Optimum pH For Activity Research Articles

Production of alkaline protease by Bacillus cereus and Bacillus polymixa in new industrial culture mediums and its immobilization

After amylases, proteases are the most useful industrial enzymes that are produced 500 tones annually. In industry, proteases are produced from bacteria and fungi. The alkaline proteases, with an optimum activity in pH: 9 - 11, have numerous applications in daily life of peoples such as food complementary of beast and poultry, confectionary, bakery, fermentation industries, leathering, detergent industry, biotransformation and so on. In this research a wild strain of Bacillus cereus andBacillus polymixa, isolated from the soils of Isfahan (Iran), showed the best alkaline protease activity in industrial mediums such as sweet sorghum extract and molasses. The maximum alkaline protease activity in productive culture mediums after 72 h in 30°C and with100 R.P.M. were measured. The results with B. polymixawere 46.7 (u/ml) in T.S.B, 57 (u/ml) in N.B, 60.22 (u/ml) in peptone solution, 67 (u/ml) in sweet sorghum solution, 216 (u/ml) in yeast extract solution, 358 (u/ml) in glucose solution, 413 (u/ml) in casamino acid solution, 418 (u/ml) in whey, 470.5 (u/ml) in casein solution, 490.5 (u/ml) in molasses, 525 (u/ml) in sweet sorghum extract and 580.5 (u/ml) in milk and with B. cereus were 34.72 (u/ml) in T.S.B, 39.85 (u/ml) in N.B, 39.1 (u/ml) in peptone solution, 58 (u/ml) in sweet sorghum solution, 200.65 (u/ml) in yeast extract solution, 363 (u/ml) in glucose solution, 191.5 (u/ml) in casamino acid solution, 302.5 (u/ml) in whey, 455.5 (u/ml) in casein solution, 485.5 (u/ml) in molasses, 510.5 (u/ml) in sweet sorghum extract and 535.5 (u/ml) in milk. The fixation experiments with alkaline protease from B. polymixa showed that this enzyme after 3 month in sawdust, perlit and silica has protected its stability and has 50, 100 and 100% of its primary activity respectively. In the case of immobilization with the same enzyme and the same bacterium, the examinations showed that the alkaline protease activity on perlit after one, two and three washing treatment were 52, 30 and 20% of its primary activity but in the case of silica were 93, 90 and 84% of its primary activity. Key words: Alkaline-protease, Bacillus polymixa, Bacillus cereus, sweet-sorghum, immobilization.

Activation of lactoperoxidase by heme‐linked protonation and heme‐independent iodide binding

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red-shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe-His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme-linked protonation site.

Polyphenol Contents and Polyphenol Oxidase Activities of Some Nigerian Kolanuts

The levels of polyphenol and activities of polyphenol oxidases in some Nigerian kolanuts were investigated. Garcina cola had the least polyphenol content of 15.60±1.70 (mg/g), while Cola nitida (red) recorded the highest value of 33.50±2.51mg/g. Polyphenol oxidase from Garcina cola had its optimum pH of activity in t he acidic region (pH 3), but the white and red species of Cola nitida had a neutral (pH 7) optimum pH. With catechol as substrate, polyphenol oxidase activity was highest in Cola nitida (white) (55.70±2.60) and lowest in Garcina cola (2.22 ± 0.04). Whilst the values of polyphenol obtained may explain the high incidence of enzymatic browning in some Nigeria kolanuts, the white cultivar of Cola nitida could be further explored as a good source of polyphenol oxidase.

Purification and Characterization of Intracellular Cellulase from Aspergillus oryzae ITCC-4857.01

Purification and characterization of intracellular cellulase produced by A. oryzae ITCC-4857.01 are reported. The enzyme was purified by ion-exchange chromatography using DEAE-cellulose followed by Gel filtration. The purification achieved was 41 fold from the crude extract with yield of 27%. The purified enzyme showed single band on poly acrylamide gel. The molecular weight as determined by SDS-PAGE and gel filtration was 38 KDa and 38.6 KDa respectively and contained only one subunit. The enzyme is glycoprotien as nature and contained 0.67% neutral sugar. The apparent Km value of the enzyme against cellulose was 0.83%. The enzyme showed the highest relative ativities on CMC followed by avicel, salicin and filter paper. The optimum pH of activity was 5.5 and very slight activity was observed at or above pH 7.5 as well as bellow pH 3.5. The optimum tempreture of the activity was 45°C and the highest activity was exhibited in 35 to 45°C. The enzyme lost their activities almost completely (95~100%) at 80 °C or above and as well as bellow 25°C.

