Decolorization and purification of crude protease from Rhizopus oryzae by activated charcoal and its electrophoretic analysis

Activated charcoal decolorized and partially purified the protease from a crude extract of solid state fermentation of wheat bran by Rhizopus oryzae. Treatment for 5 min was sufficient. Depending on the initial colour intensity of crude, the charcoal to crude extract ratio could be optimized to achieve 90% decolorization, 85% enzyme recovery, and over a 3-fold purification, even up to 20-fold variation in batch size (from 1 ml to 20 ml crude extract). Decolorization followed the Freundlich and the Langmuir models, the Freundlich constant, n, being 2.74. Partial purification was confirmed by native PAGE and the protease band identified by gelatin-PAGE. SDS-PAGE showed the protease consisted of two sub-units (about 22 and 24 kDa). List of symbols: c _o, initial solute concentration in liquid before adsorption; c ^*, equilibrium solute concentration in liquid after adsorption; k, empirical constant for Freundlich adsorption isotherm; U, unit of protease activity; v, volume of solution per unit weight of adsorbent.     

Alkaline protease from a new obligate alkalophilic isolate of Bacillus sphaericus

An obligate alkalophilic Bacillus sphaericus strain, isolated from alkaline soils in the Himalaya, produced an extracellular protease which was optimally active at 50–55 °C and pH 10.5. The enzyme was stable in presence of 500 mg chlorine l^–1 and as a detergent additive. Its stability in presence of laundry detergents was comparable to that of commercial proteases. The gelatin layer in 25 g of used X-ray films was efficiently hydrolyzed within 12 min at 50 °C, pH 11.0 and 25 U protease/ml.     

Feasibility study for decomposition of gelatin layers on X-ray films by thermostable alkaline protease from alkaliphilic Bacillus sp.

Enzymatic decomposition of gelatin layers on X-ray films and repeated utilization of enzyme for potential industrialization were investigated using thermostable alkaline protease from the alkaliphilic Bacillus sp. B21-2. The decomposition of gelatin layers at 50 °C with the mutant enzyme (Ala187 was replaced by Pro) was higher than those of the wild-type and other mutant enzymes. In the repeated experiment for every 60 min (20 U ml^–1, 50 °C), the mutant enzyme could be satisfactorily used five times while three times for the wild-type enzyme.     

Micropropagation of an Endangered Orchid Anoectochilus formosanus

A rapid and efficient procedure is outlined for in vitro clonal propagation of an elite cultivar of jewel orchid (Anoectochilus formosanus). Multiple shoot proliferation was induced in shoot tip explants on Hyponex (H3) media supplemented with 1 mg dm^–3 benzyladenine or 1 – 2 mg dm^–3 thidiazuron (TDZ). Addition of activated charcoal (1 g dm^–3) to the TDZ containing medium promoted multiple shoot formation (11.1 shoots per explant). However, the regenerated shoots had slow growth rate and failed to elongate. This problem was overcome by transferring the shoot clumps to a hormone free H3 medium supplemented with 2 % sucrose and 0.5 g dm^–3 activated charcoal. Rooting was induced in 100 % of the regenerated shoots in the same media. The plantlets were acclimatized and established in greenhouse.     

High-level expression, purification and characterization of recombinant Aspergillus oryzae alkaline protease in Pichia pastoris

The alkaline protease gene from Aspergillus oryzae was cloned, and then it was successfully expressed in the heterologous Pichia pastoris GS115 with native signal peptide or α-factor secretion signal peptide. The yield of the recombinant alkaline protease with native signal peptide was about 1.5-fold higher than that with α-factor secretion signal peptide, and the maximum yield of the recombinant alkaline protease was 513 mg/L, which was higher than other researches. The recombinant alkaline protease was purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The purified recombinant alkaline protease showed on SDS–PAGE as a single band with an apparent molecular weight of 34 kDa. The recombinant alkaline protease was identical to native alkaline protease from A. oryzae with regard to molecular weight, optimum temperature for activity, optimum pH for activity, stability to pH, and similar sensitivity to various metal ions and protease inhibitors. The native enzyme retained 61.18% of its original activity after being incubated at 50 °C for 10 min, however, the recombinant enzyme retained 56.22% of its original activity with same disposal. The work demonstrates that alkaline protease gene from A. oryzae can be expressed largely in P. pastoris without affecting its enzyme properties and the recombinant alkaline protease could be widely used in various industrial applications.     

