Pongamia pinnata seed cake: a promising and inexpensive substrate for production of protease and lipase from Bacillus pumilus SG2 on solid-state fermentation

The production of a protease and a lipase from Bacillus pumilus SG2 on solid-state fermentation using Pongamia pinnata seed cake as substrate was studied. The seed cake was proved to be a promising substrate for the bacterial growth and the enzyme production. The initial pH, incubation time and moisture content were optimized to achieve maximal enzyme production. Maximum protease production was observed at 72 h and that of the lipase at 96 h of incubation. The production of protease (9840 U/g DM) and lipase (1974 U/g DM) were maximum at pH 7.0 and at 60% moisture content. Triton X-100 (1%) was proved to be an effective extractant for the enzymes and their optimal activity was observed at alkaline pH and at 60 C. The molecular mass of the protease and lipase was 24 and 40 kDa, respectively. Both the enzymes were found to be stable detergent additives. The study demonstrated that inexpensive and easily available Pongamia seed cake could be used for production of industrially important enzymes, such as protease and lipase.     

Purification and characterization of a pineapple crown leaf thiol protease

A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

Protein storage vacuole acidification as a control of storage protein mobilization in soybeans

Soybean protease C1 (EC 3.4.21.25), the subtilisin-like serine protease that initiates the proteolysis of seed storage proteins in germinating soybean [Glycine max (L.) Merrill], was localized to the protein storage vacuoles of parenchyma cells in the cotyledons by immunoelectron microscopy. This was demonstrated not only in germination and early seedling growth as expected, but also in two stages of protein storage vacuole development during seed maturation. Thus, the plant places the proteolytic enzyme in the same compartment as the storage proteins, but is still able to accumulate those protein reserves. Since soybean protease C1 activity requires acidic conditions for activity, the hypothesis that the pH condition in the protein storage vacuole would support protease C1 activity in germination, but not in seed maturation, was tested. As hypothesized, acridine orange accumulation in the protein storage vacuole of storage parenchyma cells was detected by fluorescence confocal microscopy in seedlings before the onset of mobilization of reserve proteins as noted by SDS-PAGE. Accumulation of the dye was reversed by inclusion of the weak base methylamine to dissipate the pH gradient across the vacuolar membrane. Also as hypothesized, acridine orange did not accumulate in the protein storage vacuole of those parenchyma cells during seed maturation. These results were obtained using cells separated by pectolyase treatment and also using cotyledon slices.     

Acid Protease of Bovine Milk

Occurrence of milk acid protease in bovine casein in addition to alkaline protease was found and purification of this enzyme was achieved. The enzyme had a pH optimum at 4.0 and was most stable at pH 3.5. The molecular weight of the enzyme was 36,000 and no inhibition was observed by diisopropyl-fluorophosphate, EDTA etc. This enzyme is considered to be similar to cathepsin D.

Milk acid protease mainly hydrolyzed α_s-casein and similar change was observed in autolysis of casein at pH 5.5. It is suggested that milk acid protease may have some significance in cheese ripening.     

球形芽孢杆菌C_3—41菌株胞外蛋白酶特性

球形芽孢杆菌能够合成具杀蚊活性的蛋白晶体,该晶体在蚊中肠碱性条件下降解产生毒性,尽管球形芽孢杆菌蛋白酶与杀蚊毒素的降解无关,但它在球形芽孢杆菌杀蚊制剂的产生中有重要意义。同时球形芽孢杆菌产生的碱性蛋白酶具有潜在的医疗价值。 我们以本实验室分离的高效杀蚊菌C_3—41菌株为材料,研究了球形芽孢杆菌蛋白酶的产生特性及其理化性质,在国内尚属首次报道。     

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.     

Coupling of the Penicillium duponti acid protease to ethylene-maleic acid (1 : 1) linear copolymer. Preparation and properties of the water-soluble derivative.

The coupling of the thermostable acid protease (EC 3.4.23.-) of Penicillium duponti K 1014 to ethylene-maleic acid (1 : 1) linear copolymer in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide at pH 3.0, afforded a soluble enzyme derivative with a protein incorporation yield of 67% under optimal conditions. The protein content of the enzyme-polymer complex, the molecular weights of the reactants, and the mean value of 2.2 lysine residues per mol of enzyme found in amide linkage to the matrix, support a structure consisting of two polymer chains per mol of protease, each chain acylating a single lysine residue of the enzyme. The isoelectric point of the coupled enzyme was found to be 3,47, a value lower than that measured on the free protease (3.81). The specific activity of the bound protease against casein, at pH 3.7 and 30 degrees C, was 34% of that of the free enzyme, and at 75 degrees C increased to 70%. The increased size of the coupled enzyme resulted in an improved retention of activity by ultrafiltration membranes over that observed with free protease, alone or in admixture with ethylene-maleic acid copolymer. A water-soluble, coupled pepsin was prepared in 43% yield on protein basis by using the aminoethylmonoamide of ethylene-maleic acid copolymer and the same water-soluble carbodiimide.     