Cysteine enhances activity and stability of immobilized papain

Immobilization of papain on Sepharose 6B in the presence of different concentrations of cysteine affected the enzyme activity depending on cysteine concentration. The maximum specific activity was observed when papain was immobilized with 200 mM cysteine. The immobilization process brought significant enhancement of stability to temperature and extreme pH values with respect to free papain. After immobilization, the optimum temperature of papain activity increased by 20 degrees C (from 60 to 80 degrees C) and its optimum pH activity shifted from 6.5 to 8.0. Catalytic efficiency (k(cat)/K(m)) and specific activity of the immobilized enzyme do not significantly change after immobilization. The temperature profile of this form of immobilized papain showed a broad range of activity compared with both free and immobilized form of papain in the absence of cysteine. This significant behavior in terms of activation energy is also discussed.

Production of amylase by bacteria isolated from a cassava waste dumpsite in Minna, Niger State, Nigeria

Ten grams (10 g) of soil sample obtained from a cassava waste dumpsite in Kasuwan Gwandabe, Minna, Niger State, was bacteriologically analyzed. Onegram (1.0 g) of the sample inoculated into a liquid soluble starch medium generated reducing sugar with a concentration of 1.65 mg/ml after 72 h. Characterization of the soluble starch amylases revealed an optimum temperature of activity of 70°C. Optimum pH for activity was between 6.5 and 7.5. The most frequently occurring amylolytic bacteria were Bacillus subtilis (37.5%), followed byBacillus subtilis megaterium and Bacillus coagulans (18.75% each). The least occurring isolates were Merus and Bacillus pumilus (6.25% each). The mean zone of amylolytic activity for the isolates ranged between 2.1 mm for B. subtilisand 1.1 mm for B. pumilus. Key words: Aminolytic, enzymes, amylase, optimum, Bacillus sp.

Partial purification and characterisation of banana [Musa (AAA Group) ‘Gros Michel’] polyphenol oxidase

SummaryPolyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) ‘Gros Michel’] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S‐200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S‐200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 mm sodium metabisulphite, 2 mm l‐cysteine, 4 mm ascorbic acid, and 100 mm 4‐hexylresorcinol. The Km and Vmax of banana PPO for dopamine were 2.08 mm and 0.124 mm min−1 respectively.

Purification and characterization of keratinase from recombinant Pichia and Bacillus strains

The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 °C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K m and V max of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.

Isolation and properties of collagenolytic serine proteinase isoenzyme from king crab Paralithodes camtschatica

An electrophoretically homogeneous isoenzyme CSP-2 of collagenolytic serine proteinase has been isolated from the total preparation of king crab digestive enzymes. The molecular mass of the proteinase is 24.8 +/- 0.3 kD, pH optimum for activity is 8.5, the temperature optimum for activity is 38-40 degrees C, and the pH range of stability is 7-10. The enzyme has dual substrate specificity, but preference for positively charged amino acid residues in P(1)-position is more pronounced than in the case of the major isoenzyme. The temperature dependence of kinetic constants for synthetic substrate hydrolysis by CSP-2 has been investigated. Inhibition specificity of the enzyme is characteristic of serine proteinases but more like that of crab trypsin than that of the major CSP isoenzyme. The isolated collagenolytic proteinase also cleaves fibrinogen and fibrin and activates plasminogen. The amino acid sequence of the CSP-2 proteinase, which has been partially determined by tandem mass spectrometry, displays some similarity to the sequence of the major CSP isoenzyme.

Ceramide Generated by Sphingomyelin Hydrolysis and the Salvage Pathway Is Involved in Hypoxia/Reoxygenation-induced Bax Redistribution to Mitochondria in NT-2 Cells

Ceramide functions as an important second messenger in apoptosis signaling pathways. In this report, we show that treatment of NT-2 neuronal precursor cells with hypoxia/reoxygenation (H/R) resulted in ceramide up-regulation. This elevation in ceramide was primarily due to the actions of acid sphingomyelinase and ceramide synthase LASS 5, demonstrating the action of the salvage pathway. Hypoxia/reoxygenation treatment led to Bax translocation from the cytoplasm to mitochondria and cytochrome c release from mitochondria. Down-regulation of either acid sphingomyelinase or LASS 5-attenuated ceramide accumulation and H/R-induced Bax translocation to mitochondria. Overall, we have demonstrated that ceramide up-regulation following H/R is pertinent to Bax activation to promote cell death.

