Partial Structure of Glycopeptide Obtained from Scytalidium lignicolum Acid Protease A

The partial structure of glycopeptide moiety of new acid protease A isolated from Scytalidium lignicolum ATCC 24568 was studied by Smith degradation, methylation and partial acetolysis techniques. The main product, glycopeptide V (GP-V), obtained by Pronase digestion was composed of mannose, glucosamine, asparagine, serine and glycine in an approximate molar ratio of 10: 3: 2: 1: 1, and a possible structure was proposed as follows:     

Expression and Secretion of Scytalidopepsin B,an Acid Protease from Scytalidium lignicolum,in Yeast

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.     

Purification and Some Properties of Milk Protease

Separation of acetic acid from palm oil mill effluent (POME) to increase its concentration by an anion exchange resin was examined as a preliminary study for its recovery from POME that had been anaerobically treated by sludge from a palm oil mill. This paper concerns the acetic acid thus separated for producing bacterial polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus. It was found that sludge particles in POME strongly inhibited the adsorption of acetic acid on the anion exchange resin. Removing the sludge particles from the POME facilitated the separation of acetic acid from the POME efficiently. The concentrated acetic acid thus obtained from anaerobically treated POME could be used as a substrate in the fed-batch production of polyhydroxyalkanoate by Alcaligenes eutrophus.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Purification and Properties of Mucor pusillus Acid Protease

The protease produced by Mucor pusillus was recovered from a wheat bran medium by treatment with ammonium sulfate, ethyl alcohol, gel filtration and ion-exchange chromatography. The yield of the enzyme was 55%. The overall increase in the specific activity of the protease was 34-fold. The purified protease was most active at pH 3.8 and 5.6 against hemoglobin and casein, respectively. Optimal hydrolysis of casein was observed at 55 C. The enzyme was stable from pH 3.0 to 6.0. Enzyme inactivated by metal ions was reactivated by ethylenediaminetetraacetate and o-phenanthroline. Reducing agents and thiol poisons had no effect on the protease, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inhibit the protease, indicating the probable absence of serine in the active center. The Michaelis-Menten constant for casein was 0.357%. Electrophoretic analysis of active protein recovered by ion-exchange chromatography showed that the protease preparation was homogeneous.     

Enzymatic Hydrolysis of Esters Containing a Tetrazole Ring

The lipase‐catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4‐(5‐aryl‐2H‐tetrazol‐2yl)butan‐2‐ol and 1‐(5‐aryl‐2H‐tetrazol‐2yl)‐propan‐2‐ol and their acetates with the highest optical purities (ee = 95%‐99%) and excellent enantioselectivity (E>100). Some of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mixtures of 4‐[5‐(4‐chlorophenyl)‐2H‐tetrazol‐2‐yl)butan‐2‐ol as well as pure enantiomers of this compound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes, and A. niger. Chirality 26: 811–816, 2014. © 2014 Wiley Periodicals, Inc.     

Purification and Some Properties of a Protease from Streptomyces limosus

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.     

Chemoenzymatic synthesis and properties of Schiff bases containing (R)-1-(9-anthryl)ethylamine

Racemic 1-(9-anthryl)ethylamine (10), obtained in 70% overall yield from commercial 9-cyanoanthracene, was kinetically resolved by the Candida antarctica A lipase-catalyzed acetylation with isopropyl acetate as acyl donor, affording (R)-(+)-10 with 95.8% enantiomeric excess (e.e.) (E-value 43.5), which afforded Schiff bases (R)-4 and(R)-8. (1)H-NMR, CD, and MM2 calculations offer a consistent picture of the conformational properties of these potential ligands and an explanation for the limited enhancement of enantioselectivity in cyclopropanation of styrene by their Cu(I) complexes, as compared with previously studied ligands in this series.     

Synthesis,Enzymatic Resolution,and Stereochemical Characterization of Isoparaconic Acid Derivatives: A Combined Experimental and Theoretical Investigation

Enantiomerically enriched isoparaconic acid derivatives were obtained by kinetic enzymatic resolution. To explain the solvent dependence observed for their optical rotatory power a computational investigation of their chiroptical properties was performed. Chirality 26:640–650, 2014. © 2014 Wiley Periodicals, Inc.     

