Purification and properties of family-10 endo-1,4-beta-xylanase from Penicillium citrinum and structural organization of encoding gene

An extracellular endo-1,4-beta-xylanase was purified from the culture filtrate of a filamentous fungus, Penicillium citrinum strain FERM P-15944, grown on birch-wood xylan. The purified enzyme showed a single band on SDS-PAGE with an apparent M(r) of 31.6 kDa. The xylanase activity was optimal at pH 6.0 and 50 degrees C. Southern blot analysis indicated that the xylanase gene (xynB) was present as a single copy in the genome. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. An open reading frame of xynB was interrupted by nine introns and encoded a presumed prepropeptide of 25 amino acids and a mature protein of 302 amino acids. Sequence alignment and the constructed neighbor-joining tree showed that the P. citrinum enzyme belongs to glycoside hydrolase family 10 and is closely related to several other xylanases from penicillia and black aspergilli. The xynB cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Employing chimeric xylanases to identify regions of an alkaline xylanase participating in enzyme activity at basic pH

The xylanase A (XynA) from the alkaliphilic Bacillus halodurans C-125 and the xylanase B (XynB) from Clostridium stercorarium F9 were subdivided into four fragments at highly homologous regions present in their primary structures: an amino-terminal region (A or a), a region containing the putative proton donor (P or p), a region containing the putative catalytic nucleophile (N or n), and a carboxyl-terminal region (C or c). Six chimeric xylanases were constructed by the selective substitution of the four fragments using an overlapping PCR technique. Two of the six xylanases, APnc and Apnc (regions originating from XynA are denoted by upper case letters and those from XynB are denoted by lower case letters), were produced in Escherichia coli while the other four xylanases were obtained only as inclusion bodies. The APnc and Apnc chimeric enzymes were purified by column chromatography using Ni-NTA agarose and DEAE-Toyopearl. The respective pH and temperature stabilities of the purified enzymes were observed from pH 5.6 to 11.6 and up to 45 degrees C for APnc, and from pH 5.6 to 11.2 and up to 45 degrees C for Apnc. Thus, these enzymes were slightly less stable than the parental xylanases. An assessment of the pH-activity relationships for the chimeric xylanases employed p-nitrophenyl-beta-D-xylobioside as the substrate in determinations of the k(cat) values. The pK(a1) values for the APnc and Apnc chimeric enzymes were 4.3 and 4.2, respectively, which were almost identical to those for the parental xylanases. In contrast, the pK(a2) values obtained for APnc and Apnc were 9.1 and 8.5, respectively; these values fall between those for the parental xylanases, XynA (9.4) and XynB (7.8). These results indicate that the main regions necessary to maintain the high pK(a2) value of XynA locate in the A and P sections.     

Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

巴氏葡萄球菌TS-82类胡萝卜素裂解酶的分离纯化及其酶学特性

巴氏葡萄球菌TS-82类胡萝卜素裂解酶经强阴离子柱、高效制备液相色谱和多肽分子筛纯化得到液相级纯酶(95.6%)。该酶比活力为125 U/g,纯化倍数为446,回收率为2.39%。纯化后的类胡萝卜素裂解酶经液相色谱-质谱联用测定,得其分子质量为655.093 D。关于酶学特性,研究发现该酶对C_(40)类胡萝卜素底物的最适温度为60℃,而作用于β-阿朴-8’-胡萝卜醛的最适温度是50℃,该酶的稳定温度为50℃以下;该酶对所测定底物的最适p H值为3.0;该酶与5种底物亲和力排列为:玉米黄质虾青素β-胡萝卜素角黄质β-阿朴-8’-胡萝卜醛;Al~(3+)和Fe~(3+)是该酶的强效催化剂,Fe~(2+)是该酶的强效抑制剂;H_2O_2在低浓度范围内(0~16 mmol/L)可促进酶活性;低体积分数乙醇(4%~16%)的添加对酶活性无明显抑制作用。结果表明该酶具有很好的耐酸性和热稳定性,能够适应果酒环境,为其工业化应用提供依据。     

Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus

We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100000 x g. Purification consisted in a fractionated precipitation with ammonium sulfate and two chromatographic steps consisting of anion exchange chromatography and hydrophobic interactions chromatography. The native enzyme depicted a molecular mass of 67 kDa by gel filtration. This serine carboxypeptidase depicted an optimal pH of 8.5 and was stable at a pH ranging from 6.0 to 9.0, its optimal temperature was of 45 degrees C and was unstable at temperatures above 55 degrees C; Michaelis constant and Vmax for N-benzoyl-l-tyrosine-p-nitroanilide were of 29 microM and 612 microM/min mg of protein, respectively. The enzyme was strongly inhibited by phenylmethylsufonyl fluoride (PMSF) and, to a lesser degree, by trans-epoxysuccinyl-l-leucylamido-(4-guanidine)-butane. This study indicated that K. marxianus carboxypeptidase could be an alternative to other animal-source carboxypeptidases in the industry.     

Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719

An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp. PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and alpha-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75 degrees C. The enzyme activity was stimulated by Ca2+, but was inhibited in the presence of Fe2+ or Cu2+. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80 degrees C in the absence of Ca2+. Since phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (DCI) in addition to N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy (CBZ)-L-arginine-p-nitroanilide with the K(m) and V(max) values of 0.6 mM and 1.0 micromol.min(-1).mg protein(-1), respectively.     

Purification and characterization of a monoacylglycerol lipase from Pseudomonas sp. LP7315

A monoacylglycerol lipase (MGL) was purified from Pseudomonas sp. LP7315 by ammonium sulfate precipitation, anion-exchange chromatography, and preparative electrophoresis. The purified enzyme was homogeneous on SDS-PAGE with a molecular mass of 59 kDa. Its hydrolytic activity was confirmed to be specific for monoglycerides: the enzyme did not hydrolyze di- and triglycerides. MGL was found to be stable even after 1-h incubation at 65 degrees C. The optimum pH for monopalmitin hydrolysis was approximately 8. The hydrolytic activity depended not only on temperature and pH but also on the type of monoglyceride used. MGL also catalyzed monoglyceride synthesis at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in the glycerol phase with a low water content. The synthetic reaction proceeded at a constant rate for approximately 24 h and approximately reached an equilibrium after 48 h of reaction. The initial rate and equilibrium yield of the synthetic reaction depended on the type of fatty acid used as the substrate.     

New alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus) with high activity at low temperature: Purification and characterisation

A highly alkaline trypsin from the intestine of Grey triggerfish (Balistes capriscus), with high activity at low temperature, was purified and characterised. The enzyme was purified to homogeneity using acetone precipitation, Sephadex G-100 gel filtration and Mono Q-Sepharose anion-exchange chromatography, with a 13.9-fold increase in specific activity and 41.3% recovery. The molecular weight of the purified alkaline trypsin was estimated to be 23.2 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and size exclusion chromatography. Purified trypsin appeared as a single band on native–PAGE. Interestingly, the enzyme was highly active over a wide range of pH, from 9.0 to 11.5, with an optimum at pH 10.5, using Nα-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as a substrate. The relative activities at pH 9.0, 11.5 and 12.0 were 86.5%, 92.6% and 52.4%, respectively. The enzyme was extremely stable in the pH range 7.0–12.0. In addition, the enzyme had high activity at low and moderate temperatures with an optimum at around 40 °C and had more than 80% of its maximum activity at 20 °C. The purified enzyme was strongly inhibited by soybean trypsin inhibitor (SBTI) and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. The enzyme showed extreme stability towards oxidising agents, retaining about 87% and 80% of its initial activity after 1 h incubation at 40 °C in the presence of 1% sodium perborate and 1% H₂O₂, respectively. In addition, the enzyme showed excellent stability and compatibility with some commercial solid detergents.     