A pseudo-β-glucosidase in Arabidopsis thaliana: Correction by site-directed mutagenesis, heterologous expression, purification, and characterization

Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature beta-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZalphaB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant beta-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0-8.5. The purified recombinant beta-glucosidase is effectively active on para-/ortho-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with K(m) values of 1.9, 2.1, 0.78 mM and k(cat) values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-beta-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of beta-glucosidases and mutated recombinant enzyme is discussed.

Production of laccase and xylanase from Coriolus versicolor grown on tomato pomace and their chromatographic behaviour on immobilized metal chelates

A strain of Coriolus versicolor was grown in tomato pomace as sole carbon source for the production of laccase and xylanase. This culture exhibited a peak of laccase (362 U/L of fermentation broth) and xylanase (2565 U/L of fermentation broth) activities on the 3rd and 14th day of cultures with a specific activity of 3.7 and 11.7 U/mg protein, respectively. Differential chromatographic behaviour of xylanase and laccase from C. versicolor was investigated on immobilized metal chelates. The effect of pH, length of spacer arm, the presence of imidazole and nature of metal ion was studied for enzyme adsorption on immobilized metal affinity chromatography (IMAC). PDB survey of solvent accessible histidine residues in laccase and xylanase families was carried out by using bioinformatic tools. A one-step purification for laccase from C. versicolor was devised by using Sepharose 6B–EPI 30–IDA–Cu(II) and the purified enzyme was obtained with a specific activity of about 15.0 U/mg protein, a final recovery of enzyme activity of about 60% and a purification factor of about 10. The purified preparation of laccases A exhibited an optimum pH of activity of 5.0 and 3.0 with o-dianisidine and ABTS as substrates, respectively. The optimum temperature of activity for this enzyme was found to be 80 °C in acetate buffer at pH 4.5 whereas the half-life ( t 1/2) of 19.4 ± 2.2 h and 0.50 ± 0.012 h was obtained at 45 and 60 °C, respectively. The kinetic parameters ( V max, K m, K cat and K cat/ K m) of the purified enzyme were also obtained with o-dianisidine, guaiacol and ABTS as substrates. By using selective experimental conditions in IMAC, it was possible to separate successfully laccase isoenzymes into two groups, one with low and the other with high p I values. Kinetic characterization of both groups of isoenzymes was also carried out.

Inhibition of Mg 2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ∼4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg2+ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg2+directly, can nonetheless inhibit Mg2+ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg2+ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.

The significance and effect of tandem repeats within theMycobacterium tuberculosis leuAgene on α-isopropylmalate synthase

The 57-bp tandem repeats located in the Mycobacterium tuberculosis leuA gene code for the alpha-isopropylmalate synthase (alpha-IPMS). It is unique to this pathogen. It was previously demonstrated that the leuA-coding sequence Rv3710, containing the tandem repeats, can be translated to an active alpha-IPMS. The objective of the present study was to investigate the significance and effect of the two 57-bp tandem repeats upon gene expression and the general properties of alpha-IPMS. The putative M. tuberculosis H37Rv leuA gene with and without the tandem repeats was cloned by PCR and expressed in an Escherichia coli host. The enzyme product was studied for general properties, comparing that from a native leuA gene containing two repeats and that from the 57-bp tandem repeats deletion mutant. Upon deletion of the two 57-bp tandem repeats, the expression level of leuA from M. tuberculosis H37Rv was comparable with that of the native form. The general properties of the two types of enzymes were similar. They were both functional with the same range of optimal temperature and optimal pH for activity and with similar enzyme stability. Deletion of the repeats had no detectable effect on leuA expression level or the general properties of the enzyme product.

A purple acidophilic di-ferric DNA ligase from Ferroplasma.

We describe here an extraordinary purple-colored DNA ligase, LigFa, from the acidophilic ferrous iron-oxidizing archaeon Ferroplasma acidiphilum, a di-ferric enzyme with an extremely low pH activity optimum. Unlike any other DNA ligase studied to date, LigFa contains two Fe(3+)-tyrosinate centers and lacks any requirement for either Mg(2+) or K(+) for activity. DNA ligases from closest phylogenetic and ecophysiological relatives have normal pH optima (6.0-7.5), lack iron, and require Mg(2+)/K(+) for activity. Ferric iron retention is pH-dependent, with release resulting in partial protein unfolding and loss of activity. Reduction of the Fe(3+) to Fe(2+) results in an 80% decrease in DNA substrate binding and an increase in the pH activity optimum to 5.0. DNA binding induces significant conformational change around the iron site(s), suggesting that the ferric irons of LigFa act both as structure organizing and stabilizing elements and as Lewis acids facilitating DNA binding at low pH.

