Molecular cloning,nucleotide sequence and expression of the structural gene for a thermostable alkaline protease from Bacillus sp. no. AH-101

Summary Alkaliphilic Bacillus sp. no. AH-101 produces an extremely thermostable alkaline serine protease that has a high optimum pH (pH 12–13) and shows keratinolytic activity. The gene encoding this protease was cloned in Escherichia coli and expressed in B. subtilis. The cloned protease was identical to the AH-101 protease in its optimum pH and thermostability at high alkaline pH. An open reading frame of 1083 bases, identified as the protease gene, was preceded by a putative Shine-Dalgarno sequence (AAAGGAGG) with a spacing of 11 bases. The deduced amino acid sequence revealed a pre-pro-peptide of 93 residues followed by the mature protease comprising 268 residues. AH-101 protease showed slightly higher homology to alkaline proteases from alkaliphilic bacilli (61.2% and 65.3%) than to those from neutrophilic bacilli (54.9–56.7%). Also AH-101 protease and other proteases from alkaliphilic bacilli shared common amino acid changes and a four amino acid deletion when compared to the proteases from neutrophilic bacilli. AH-101 protease, however, was distinct among the proteases from alkaliphilic bacilli in showing the lowest homology to the others.Correspondence to: H. Takami     

Characterization of an alkaline protease from Bacillus sp. no. AH-101

Summary The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes. Offprint requests to: H. Takami     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

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.     

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.     

Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2

An obligatory alkalophilic Bacillus sp. P-2, which produced a thermostable alkaline protease was isolated by selective screening from water samples. Protease production at 30 °C in static conditions was highest (66 U/ml) when glucose (1% w/v) was used with combination of yeast extract and peptone (0.25% w/v, each), in the basal medium. Protease production by Bacillus sp. P-2 was suppressed up to 90% when inorganic nitrogen sources were supplemented in the production medium. Among the various agro-byproducts used in different growth systems (solid state, submerged fermentation and biphasic system), wheat bran was found to be the best in terms of maximum enhancement of protease yield as compared to rice bran and sunflower seed cake. The protease was optimally active at pH 9.6, retaining more than 80% of its activity in the pH range of 7–10. The optimum temperature for maximum protease activity was 90 °C. The enzyme was stable at 90 °C for more than 1h and retained 95 and 37% of its activity at 99 °C and 121 °C, respectively, after 1 h. The half-life of protease at 121 °C was 47 min.     

Purification and Characterization of Alkaline Proteinase from AlkalophilicBacillussp.

Alkaline proteinase was purified from Bacillussp. isolated from soil. The pH optimum was 11.5 at 37°C. Calcium divalent cation was effective in stabilizing the enzyme, especially at higher temperatures. The proteolytic activity was inhibited by the specific serine proteinase inhibitor PMSF (phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, and Hg. The enzyme was stable in the presence of detergents, such as Triton-X100, Tween-80, SDS (sodium dodecyl sulfate), and EDTA (ethylenediaminetetraacetic acid), at pH 11.5 and 37°C for 30 min. The optimum pH was 11.5 at 37°C, and the optimum temperature was 62°C at pH 11.5.     

Utility of alkaline protease from marine shipworm bacterium in industrial cleansing applications

Summary An alkaline protease, previously isolated from a symbiotic bacterium found in the gland of Deshayes of marine shipworm, was evaluated as a cleansing additive. The protease nearly doubled the cleaning power of a standard phosphate detergent at temperatures up to 50°C as determined by fabric swatch assays. The enzyme was, however, ineffective at 70°C. In both fresh and seawater, it was also an efficient presoak. The cleaning power of a non-phosphate detergent was significantly improved by added protease, independent of the pH range 10 to 12. The enzyme degraded lysozyme, the major protein contaminant of contact lenses, more extensively than subtilisin and was effective in solutions containing hydrogen peroxide, often employed to sterilize lenses. The protease was unusually stable in sodium perborate, as well as hydrogen peroxide, and retained good activity in the presence of sodium hypochloride.     