Purification and some characteristics of a monomeric alanine racemase from an extreme thermophile, Thermus thermophilus

We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about 38,000. The enzyme was most active at pH 8 and 75 degrees C, and remained active after incubation at 80 degrees C for 30 min.     

Purification and characterization of polyphenol oxidase from Trametes sp. MS39401

Two polyphenol oxidases (EC 1.14.18.1), P-1 and P-2, were purified as electrophoretically homogeneous proteins from the culture filtrate of Trametes sp. MS39401 by acetone precipitation and column chromatographies on DEAE-Sephadex A-50, Sephadex G-150 and hydroxylapatite. P-1 was purified 34-fold with a yield of 4.2%, while P-2 was purified 37-fold with a yield of 20.7%. The molecular masses of P-1 and P-2 were estimated to be 61 kDa and 90 kDa, respectively, by gel filtration. The isoelectric points of P-1 and P-2 were 3.4 and 2.7, respectively. The optimum pH range of both enzymes was 4.5-5.0 at 45 degrees C. The optimum temperature of both enzymes was 55 degrees C at pH 5.0. P-1 was stable at pH 5.0-7.5 and temperatures up to 60 degrees C. P-2 was stable at pH 3.0-7.5 and temperatures up to 50 degrees C. The thermostability of P-1 was comparable to that of the PM1 laccase of basidiomycetes, which was reported to be the most stable among basidiomycete laccases. Both enzymes were active toward various phenolic compounds and aminophenols. However, they lacked activity toward l-tyrosine. The K(m) values for (+)-catechin were 0.19 mM for P-1 and 0.67 mM for P-2. Both enzymes were appreciably inactivated by Hg(2+) and Sn(2+). Significant activation of neither enzyme was observed in the presence of metal ions and reagents. Both enzymes were significantly inhibited by copper-chelating agents, reducing agents and N-bromosuccinimide. Carbon monoxide caused appreciable inactivation of neither enzyme, so it is suggested that P-1 and P-2 belong to the group of laccases.     

Purification and Characteristics of Feruloyl Esterase from Aspergillus awamori G-2 Strain

ABSTRACT:  For food industry production processes and other uses, a mold that produces high levels of feruloyl esterase was obtained from laboratory mold collections and other sources. It was Aspergillus awamori G-2 that produces high levels of feruloyl esterase. The feruloyl esterase was purified using ion-exchange chromatography, size-exclusion chromatography, and HPLC chromatography. The enzyme was identified as a monomer protein using size-exclusion chromatography. Its optimum temperature and pH were, respectively, 40 °C and pH 5. Its activity was stable at pH 3 to 5. The enzyme was combined with xylan and starch, but it was absorbed by cellulose. The km of the feruloyl esterase was 0.0019% (0.01 mM). The enzyme showed stable activity at pH 3 and 50 °C, making this enzyme useful for food production.     

Leucine aminopeptidase from Streptomyces hygroscopicus is controlled by a low molecular weight inhibitor

In culture filtrate of Streptomyces hygroscopicus a producer of polyketide antibiotics, a leucine aminopeptidase and its autogenous inhibitor were detected. The leucine aminopeptidase was purified 4573-fold with yield of 82% by combination of ion exchange and hydrophobic chromatography. The enzyme is monomeric with a molecular mass of 51 kDa determined by gel chromatography and 67 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal activity was at pH 8.0 and 40 degrees C. The pI of leucine aminopeptidase is 8.2. The enzyme is strongly inhibited by 1,10-phenantroline, amastatin and dithiothreitol. Atomic absorption spectrometry indicated 2 mols of ion zinc per mol of enzyme. The enzyme is stable at up to 70 degrees C. Leucine aminopeptidase prefers leucine and methionine as N-terminal amino acids. Activity of leucine aminopeptidase is strongly modulated by an autogenous low-molecular weight inhibitor during fermentation, especially during periods of intensive antibiotic production.     

