Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.     

Purification and partial characterization of three extracellular cellulases from Cellulomonas sp.

Three extracellular cellulases have been purified from cultures of Cellulomonas. One was found in solution in the cell-free supernatant and two others were found to be bound to the cellulose added as a carbon source. The free enzyme and one of the cellulose-bound enzymes bind to Sephadex. The two cellulose-bound enzymes are glycosylated. The three enzymes behave as endocellulases towards soluble carboxymethyl-cellulose and have little activity on cellulose powder.     

Production and partial characterization of lipases from a newly isolated Penicillium sp. using experimental design

Aims:  The objective of this work was to investigate the lipase production by a newly isolated Penicillium sp . , using experimental design technique, in submerged fermentation using a medium based on peptone, yeast extract, NaCl and olive oil, as well as to characterize the crude enzymatic extracts obtained.
Methods and Results:  Lipase activity values of 9·5 U ml⁻¹ in 96 h of fermentation was obtained at the maximized operational conditions of peptone, yeast extract, NaCl and olive oil concentrations (g l⁻¹) of 20·0, 5·0, 5·0 and of 10·0 respectively. The partial characterization of crude enzymatic extract obtained by submerged fermentation showed optimum activity at pH range from 4·9 to 5·5 and temperature from 37°C to 42°C. The crude extract maintained its initial activity at freezing temperatures up to 100 days.
Conclusions:  A newly isolated strain of Penicillium sp . used in this work yielded good lipase activities compared to the literature.
Significance and Impact of the Study:  The growing interest in lipase production is related to the potential biotechnological applications that these enzymes present. New lipase producers are relevant to finding enzymes with different catalytic properties of commercial interest could be obtained, without using genetically modified organisms (GMO).     

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15 °C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9 kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40 °C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10–90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (ee_p 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

     

Purification and characterization of lipases from wheat germ

A method of isolation and purification of lipase (EC 3.1.1.3) from the germ of wheat (Triticum aestivum) is described. Electrophoretically homogeneous preparation of the enzyme (specific activity, 622.5 x x 10(-3) mumol/min per mg protein) was obtained after purification in 61 times. The molecular weight of the enzyme, determined by gel chromatography, was 143 +/- 2 kDa. The optimal conditions for the enzyme were 37 degrees and pH 8.0. Homogeneous preparation of the lipase exhibited high thermal stability: over 20% of original activity was retained after incubation of the preparation at high temperatures (60-90 degrees) for 1 h at pH 8.0.     

Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and pI were approximately 40 and 50 kDa, and approximately 4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 degrees C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0-7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 degrees C. However, EG II was active over pH 4.0-6.0 and temperature 40-80 degrees C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-beta-glucan as compared to CMC. Km values of EG I and EG II for barley-beta-glucan were lower than CMC. Turn over number (K(cat)) and catalytic efficiency (K(cat)/Km) values of both the endoglucanases were higher with barley-beta-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD.     

Separation and characterization of 61- and 57-kDa lipases from Geotrichum candidum ATCC 66592.

Two lipolytic proteins (61 and 57 kDa) present in a Sephadex G-100 fraction of extracellular lipase from Geotrichum candidum ATCC 66592 were separated using high-performance liquid chromatography. Crossed electrofocusing immunoelectrophoresis was used to demonstrate that the 61-kDa lipase fraction contained two forms of lipase with pI 4.5 and 4.7. However, when deglycosylated with endoglycosidase H, the two forms gained an identical pI, 4.6. The 57-kDa lipase fraction contained one form of lipase with pI close to 4.5. Although the 61- and 57-kDa lipases were immunologically identical, the substrate specificity differed. Thus, the 61-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was 60% of the initial velocity of hydrolysis of oleic acid methyl ester, whereas the 57-kDa lipase hydrolysed palmitic acid methyl ester at an initial velocity of hydrolysis that was only 7% of the initial velocity of hydrolysis of oleic acid methyl ester.     

Purification and properties of two lipases from pig adipose tissue.

Two lipases were purified from pig adipose tissue after delipidation by a mild and effective procedure using mixtures of chloroform and butanol. This was followed by hydrophobic adsorption chromatography on aminohexyl-Sepharose 4B coupled with octanoic acid, gel filtration on Sephadex G-100, and isoelectric focusing. Two electrophoretically and chromatographically pure enzymes were obtained, which had the same molecular weight (60 000 +/- 3000) and specific activity, and almost identical amino acid compositions; the isoelectric points, i.e. 5.2 and 5.5, differed.     

Purification and partial characterization of two basic proteins from human peripheral nerve.

