max values for azocasein were 1 mg ml^?1 and 43 μg min^?1 ml^?1, respectively.