Decolorization method of crude alkaline protease preparation produced from an alkalophilic <Emphasis Type="Italic">Bacillus clausii</Emphasis>

Diaion HPA75 decolorized efficiently the crude preparation of novel alkaline protease produced by Bacillus clausii. The optimum concentrations of HPA75 and contact time for efficient decolorization were determined to be approximately 6 ∼ 10% (w/v) and 8 h, respectively. Color removal efficiency was improved at alkaline pH, and 21% color intensity was retained with a protease yield of 99.7% at pH 11. By using highly concentrated samples, a pattern of decolorization was achieved that was similar to that produced by unconcentrated enzyme preparations. After treatment with 6% HPA75 for 8 h, the residual color intensity was approximately 20% with a protease yield of nearly 100%. Used HPA75 could be regenerated easily, and the regenerated HPA75 was as effective as the fresh HPA75 for decolorization and protease recovery. The regeneration efficiency of the used Diaion HPA75 was greater than 90% until it was used four times. Considering these results, we suggest Diaion HPA75 is suitable for color removal applications, producing high protease yields from fermented broth.     

Adsorption of Bovine Serum Albumin from salt solution onto activated carbon

One of the wastewaters from tanning industry (soak liquor) contains 0.4 g/l of dissolved protein. During coagulation and flocculation 41 % of protein was removed. A suggestion has been made to remove the residual protein by adsorption technique. The optimum conditions for adsorption of Bovine Serum Albumin (BSA) on rice bran based activated carbon (RBAC) have been determined. Maximum adsorption of BSA took place at pH 7.O. Ionic strength was found to have influence on the adsorption behaviour. Adsorption capacity of BSA onto charcoal surface decreased with increase in temperature. Enthalpy of adsorption in all cases was found to be within –19 to –57 kJ/mole, indicating exothermic nature of the process. Applicability of adsorption technique to the removal of dissolved protein from soak water has been studied. The maximum removal of protein occurred at pH 7.0 and the ratio of protein removed to weight of adsorbent was 3.22×10^–3 g/g.     

Production of extremely thermostable alkaline protease from Bacillus sp. no. AH-101

Summary An alkalophilic Bacillus sp. no. AH-101, which produced extremely thermostable alkaline protease, was isolated among 200 soil samples. The enzyme production reached its maximum level of 1500 units/ml after about 24 h in alkaline medium (pH 9.5). The enzyme was most active toward casein at pH 12–13 and stable to 10 min incubation at 60° C from pH 5–13. Calcium ions were effective in stabilizing the enzyme especially at higher temperatures. The optimum and stable temperatures were about 80° C and below about 70° C respectively in the presence of 5 mM calcium ions. The enzyme was completely inactivated by phenylmethane sulphonyl fluoride, but little affected by ethylenediaminetetraacetic acid, urea, sodium dodecylbenzenesulphonate and sodium dodecyl sulphate. The molecular weight and sedimentation constant were approximately 30 000 and 3.0S respectively, and the isoelectric point was at pH 9.2. These results indicte that no. AH-101 alkaline protease is more stable against both temperature and highly alkaline conditions than any other protease so far reported.     

Effect of Sucrose Concentration, Charcoal, and Indole-3-Butyric Acid on Germination of Abies Numidica Somatic Embryos

The dependency of radicle elongation in Abies numidica somatic embryos on germination media has been studied. No significant differences were detected between the Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) medium. The addition of 10 g dm^–3 activated charcoal or 0.05 mg dm^–3 indole-3-butyric acid (IBA) into both media had positive influence on embryo germination. Difference between activated charcoal and IBA effects were significant. The high rooting percentage (85 %) was recorded on half SH medium with 10 g dm^–3 sucrose and activated charcoal. After IBA addition rooting percentage was increased to 95 %. During 7 months 73 % of plantlets survived transfer to soil and in 54 % of plantlets shoot growth was observed.     