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources

Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55°C, and, in the substratum absence, the thermostability was for 1h at 50°C. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65°C was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol(-1). The hydrolysis of different substrate showed the enzyme's preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.

PURIFICATION AND CHARACTERIZATION OF A CHITINASE FROM PEANUT (ARACHIS HYPOGAEA L.)

ABSTRACT A chitinase was isolated from peanut (Arachis hypogaea L.) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, and high-performance liquid chromatography on POROS 20 HQ. There was a 133-fold increase in specific activity of purified chitinase compared with that of the crude extract. The protein exhibited a molecular mass of 34.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and nonreducing conditions, indicating that it is a monomeric protein. The isoelectric point was 5.1 by isoelectric focusing electrophoresis. Optimal pH activity was 5.4 and optimal temperature was 40–50C. The enzyme was stable below 55C, but was rapidly inactivated when incubated at temperatures above 60C. These results demonstrated that the purified protein was a kind of relatively thermostable chitinase from the peanut seeds. PRACTICAL APPLICATIONS This paper describes the isolation and characterization of a chitinase from Arachis hypogaea L. seeds. Chitinases are listed as the class of pathogenesis-related proteins, and they have become a popular research field because of their resistance to plant disease. The research we are presenting should help in understanding chitinase functions in plant defense mechanisms. A relatively thermostable enzyme newly obtained, which was stable below 55C, would certainly be significant for its utilization both in enzymology and in plant defense. In particular, a relatively heat-resistant enzyme from A. hypogaea L. seeds would certainly be helpful for elaborating strategies for constitutive expression of chitinase genes and their manipulation and general utilization in plant defense mechanisms through more recent techniques of genetic manipulation.

Production and characterization of glucoamylase from fungus Aspergillus awamori expressed in yeast Saccharomyces cerevisiae using different carbon sources.

Glucoamylase is widely used in the food industry to produce high glucose syrup, and also in fermentation processes for production beer and ethanol. In this work the productivity of the glucoamylase of Aspergillus awamori expressed by the yeast Saccharomyces cerevisiae, produced in submerged fermentation using different starches, was evaluated and characterized physico-chemically. The enzyme presented high specific activity, 13.8 U/mgprotein or 2.9 U/mgbiomass, after 48 h of fermentation using soluble starch as substrate. Glucoamylase presented optimum activity at temperature of 55°C, and, in the substratum absence, the thermostability was for 1h at 50°C. The optimum pH of activity was pH 3.5 - 4.0 and the pH stability between 5.0 and 7.0. The half life at 65°C was at 30.2 min, and the thermal energy of denaturation was 234.3 KJ mol-1. The hydrolysis of different substrate showed the enzyme’s preference for the substrate with a larger polymerization degree. The gelatinized corn starch was the substratum most susceptible to the enzymatic action.

Purification and Characterization of Alcohol Dehydrogenase from Gluconobacter suboxydans

Purification and characterization of alcohol dehydrogenase (ADH) from Gluconobacter suboxydans was done in order to biotechnological and industrial application. Solubilization of enzyme from bacterial membrane fraction by Triton X-100 and subsequent fractionation on DEAE-Sephadex A-50 and Hydroxyapatite was successful in enzyme purification. Enzyme assay reaction mixture contained potassium ferricyanide 0.1 M, McIlvaine buffer 0.1 M (pH 5.5), Triton X-100 10%, ethanol 1 M and enzyme solution. The purified ADH Optimum pH activity was 5.5. The enzyme was in maximum stability in pH 5.8. The substrate specificity of the enzyme was determined using the same enzyme assay method as described above, except that various substrates (100 mM) were used instead of ethanol. The relative activity of the ADH for ethanol was higher than the others. The effects of metal ions and inhibitors on the activity of the enzyme were examined by measuring the activity using the same assay method as described above. Activity of purified enzyme was increased in the presence of Ca(+2) and was decreased in presence the of ethylenediamine tetra acetic acid (EDTA). Because the proper structure and function of the enzyme is related to structural Ca(+2) and EDTA can chelate Ca(+2). An apparent Michaelis constant for ethanol were examined to be 1.7 x 10(-3) M for ethanol as substrate.

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 513mg/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 34kDa. 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 10min, 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.