Screening and characterization of koji molds producing saline-tolerant protease

Three mold strains isolated from soil in the Taipei area of Taiwan were compared with a commercial strain of Aspergillus oryzae for their proteolytic activities in an 18% NaCl aqueous solution system. Among these strains, the one subsequently identified and designated as Aspergillus sp. FC-10 produced protease with superior saline tolerance. In aflatoxin tests, this strain did not generate detectable aflatoxin after growing on steamed grain polished rice substrate for 24 days. Two types of extracellular proteases were preliminary fractionated by column chromatography on DEAE Sepharose CL-6B. Proteolytic activity of the nonadsorbed protease (P-I) was reduced to 9.4% in the 18% NaCl solution compared to its original activity determined in the buffer solution. However, the adsorbed protease (P-II) was particularly salt tolerant and stable, with 50% proteolytic activity retained throughout the 6-h stability test in 18% NaCl solution. Journal of Industrial Microbiology & Biotechnology (2001) 26, 230–234. Received 06 June 2000/ Accepted in revised form 25 January 2001     

Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold.

A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.     

Survival strategy of the salt-tolerant lactic acid bacterium,Tetragenococcus halophilus,to counteract koji mold,Aspergillus oryzae,in soy sauce brewing

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.     

Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold

A soy sauce koji mold, Aspergillus flavus var. columnaris Raper and Fennel (ATCC 44310), was treated with UV irradiation to obtain mutant strains possessing high protease activities, high amylase activities, and light-colored conidia. Selected mutant strains were tested for toxicity, and some were found acutely toxic to weanling rats, although all were negative for aflatoxin production.     

Purification and characterization of a solvent and detergent-stable novel protease from Bacillus cereus

A protease-producing bacterium was isolated from slaughterhouse waste samples, Hyderabad, India. It was related to Bacillus cereus on the basis of 16S rRNA gene sequencing and biochemical properties. The protease was purified to homogeneity using ammonium sulfate precipitation, and ion exchange chromatography with a fold purification of 1.8 and a recovery of 49%. The enzyme had a relative molecular weight of 28 kDa, pH and temperature optima for this protease were 10 and 60 °C. The activity was stable between a pH range of 7.0 and 12.0. The activity was inhibited by EDTA and enhanced (four-fold) by Cu²⁺ ions indicating the presence of metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents and anionic surfactants. The enzyme also showed stability in the presence of organic solvents.     

Purification and characterization of a novel metalloprotease isolated from Aeromonas caviae

A novel protease produced by Aeromonas caviae was purified and characterized. The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS-polyacrylamide gel electrophoresis. The protease activity was not inhibited completely by heating at 100 degrees C for 15 min. The proteolytic activities were inhibited by metalloprotease inhibitor. The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN. AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test. A high concentration of the protease showed cytotoxic activity against Vero cells.     

Purification and characterization of a novel fibrinolytic protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. CPCC 480097

A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45°C and 8.5, respectively. Fu-P cleaved the α-chain of fibrin (ogen) with high efficiency, and the β-chain and γ-γ (γ-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S–S-G-T-P-A-T-I-R-V-L-V–V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.     

Purification and biochemical characterization of a protease secreted by the <Emphasis Type="Italic">Salinivibrio</Emphasis> sp. strain AF-2004 and its behavior in organic solvents

A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.     

Cloning and characterization of a chitosanase gene from the koji mold Aspergillus oryzae strain IAM 2660

A genomic copy of the gene coding for chitosanase (csnA) was isolated from Aspergillus oryzae IAM 2660. A. oryzae csnA contains an open reading frame that encodes a polypeptide of 245 amino acids with a calculated molecular mass of 26,500 Da. The deduced amino acid sequence of A. oryzae csnA indicates extensive similarities to those of other fungal chitosanases.     

Cloning, expression and characterization of a novel salt-tolerant xylanase from Bacillus sp. SN5

A xylanase gene (xyn10A) was cloned from Bacillus sp. SN5 and expressed in Escherichia coli. It encoded a 348-residue polypeptide of ~45?kDa. The deduced amino acid sequence had 68?% identity with the endo-1,4-beta-xylanase from Paenibacillus lactis 154 that belonged to family 10 of the glycoside hydrolases. Purified recombinant Xyn10A had maximum activity at 40?°C and pH 7.0, with the specific activity of 105?U/mg and a Km of 0.6?mg/ml for beechwood xylan. Xyn10A retained more than 80?% activity between 25 and 45?°C and 29?% activity at 5?°C. It exhibited the highest activity (134?%) in 0.5?M NaCl and still retained 90?% activity in 2.5?M NaCl. It retained about 87?% activity after incubation in 2?M NaCl for 24?h. The cold-active and halo-tolerant properties of Xyn10A make it promising for application in the food industry, especially in the processing of saline food and sea food.     

Purification and characterization of a serine protease secreted by Brevibacillus sp. KH3 for reducing waste activated sludge and biofilm formation

A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 °C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50 °C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.     

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum)

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20?mM tris-CaCl₂ buffer (pH 8.2) with a flow rate of 0.5?mL min^?1. The molecular weight and purity of ～23?kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697?U?mg^?1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015     

Cloning and characterization of a gene coding for a major extracellular chitosanase from the koji mold Aspergillus oryzae

A gene coding for a major extracellular chitosanase was isolated from Aspergillus oryzae IAM2660. It had a multi-domain structure composed of a signal peptide, a catalytic domain, Thr- and Pro-rich linkers, and repeated peptides (the R3 domain) from the N-terminus. The R3 domain bound to insoluble powder chitosan, but it did not promote the hydrolysis rate of the chitosanase to any extent.     

Aspergillus sp. RSD生淀粉糖化酶的分离纯化及酶学性质

【目的】纯化得到一种生淀粉糖化酶,并对其酶学性质进行分析。【方法】从曲霉RSD发酵液中,经过硫酸铵分级盐析,HiPrep DEAE FF16/10弱阴离子交换层析,凝胶过滤层析,Hiprep 16/10 source 30S阳离子交换层析最终纯化出一种电泳纯的生淀粉酶。【结果】粗酶液纯化倍数为12.65倍,活力回收率为9.02%,SDS-PAGE结果显示该酶的相对分子质量约为82 kD。对该酶的酶学性质分析结果表明,该酶最适作用温度为50°C,在50°C以下稳定性很好,对高温较为敏感;最适作用pH为4.5,在pH 3.5-7.0范围内酶活力较为稳定,在40°C、pH 4.6条件下以可溶性淀粉为底物时的Km值和Vmax值分别为7.44 g/L和1.45 g/(L·min);金属离子对酶活性的影响试验表明,Fe2+对该酶具有显著激活效果,EDTA、Cu2+、K+对该酶酶活力有不同程度的抑制作用;底物特异性研究表明该酶对麦芽糊精具有较高酶活力。【结论】与市售糖化酶及生淀粉糖化酶相比,该酶对生淀粉的降解能力更高,在工业应用上有较好的前景。     

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.     

Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi

An alpha-mannosidase differing from 1,2-alpha-mannosidase was found to occur in Aspergillus saitoi. By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.     

Purification and characterization of chitosanase and Exo-beta-D-glucosaminidase from a Koji mold, Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa exo-beta-D-glucosaminidase,enzyme,named released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

Purification and characterization of a propanol-tolerant neutral protease from Bacillus sp. ZG20

Abstract

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated K_m and V_max values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.