Purification and Some Properties of Extracellular Protease of Euglena gracilis Z

The extracellular protease of Euglena gracilis z was purified to a single protein. It was an endopeptidase as found by the Nunokawa’s method, and showed optimum pH for the proteinase, esterase and amidase activities at 7.3, 7.0 and 6.3, respectively. It had a molecular weight of 41,000 and isoelectric point of 8.3. The bleached mutant of E. gracilis produced higher activity of extracellular protease than the wild strain, and supplementation of peptone to the growth medium augmented the enzyme production in both green and bleached cells. The Euglena extracellular protease was markedly inhibited by diisopropylfluorophosphate and Streptomyces subtilicin inhibitor, and to lesser extents by EDTA and p-chloromercuribenzoate. The enzyme was potentiated by some sulfhydryl compounds, activated greatly by Fe²⁺ and stabilized by Ca²⁺ and K⁺.     

Purification and Some Properties of An Aldehyde Oxidase from Streptomyces Rimosus ATCC10970

Summary An aldehyde oxidase was purified from a cell-free extract of Streptomyces rimosus ATCC10970 to an electrophoretically homogeneous state. The molecular mass of the native enzyme was estimated to be 150 kDa by a gel filtration. SDS-polyacryamide gel electrophoresis showed that the enzyme consisted of three non-identical subunits with molecular masses of 79, 39 and 23 kDa. The absorption spectrum revealed a distinctive feature as an enzyme belonging to the xanthine oxidase family with maxima at 277, 325, 365, 415, 450, 480, and 550 nm. A variety of aliphatic and aromatic aldehydes were oxidized, but nitrogen-containing heterocyclic compounds were not. Among the substrates tested, n-heptanal was most rapidly acted on. Its optimum pH and temperature were pH 7.0 and 30 °C, respectively.     

Partial Purification and Some Properties of Tyrosine Phenol-Lyase from Aeromonas phenologenes ATCC 29063

Tyrosine phenol-lyase was purified 32-fold from Aeromonas phenologenes ATCC 29063, the organism that produces phenol in refrigerated haddock. The purification procedure included ammonium sulfate fractionation, protamine sulfate treatment, and column chromatography with Sephadex G-200, diethyl-aminoethyl-cellulose, and hydroxyapatite. The enzyme was found to be thermally inactivated at temperatures above 40 degrees C. The optimum pH of the enzyme was found to be pH 8.5. The Michaelis constants for l-tyrosine and pyridoxal phosphate were 2.3 x 10 M and 3.2 x 10 M, respectively. The molecular weight of tyrosine phenol-lyase was found by gel filtration and electrophoresis to be approximately 380,000.     

Two Components of Cell-bound Isopullulanase from Aspergillus niger ATCC 9642—Their Purification and Enzymatic Properties

Cell-bound isopullulanase (pullulan 4-glucanohydrolase: EC 3.2.1.57, IPU) from Aspergillus niger ATCC 9642 [Y. Sakano et al, Denpun Kagaku, 37, 39–41 (1990)] was separated into two active components, IPU F1 (pI = 5.0) and IPU F2 (pI = 4.9), using a Mono-P HR 5/20 column. The substrate specificity on pullulan and panose, specific activity, optimum pH, pH stability, and susceptibility to certain chemical reagents were similar between IPU F1 and IPU F2. IPU F1 and F2 had an identical N-terminal amino acid sequence, A-V-T-A-D-N-S-Q-L-L-. However, IPU F1 contained more total carbohydrate (15.3%) than IPU F2 (12.4%). SDS–polyacrylamide gel electrophoresis showed that the molecular weight of IPU F1 (71,000) was greater than that of IPU F2 (69,000). After deglycosylation of IPU F1 and F2 with peptide-N-glycosidase F, the molecular weights of IPU F1 and F2 became 59,000.     