Continuous decolorization of bleached kraft effluents byCoriolus versicolor in the form of pellets

Summary Continuous decolorization of kraft black liquor by mycelial pellets ofCoriolus versicolor in the presence of glucose as co-substrate is discussed. A linear relationship was developed between the rate of decolorization and the liquor concentration. The rate constant was equal to 0.00961 gmyc^–1 h^–` at 22°C. During the continuous experiments the pellets exhibited no apparent loss of activity when the liquor concentration was in the range of 400 CU l^–1 to 5000 CU l^–1. However, in the repeated batch experiments a loss of activity was observed which was dependent on the initial liquor concentration. The half-life of pellets was equal to 4.7, 9.4 and 20.2 days for the initial liquor concentration of 1380, 31 780 and 6990 CU l^–1, respectively. The production of the extracellular enzyme, laccase, was followed but could not be used as an indicator of the ligninolytic activity. The involvement of the intracellular enzymes ofC. versicolor in the decolorization process is described.     

Protease production by a thermophilic Bacillus species (P-001A) which degrades various kinds of fibrous proteins

An aerobic Gram-positive sporeforming bacterium was isolated from an alkaline hot spring at Wondo Genet, Ethiopia. In an optimized culture medium it produced maximum activity of protease at 55°C and pH 9.5. The protease activity against casein was 65 units/ml. Enzyme activity was detected between 30–70°C and pH of 4.5–11.5. The enzyme had a half-life of 55 and 30 min at 60° and 70°C, respectively. The isolate hydrolysed 90, 60 and 50% of the skin, feather and horn used in the optimized medium within 120 h.     

Fast and efficient adsorption/desorption of protein by a novel magnetic nano-adsorbent

A novel magnetic nano-adsorbent was prepared by covalently binding polyacrylic acid (PAA) on Fe₃O₄ superparamagnetic nanoparticles (13.2 nm) via carbodiimide activation. The maximum weight ratio of PAA to Fe₃O₄ was 0.12 (i.e., average of two PAA molecules on a magnetic nanoparticle). The magnetic nano-adsorbent possessed a high ionic exchange capacity of 1.64 meq g^–1 and was efficient for the recovery of lysozyme. The lysozyme could be completely adsorbed in 0.1 M phosphate buffer at pH 3–5 and completely desorbed in NaSCN solution (>1 M) within 1 min, and retained 95% activity after adsorption/desorption. In addition, the adsorption behavior followed the Langmuir adsorption isotherm with a maximum adsorption amount of 0.224 mg mg^–1 and a Langmuir adsorption equilibrium constant of 10 ml mg^–1 at 25 °C. The change of enthalpy at 15–35 °C was –4.2 kJ ml mol^–1 mg^–1.     

Biosorption of reactive dyes by the mycelium pellets of a new isolate of Penicillium oxalicum

Three reactive dyes were rapidly adsorbed by the mycelium pellets of Penicillium oxalicum. Dye removal of Reactive Blue 19 was up to 60% in 10 min and 91% in 80 min. Dye adsorption isotherms fitted Langmuir model well and the maximum adsorption capacities at 20 °C were calculated to be 160 mg g^–1 for Reactive Blue 19, 122 mg g^–1 for Reactive Red 241 and 137 mg g^–1 for Reactive Yellow 145, respectively. The pellets exhibited a high dye adsorption capacity (80–180 mg g^–1) for all of the 3 dyes over a wide pH range (pH 2–10), and the maximum adsorption was obtained at pH 2. The adsorption capacity was mildly increased by increasing salinity.     

Properties of extracellular proteases from three psychrotolerant Stenotrophomonas maltophilia isolated from Antarctic soil

Three Antarctic psychrotolerant Stenotrophomonas maltophilia were isolated and the characteristics of their extracellular serine proteases were described. The isolates were able to grow at 14 and 34°C, but grew better between 20 and 28°C. The highest protease secretion was reached at 20–24°C. The purified enzyme preparations had maximal activity at 55–60°C and alkaline pH. They showed high pH stability, retaining more than 60% of residual activity after 3 h of incubation at a pH range of 4–12. The thermal stability was slightly lower compared with a commercial mesophilic protease, with 74–79% residual activity after 90 min at 40°C and 50% inactivation at 50°C between 43 and 69 min. These properties suggest that the Antarctic isolates could be adapted to cold by means of synthesising more enzymes with high activity but that the proteases they produce are not truly cold-active, being more similar to mesophilic enzymes.     

Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii

A detoxification method using activated charcoal with concentrated rice straw hemicellulosic hydrolysate improved the conversion of xylose to xylitol by the yeast Candida guilliermondii by 22%. This was achieved when the hydrolysate:charcoal ratio was 40 g g^–1, resulting in removal of 27% of phenolic compounds. Under this condition, the xylitol yield factor (0.72 g g^–1) and volumetric productivity (0.61 g l^–1 h^–1) were close to those attained in a semi-defined medium simulating hydrolysate sugars.     

Medium-term conservation of mature embryos of coconut

Optimal conditions for the medium-term conservation of zygotic embryos of coconut were developed. After 6 months of storage on a medium devoid of sucrose and containing 2 g l^–1 activated charcoal, 100% of the embryos developed into whole plantlets within 5 months after transfer to the recovery medium. After a 12-month storage period on medium containing 15 g l^–1 sucrose and devoid of activated charcoal, 51% of the embryos germinated within 2 months after transfer to the recovery medium.     

Effect of particle size of activated charcoal on separation of Triton X-100 from protein, liver cytosol, and lipoxygenase extracts

Triton X-100 was almost completely removed from bovine serum albumin solutions, BALB/c mouse liver extracts, and avocado peel lipoxygenase extracts by stirring the samples for 30 min in the presence of 250-350 mesh activated charcoal. The procedure did not remove protein significantly and did not reduce enzyme activity. At higher charcoal particle sizes, the efficiency of Triton adsorption was decreased and protein adsorption was increased. ElevateD temperatures enhanced Triton and protein adsorption. Adsorption on activated charcoal of 250-350 mesh is a simple and rapid procedure for Triton removal at a ratio of 0.23 g Triton X-100 per gram of activated charcoal.     

Effect of digitonin on membrane-bound and chitosomal chitin synthetase activity in protoplasts from yeast cells ofCandida albicans

The effect of digitonin on chitin synthetase present in membrane (MMF) and cytoplasmic fractions (chitosomes) (CF) fromC. albicans yeast protoplasts has been determined. The zymogen is preferentially, but not exclusively, solubilized by digitonin from MMF. Centrifugation of distinct solubilized preparations, containing either zymogen,in vivo active enzyme and/or trypsin activated enzyme, on linear sucrose gradients suggests that both zymogen and trypsin activated enzyme sediment slightly slower than the active enzyme, pointing out differences between the activation processesin vivo andin vitro or, alternatively, that both enzyme activities (activein vivo and zymogenic) correspond to different gene products. The detection of a zymogenic activity under certain conditions (0.5 mg ml^–1 of digitonin and 64 µg ml^–1 of trypsin) also suggests the existence of more than one pool of zymogenic enzyme in the MMF. Digitonin sensitizes the chitosomal (CF) proenzyme to trypsin: activation is enhanced by low digitonin concentrations in the presence of 8 µg ml^–1 of protease, whereas activity strongly decreases in the presence of 64 µg ml^–1 of trypsin. Digitonin does not produce zymogen activationper se in absence of exogenous protease. Furthermore, chitosome structure is modified into particles with low buoyant densities.     

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Summary Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml^–1) precipitated 1.2 g l^–1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.Offprint requests to: R. G. Burns     

Characterization of an alkaline serine protease from an alkaline-resistant Pseudomonas sp.: Cloning and expression of the protease gene in Escherichia coli

Summary A gene, aprP, encoding an extracellular alkaline serine protease from a newly isolated Pseudomonas sp. KFCC 10818 was cloned and characterized. Nucleotide sequence analysis revealed an open reading frame of 1,266 nucleotides which could encode a polypeptide comprised of 422 amino acids. The C-terminal 283 residues showed an overall sequence homology with the subtilisin-type serine proteases. When expressed in E. coli, the alkaline protease, AprP, was released to the culture medium. The purified AprP was most active at pH 11. The k _Cat/K _m value of this enzyme was 9.2 × 10³ S^–1mM^–1, which is much higher than those of subtilisins.