InhA-like protease secreted by Bacillus sp. S17110 inhabited in turban shell

A strain producing a potent protease was isolated from turban shell. The strain was identified as Bacillus sp. S17110 based on phylogenetic analysis. The enzyme was purified from culture supernatant of Bacillus sp. S17110 to homogeneity by ammonium sulfate precipitation, SP-Sepharose, and DEAE-Sepharose anion exchange chromatography. Protease activity of the purified protein against casein was found to be stable at pH 7 to pH 10 and around 50 degrees . Approximately 70% of proteolytic activity of the enzyme was detected either in the presence of 100 mM SDS or Tween 20. The enzyme activity was enhanced in the presence of Ca2+, Zn2+, Mg2+, but was inhibited by EDTA, indicating that it requires metal for its activity. The purified enzyme was found to be a monomeric protein with a molecular mass of 75 kDa, as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The purified enzyme was analyzed through peptide fingerprint mass spectra generated from matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) and a BLAST search, and identified as immune inhibitor A (inhA) deduced from nucleotide sequence of B. cereus G9241. Since InhA was identified as protease that cleave antibacterial proteins found in insect, inhA-like protease purified from Bacillus sp. S17110 might be pathogenic to sea invertebrates.     

Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean beta-conglycinin seed proteins

The protease that degrades the beta subunit of the soybean (Glycine max (L.) Merrill) storage protein beta-conglycinin was purified from the cotyledons of seedlings grown for 12 days. The enzyme was named protease C2 because it is the second enzyme to cleave the beta-conglycinin storage protein, the first (protease C1) being one that degrades only the alpha' and alpha subunits of the storage protein to products similar in size and sequence to the remaining intact beta subunit. Protease C2 activity is not evident in vivo until 4 days after imbibition of the seed. The 31 kDa enzyme is a cysteine protease with a pH optimum with beta-conglycinin as substrate of 5.5. The action of protease C2 on native beta-conglycinin produces a set of large fragments (52-46 kDa in size) and small fragments (29-25 kDa). This is consistent with cleavage of all beta-conglycinin subunits at the region linking their N- and C-domains. Protease C2 also cleaves phaseolin, the Phaseolus vulgaris vicilin homologous to beta-conglycinin, to fragments in the 25-28 kDa range. N-Terminal sequences of isolated beta-conglycinin and phaseolin products show that protease C2 cleaves at a bond within a very mobile surface loop connecting the two compact structural domains of each subunit. The protease C2 cleavage specificity appears to be dictated by the substrate's three-dimensional structure rather than a specificity for a particular amino acid or sequence.     

产碱性蛋白酶芽孢杆菌的鉴定

通过测量比较在碱性蛋白平板上产生的蛋白水解圈直径,从土壤中筛选到一株高产蛋白酶菌株Bacillus sp.HFBL0079,根据生理生化特性、16S rDNA序列,鉴定为B.amyloliquefaciens。其最适培养温度为35°C-37°C,最适生长pH 8.0,在特定培养条件下16 h达到稳定期,菌体生长和蛋白酶合成同步进行。以大豆分离蛋白为氮源时发酵液具有最高酶活。发酵液在pH 10时具有最高酶活,表明为碱性蛋白酶。该菌株产生的碱性蛋白酶可水解多种天然蛋白质,对胶原蛋白水解度高于其他蛋白质,对羽毛角蛋白也有一定水解能力,提示该酶具有一定新颖性。     

A novel extracellular subtilisin-like protease from the hyperthermophile<Emphasis Type="Italic"> Aeropyrum pernix</Emphasis> K1: biochemical properties,cloning, and expression

A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0-12.0) and temperature (60-120 degrees C) with maximal activity at 90 degrees C and between pH 8.0 and 9.0. In the presence of 1 mM CaCl(2) the activity, as a function of the temperature, reached a maximum at 90 degrees C but at 120 degrees C the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione- S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.     

Enzymatic properties of a novel highly active and chelator resistant protease from a Pseudomonas aeruginosa PD100

Pseudomonas aeruginosa PD100 capable of producing an extracellular protease was isolated from the soil collected from local area (garbage site) from Shivage market in Pune, India. The purified protease showed a single band on native and SDS-PAGE with a molecular weight of 36 kDa on SDS-PAGE. The optimum pH value and temperature range were found to be 8 and 55–60 °C, respectively. The enzyme exhibited broad range of substrate specificity with higher activity for collagen. The enzyme was inhibited with low concentration of Ag²⁺, Ni²⁺, and Cu²⁺. β-Mercaptoethanol was able to inactivate the enzyme at 2.5 mM, suggesting that disulfide bond(s) play a critical role in the enzyme activity. Studies with inhibitors showed that different classes of protease inhibitors, known to inhibit specific proteases, could not inhibit the activity of this protease. Amino acid modification studies data and pK_a values showed that Cys, His and Trp were involved in the protease activity. P. aeruginosa PD100 produces one form of protease with some different properties as compared to other reported proteases from P. aeruginosa strains. With respect to properties of the purified protease such as pH optimum, temperature stability with capability to degrade different proteins, high stability in the presences of detergents and chemicals, and metal ions independency, suggesting that it has great potential for different applications.     