Purification and Some Properties of Acid Invertase of Persimmon Fruits

Single cells were prepared from mesocarp tissue of ripe persimmon (Diospyros kaki cv. Fuyu) fruits, and inter- or intracellular localization of acid invertase (AI, EC 3.2.1.26) was studied. AI was localized in the intercellular fraction (cell wall fraction). AI was isolated and purified from the cell wall fraction of ripe persimmon fruits by column chromatography on SE-53 cellulose and Toyopearl HW 55F. The specific activity of purified AI was 570 units per mg protein at 30°C. The molecular mass of AI was estimated to be 44 kDa by gel filtration over Sephacryl S-200 and 70 kDa by SDS–PAGE. The optimum pH of the activity for sucrose was 4.25. The purified enzyme hydrolyzed sucrose and raffinose but not melibiose. The enzyme had a K_m of 3.2 mM for sucrose and a K_m of 2.6 mM for raffinose. Silver nitrate (5 μM), HgCI₂ (2 μM), p-chloromercuribenzoate (100mM), pyridoxamine (10mM), and pyridoxine (2.5mM) inhibited AI activity by 95, 85, 100, 41, and 300%, respectively.     

乳酸菌Enterococcuse faecalis TN-9低温蛋白酶的提纯及性质

对产自乳酸菌Enterococcuze fecalis TN-9的蛋白酶,进行了硫酸铵沉淀,DEAE—Sephadex A-25以及DEAE Cellulofine A-500离子交换层析的3步纯化和特性研究。纯化酶Native PAGE显示1条蛋白带。SDSPAGE和凝胶层析分子量分别为30ku及69ku。纯化酶最适作用温度为30℃,最适作用PH为7．5～8．0,在pH6．0～9．5和45℃以下条件下稳定,在0℃下显示了6．1％的相对活性,60℃以上热处理完全失去酶活。该酶被EDTA-2Na,Hg^2＋、Cu^2＋、Ni^2＋、Ag^2＋、Co^2＋及Pepstatin A不完全抑制。Zn^2＋对蛋白酶具有明显的激活作用。纯化酶作用于偶氮酪蛋白的Km和Vmax分别为0．098％和72mg／（h·mg）。该酶为N末端VGSEVTLKNS的明胶酶（Gelatinase）的一种,性质属于低温蛋白酶。     

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.     

Kinetic resolution of (±)-diethyl- and dibenzyl hydroxy(phenyl)methanephosphonates and their acyl derivatives with lipases

Abstract

A wide variety of commercially available lipases and microbial whole cells were tested for biotransformations of (±)-diethyl and dibenzyl hydroxyl(phenyl)methanephosphonates. Biocatalytic hydrolysis of acylated hydroxyphosphonates by whole cells of Bacillus subtilis gave optically active compounds with 95%ee _S. Enantioselectivities obtained when using commercially available enzymatic preparations were less satisfactory, leading to both compounds with an enantiomeric excess in the range 15 35%. Screening lipases for their ability to acylate these phosphonates or to hydrolyze their acylated derivatives enabled selection of enzymes and organisms suitable for use in both processes.     

Enzymatic and chromatographic chiral discrimination of racemic (thio)glycidyl esters: Part I. A chemoenzymatic approach to both enantiomers of thioglycidyl esters

Both hitherto unknown (+)-(R)- and (?)-(S)-thioglycidyl esters, (R)-( 2 ) and (S)-( 2 ), have been synthesized with different high enantiomeric excesses (ee) by two routes from the corresponding rac-glycidyl esters rac-( 1 ). The first includes a porcine pancreatic lipase (PPL)-mediated kinetic resolution of these esters followed by sulfuration with practically complete inversion to the (+)-(R)-enantiomer (+)-(R)-( 2 ) (36–86% ee). (?)-(S)-Thioglycidyl esters (?)-(S)-( 2 ) are obtained by the reverse reaction sequence (43–80% ee). In the latter case the hydrolysis rate is lower than that of analogous glycidyl esters. Moreover, the dependence of enantiomeric excess on the size of the acyl-group is of the opposite tendency. Therefore, in both cases suitable selection of the acid residue gives rise to maximum enantioselectivity. The irreversible lipase-catalyzed acylation of rac-glycidol and rac-thioglycidol, however, was found to be a less suitable alternative. The enantiomeric excess of recovered homochiral esters was determined by chiral chromatography using modified cellulose stationary phases (OB, OD). © 1993 Wiley-Liss, Inc.