Two basic proteins, denoted P1 and P2 protein, were purified from human sciatic nerve. The isolation was achieved by the following steps: delipidation with chloroform/methanol mixtures, dry acetone and dry ether; acid extraction at pH 2; ion exchange chromatography on QAE-Sephadex A-25 and gel filtration on Sephadex G-100. P1, P2 proteins and the basic protein of the central nervous system have been shown to have different electrophoretic mobility, and each of the two peripheral basic proteins was shown to be homogeneous by disc electrophoresis. The molecular weight of P1 protein is around 14 100 and that of P2 protein is around 12 200, as determined by ultracentrifugal analysis. There was some difference in the amino acid composition of human P1 and P2 protein, and a marked difference between their composition and the composition of central basic protein and bovine peripheral P1 and P2 proteins which were described previously. When injected to animals, P1 protein induced only experimental allergic neuritis while P2 protein induced both mild experimental allergic neuritis and experimental allergic encephalomyelitis. Thus, the human P1 protein is similar to the bovine P1 protein and human P2 protein is similar to bovine P2 protein, concerning their electrophoretic mobilities, molecular weights and biological properties.     

Purification and characterization of a new bacteriocin isolated from a Carnobacterium sp.

A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, was purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin UI49 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin UI49 belongs to the class of bacteriocins termed lantibiotics.     

Purification and characterization of a new laccase from Shiraia sp.SUPER-H168

A new laccase from Shiraia sp.SUPER-H168 was purified by ion exchange column chromatography and gel permeation chromatography and the apparent molecular mass of this enzyme was 70.78 kDa, as determined by MALDI/TOF-MS. The optimum pH value of the purified laccase was 4, 6, 5.5 and 3 with 2,6-dimethoxyphenol (DMP), syringaldazine, guaiacol and 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as substrates, respectively. The optimum temperature of the purified laccase was 50 °C using DMP, syringaldazine and guaiacol as substrates, but 60 °C for ABTS. Inhibitors and metal ions of SDS, NaN₃, Ag⁺ and Fe³⁺ showed inhibition on enzyme activity of 10.22%, 7.86%, 8.13% and 67.50%, respectively. Fe²⁺ completely inhibited the purified laccase. The K_cat/K_m values of the purified laccase toward DMP, ABTS guaiacol and syringaldazine were 3.99 × 10⁶, 3.74 × 10⁷, 8.01 × 10⁴ and 2.35 × 10⁷ mol^?1 L S^?1, respectively. The N-terminal amino acid sequence of the purified laccase showed 36.4% similarity to Pleurotus ostrestus. Approximately 66% of the Acid Blue 129 (100 mg L^?1) was decolorized by 2.5 U of the purified laccase after a 120 min incubation at 50 °C. Acid Red 1 (20 mg L^?1) and Reactive Black 5 (50 mg L^?1) were decolorized by the purified laccase after the addition of Acid Blue 129 (100 mg L^?1).     

New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases

A Photobacterium strain, M37, showing lipolytic activity, was previously isolated from an intertidal flat of the Yellow Sea in Korea and identified as Photobacterium lipolyticum sp. nov. In the present study, the corresponding gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,023 bp) corresponded to a protein of 340 amino acid residues with a molecular weight of 38,026. No sequence similarity was found with any known bacterial lipases/esterases; instead, the most similar enzymes were several filamentous fungal lipases. Although the similarity was very low (less than 16%), there were many conserved regions over the entire sequence and N-terminal oxyanion hole (RG) region, a signature sequence of filamentous fungal lipases. The novel protein M37 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18°C. The soluble protein exhibited lipase activity in a pH-stat assay using an olive oil emulsion. The M37 lipase also displayed a maximum activity at 25°C and maintained its activity at a low temperature range (5–25°C) with an activation energy (E _a) of 2.07 kcal/mol. Accordingly, these results indicate that the M37 lipase from P. lipolyticum sp. nov. is a new cold-adapted enzyme.     

DNA ligases from rat liver. Purification and partial characterization of two molecular forms

The differential ability of mammalian DNA ligases to use oligo(dT).poly(rA) as a substrate has been used to detect, and thereby extensively purify, two immunologically distinct forms of DNA ligase from rat liver. The activity of DNA ligase I, which is unable to use this template, is uniquely increased during liver regeneration, while that of DNA ligase II remains at a low level. Both enzymes require ATP and Mg2+ for activity and form an adenylylated intermediate which is stable and reactive. After SDS-PAGE, such radiolabeled complexes correspond to polypeptides of 130,000 and 80,000 Da for DNA ligase I and to 100,000 Da for DNA ligase II. That these labeled polypeptides do indeed correspond to active polypeptides of two different forms of DNA ligase is shown by the removal of the radiolabeled AMP, only when the intermediate is incubated with an appropriate substrate. In contrast to other eukaryotic DNA ligases, rat liver DNA ligase II has a lower Km for ATP (1.2 X 10(-5) M) than DNA ligase I (6 X 10(-5) M). Also, DNA ligase II can use ATP alpha S as a cofactor in the ligation reaction much more efficiently than DNA ligase I, further discriminating the ATP binding sites of these enzymes. Finally, antibodies raised against the 130,000-Da polypeptide of DNA ligase I specifically recognize this species in an immunoblot and inhibit only the activity of DNA ligase I.     

Multiple crystal forms of lipases from Geotrichum candidum

Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified.     