Bacillus stearothermophilus KP 1236 neutral protease with strong thermostability comparable to thermolysin

Summary An extracellular neutral protease, of Bacillus stearothermophilus KP 1236 (a soil isolate) able to grow at 39°–71°C was purified to homogeneity. The molecular weight, sedimentation coefficient in water at 20°C, and isoelectric point were estimated as 33,000, 3.46 S and 7.5, respectively. The enzyme was most active at 80°C and pH 7.0. The activity was stable for 10 min up to 80°C at pH 7.5 and for 18 h at 60°C over pH 6.0–8.8. The enzyme and thermolysin (microbial metalloproteinase, EC 3.4.24.4) shared their antigenic determinants in part.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan on Sapporo, July 30, 1985 (Abstracts, p. 333)     

Extracellular alkaline protease of newly isolatedRhizopus oryzae

Summary A fungal isolate identified asRhizopus oryzae, produces an extracellular alkaline serine protease. Maximum protease formation was after six days in shake flask culture at two different conditions of pH and temperature optimum (pH 5 at 30°C and pH 10 at 37°C). AgNO₃ and Tween 80 increased protease synthesis. The enzyme is stable between pH 3 and pH 11 and has a temperature optimum of 60°C.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

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.     

Red cell hydrolases

Summary A 0.1% Triton X-100 extract of human erythrocyte plasma membranes contained high proteolytic activity as determined by a very sensitive assay utilizing³H-acetylated hemoglobin (162 cpm/pmole) as a substrate. Two proteolytic enzymes having optimum activity at pH 3.4 and pH 7.4 were isolated from Sephadex G-100. The protease active at pH 3.4 was 75 times as active as the pH 7.4 enzyme and it was purified 182-fold over the original homogenate and characterized. A linear relationship for activity versus time and activity versus concentration of enzyme was found. The optimum temperature was 37°C and theK _m was 1×10^–5 m hemoglobin. No enzyme activation was observed with any cation studied and EDTA had no inhibitory effect; (10mm Fe⁺³ and Hg⁺² were inhibitory). The pH 3.4 protease was stable indefinitely at –20°C in 0.1% Triton X-100. Gel electrophoresis was performed on a sodium dodecylsulfate-mercaptoethanol enzyme preparation and two protein bands (mol. wt. 33,000 and 54,000) were evident for the Sephadex G-200 eluate containing the pH 3.4 protease.     

Pectin lyase from Aspergillus sp. CH-Y-1043

Aspergillus sp. CH-Y-1043 synthesizes pectin lyase when grown on citrus pectin at 37° C. Production is favoured by increased esterification degree of the pectin used as carbon source. This enzyme displays higher activity at pH values of 8.5–8.8 and temperatures of 40–45° C. The optimal substrate for the enzyme was highly esterified pectin and no enzymatic activity was registered on polygalacturonic acid. The activity is stimulated by, though not dependent on, divalent cations (Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺ and Co²⁺) and inhibited by Zn²⁺, and it is not sensitive to the addition of EDTA. The enzyme is very stable when exposed to pH variations: at 4° C it preserves more than 95% of its activity at pHs ranging from 2.0 to 10.0, and at 30° C stability is preserved at pHs ranging from 4.0 to 8.0. At a constant pH of 5.0, the enzyme conserves its stability at temperatures ranging from 4 to 50° C and at pH 8.0 sensitivity to temperature increased. The results on the endo-exo nature of the enzyme suggest that this is an exo-pectin lyase. Correspondence to: G. Aguilar     

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.     

Application of thermostable xylanase of Dictyoglomus sp. in enzymatic treatment of kraft pulps

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improved pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90°C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80°C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.Correspondence to: M. Rättö     

A hyper-thermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation

A hyper-thermostable, alkaline lipase from a newly-isolated, mesophilic Pseudomonas sp. was optimal at pH 11 and at 90 °C. It had a half-life of more than 13 h at 90 °C. It was activated by 30% when heated at 90 °C for 2 h. The enzyme had a greater affinity for mustard oil (K _m=40 mg ml^–1) than for olive oil (K _m=140 mg ml^–1).     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

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     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.