Purification and characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver

Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver.     

Purification and Properties of a Proteolytic Enzyme,Cathepsin D,from Chicken Muscle

Cathepsin D was isolated from crude extract of chicken muscle by the purification procedures of acid- and heat-treatments, ammonium sulfate fractionation, DEAE-Sephadex A-50 column chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 3700 fold and homogeneous in disc-electrophoretic analysis. The molecular weight was found to be about 36,000 and the isoelectric point to be pH 7.3. The best substrate for this enzyme was 6 m urea-denatured casein, and its activity was maximal at pH 3.5 and 40°C. This enzyme was most stable between pH 4 and 5, and its stability was affected by cupric ion. The enzyme activity was markedly inhibited by sodium laurylsulfate and oxidizing agents such as potassium permanganate, N-bromosuccinimide and iodine, and was slightly activated by hydrogen peroxide. The purified cathepsin D was found to be fairly similar to the acid protease from lotus seed, previously reported by the authors.     

Separation and comparative characterization of the cationic protease and anionic protease from the culture medium of Thermoactinomyces vulgaris

The anionic protease component which frequently contaminates preparations of routinely isolated cationic protease (thermitase) from Thermoactinomyces vulgaris was purified, virtually to homogeneity, by rechromatography on controlled pore glass (CPG-10). Starting materials were column eluates with anionic protease, contaminated with residual thermitase activity. The purified anionic enzyme shares several properties with thermitase, such as size, sensitivity against phenylmethanesulfonyl fluoride and Hg2+, UV-spectral, immunological and pH behavior. On the other hand, the isoelectric point (at pH 6.5), temperature dependence (more heat stable) and enzymatic activity (less active) of anionic protease differ significantly from thermitase. At pH 8 or 6 and 25 degrees or 4 degrees C anionic protease is hydrolysed completely by thermitase. Like other protein substrates, anionic protease simultaneously acts as a stabilizer for thermitase. In contrast to thermitase, the anionic enzyme partially changes spontaneously during long-term storage at 4 degrees C and pH 6 to a cationic protein species endowed with proteolytic activity.     

Characterization of an Extracellular Protease from the Insect Pathogen Xenorhabdus luminescens

Xenorhabdus luminescens Hm cultured in gelatin broth produced a single extracellular protease. The protease was purified by a factor of 500 and characterized as a monomeric protein with an approximate molecular weight of 61,000. On the basis of inhibitor studies and its pH optimum, the protease was classified as an alkaline metalloprotease with a pH optimum near 8; the isoelectric point of the enzyme is 4.2 +/- 0.2. The protease may be a major factor in the ecology of X. luminescens, which is carried as a symbiom of some parasitic nematodes.     

Purification and Some Properties of a Protease from the Sarcocarp of Musk Melon Fruit

A protease has been purified from sarcocarp of musk melon, Cucumis melo ssp. melo var. reticulatus Naud. Earl’s Favourite. The protease was mostly present in the placenta part of the fruit and next in the inside mesocarp. The molecular mass of the enzyme was estimated to be about 62kDa on SDS-PAGE. The enzyme had a carbohydrate moiety. The optimum pH of the enzyme was 11 at 35°C using casein as a substrate. The enzyme was stable between pH 6 and 11. The enzyme was strongly inhibited by diisopropyl fluorophosphate, but was not inhibited by EDTA or cysteine protease inhibitors. From the digestion of Ala-Ala-Pro-X-pNA (X = Phe, Leu, Val, Ala, Gly, Lys, Glu, Pro, and diaminopropionic acid (Dap) substrates the specificity of the protease was found to be approximately broad, but the preferential cleavage sites were C-terminal sites of hydrophobic or acidic amino acid residues at P, position. It was proved that the enzymatic properties of musk melon protease are similar to those of cucumisin [EC 3.4.21.25]. The enzyme was not inhibited by typical proteinous inhibitors such as STI or ovomucoid. Therefore, this enzyme seems to be a useful protease for the food industries.     

雅致放射毛霉AS3.2778碱性蛋白酶的纯化及性质研究

利用盐析,离子交换,疏水层析及凝胶过滤的方法从雅致放射毛霉AS3.2778的发酵麸曲中分离纯化出一碱性蛋白酶,其纯化提高了22.7倍,酶活回收率16.1%,最终比酶活可达到6094u/mg。电泳分析发现,该蛋白酶是一单体蛋白,其分子量大约在32KDa。性质分析表明：该蛋白酶在60℃、pH8.5～10.5具有最大催化活性;在40℃以下,pH6.0～9.0的范围有很好的稳定性;1mM的PMSF可以完全抑制其活性,显示该蛋白酶属于丝氨酸蛋白酶家族。底物专一性的研究发现,该蛋白酶有相当广泛的肽键选择性,对绝大多数由疏水性氨基酸（尤其是亮氨酸）构成的肽键有很强的水解能力。     

Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.