Adenosylcobalamin-dependent ribonucleotide reductase from the blue-green alga, Anabaena sp. Purification and partial characterization

The ribonucleotide reductase from Anabaena 7119 has been purified approximately 60- to 80-fold by conventional techniques and adsorption to the affinity medium, Matrix Gel Red A. The enzyme from Anabaena resembles the adenosylcobalamin-dependent reductase from Lactobacillus leichmannii, in that it is a small molecule (molecular weight 72,000) with no subunit structure as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Unlike its prototype, the Anabaena reductase is absolutely dependent on a divalent cation for activity, Ca2+ being the most effective. In addition, the Anabaena reductase shows a simple pattern of alloteric control by deoxyribonucleotides. CTP reduction is stimulated by dATP, GTP by dTTP, and ATP by dGTP. No reduction is observed in the absence of effectors, and none of the effectors inhibits enzyme activity. Thus, the Anabaena ribonucleotide reductase can be more easily studied by kinetic analysis than the Lactobacillus enzyme, and should provide additional information as to the mechanism of action of this enzyme in a photosynthetic organism.     

Purification and partial characterization of two lectins from the cactus Machaerocereus eruca

Two lectins (MEAI and MEAII) were isolated from the cactus Machaerocereus eruca by affinity chromatography on mucin-Sepharose and partially characterized with respect to their biochemical and carbohydrate binding properties. Both are oligomeric glycoproteins consisting of 35 kDa monomers. Amino acid analysis indicates that both lectins have similar composition with high amounts of glycine, glutamic acid and serine. MEAI and MEAII contain approximately 36 and 24% (w/w) of carbohydrates, respectively. They agglutinate erythrocytes from several animal species. Binding specificity was directed to galactose-containing oligosaccharides and glycopeptides. The M. eruca lectins are the first lectins to be isolated from a species belonging to the plant family of Cactaceae.     

Purification and partial characterization of two azoreductases from Shigella dysenteriae Type 1

Two azoreductases (I and II) were purified to homogeneity from extracts of Shigella dysenteriae (type 1). Azoreductase I was a dimer of identical subunits of M(r) 28,000, whereas azoreductase II was a monomer of 11,000 M(r). Both were flavoproteins, each containing 1 mol of FMN per mol enzyme. Both NADH and NADPH functioned as electron donors for the azoreductases. Azoreductase I used Ponceau SX, Tartrazine, Amaranth and Orange II as substrates. Azoreductase II utilized all the dyes except Amaranth.     

Purification and characterization of novel raw-starch-digesting and cold-adapted alpha-amylases from Eisenia foetida

Novel raw-starch-digesting and cold-adapted alpha-amylases (Amy I and Amy II) from the earthworm Eisenia foetida were purified to electrophoretically homogeneous states. The molecular weights of both purified enzymes were estimated to be 60,000 by SDS-PAGE. The enzymes were most active at pH 5.5 and 50 degrees C and stable at pH 7.0-9.0 and 50-60 degrees C. Both Amy I and II exhibited activities at 10 degrees C. The enzymes were inhibited by metal ions Cu(2+), Fe(2+), and Hg(2+), and hydrolyzed raw starch into glucose, maltose and maltotriose as end products.     

响应面法优化Burkholderiasp．SYBCLIP—Y发酵产低温脂肪酶条件的研究

用响应面法对Burkholderiasp．SYBCLIP—Y液体发酵产低温脂肪酶的发酵条件进行了快速优化。首先利用Plackett—Burman设计对影响其产酶相关因素进行评估并筛选出具有显著效应的三个因素：牛肉膏,橄榄油,TritonX-100;用最陡爬坡路径逼近最大产酶区域后,利用响应面中心组合设计对显著因素进行优化,确定出牛肉膏,橄榄油,TritonX-100的最佳浓度分别为：牛肉膏31．8g／L、橄榄油21mL／L、TritonX-10036．55mL／L,优化后脂肪酶的酶活达到61．52U／mL,是优化前的2．62倍。     

Purification and characterization of two small heat shock proteins from Anabaena sp. PCC 7120

Two small heat shock proteins (sHsps), Hsp17.8 and Hsp17.1, were identified in the cyanobacterium Anabaena sp. PCC 7120. Recombinant Hsp17.8 and Hsp17.1 were overexpressed in Escherichia coli and characterized here. Hsp17.8 was purified by sequential chromatography on DEAE-Sepharose and Superose 6 10/300 column, and Hsp17.1 was purified by Superose 6 10/300 column in 4M urea. Size exclusion chromatography demonstrated that both purified proteins form large oligomers approximately 420kDa and 410kDa, respectively. Both Hsp17.8 and Hsp17.1 showed chaperone-like activity to protect citrate synthase (CS) from thermal aggregation at 43 degrees C. Furthermore, both proteins were found to form complexes with denatured CS at 45 degrees C. Our study also demonstrated that despite a high degree of sequence homology and similar subunit size, Hsp17.1 showed higher hydrophobicity indicated by 8-anilino-1-naphthalene sulfonate fluorescence and thus greater chaperone-like activity. This is the first report of characterization and comparison of an sHsp system containing two chaperones in cyanobacteria.