A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

A thermostable histamine oxidase (EC 1.4.3.-) was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The enzyme was purified about 715-fold over the cell free extracts with a yield of 55% by ammonium sulfate fractionation and various column chromatographies. The purified enzyme was homogeneous on polyacrylamide gel-electrophoresis (native-PAGE). When the enzyme was kept at 65 degrees C and 70 degrees C for 10 min, the activity was fully stable at 65 degrees C and decreased to 9% of the initial level at 70 degrees C. The enzyme was very thermostable. The optimum pH for histamine oxidase activity was found to be at 9.0, and the enzyme was stable over the pH range of 6 to 9. The purified enzyme showed a single protein band on SDS-PAGE and its molecular mass was estimated to be about 81 kDa. The enzyme showed potent activity toward histamine, whereas it was inactive toward putrescine, cadaverine, spermine, and spermidine. Histamine oxidase was inhibited by N,N-diethyldithiocarbamate (DDTC). The inactive enzyme was restored with Cu2+ to 65% of the initial activity, but Cu+ did not enhance the enzyme activity. It is suggested that Cu2+ is essential for expression of histamine oxidase activity. The enzyme was a copper-containing protein having one atom of copper per mol of the enzyme protein as a result of atomic absorption analysis. The N-terminal amino acid sequence of the purified enzyme was different from that of histamine oxidase from Arthrobacter globiformis IFO12137.     

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.     

Characterization of Clostridium paraputrificum chitinase A from a recombinant Escherichia coli

Clostridium paraputrificum chitinase A (ChiA) was purified from a recombinant Escherichia coli. ChiA was active toward chitin from crab shells, colloidal chitin, glycol chitin, and 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside [4-MU-(GlcNAc)2]. ChiA showed maximum activity at pH 6.0 and its optimum temperature was 45 degrees C. ChiA was stable between pH 6.0 and 9.0 and at temperatures up to 40 degrees C. The K(m) and V(max) values of ChiA for 4-MU-(GlcNAc)2 were estimated to be 6.9 microM and 43 micromol/min/mg, respectively. Thin-layer chromatography indicated that ChiA hydrolyzes chitooligosaccharides to mainly chitobiose. ChiA was found to adsorb not only chitinous polymers but also cellulosic polymers.     

海栖热袍菌α-葡萄糖苷酶基因aglA的克隆、表达及酶学性质

主要研究了在大肠杆菌中克隆和表达海栖热袍菌(Thermotoga maritima)的一个α-葡萄糖苷酶(TM1834).通过PCR方法克隆编码T.maritima的一个α-葡萄糖苷酶基因aglA,构建重组质粒pHsh-AglA,电击转化Escherichia coli JM109,通过热激诱导高效表达.SDS-PAGE检测出蛋白相对分子质量约55 000,经热处理,阴离子交换层析和疏水层析纯化后的α-葡萄糖苷酶最适反应温度为90℃,最适反应pH为7.5,在pH 6.5～8.5,温度65～100℃之间酶活仍达到50％以上.在辅助因子NAD+,Mn2+和还原剂DTT的存在条件下达到最高酶活.     

Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614

A dimeric, 90 kDa subunit intracellular proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromatography on hydroxyapatite, Sephacryl 200, Phenyl Superose and Mono Q. The enzyme was specific on Pro-p-nitroanilide and Pro-X dipeptides. It hydrolyzed 2 fragments of hormone oligopeptides with an N-terminal proline: bradykinin, f2-7 and substance P, f4-11. A number of oligopeptides containing 5-11 amino acids residues and proline at the penultimate position from N-terminus or other internal position were not hydrolyzed. The enzyme was most active at pH 7-7.5 and at 37-40 degrees C but it retained 9% of maximal activity at pH 5.5 and >12% of maximal activity at 10 or 60 degrees C. The enzyme was inhibited strongly by the serine protease inhibitor 3,4-dichloroisocoumarin, and stimulated markedly by 1 mol/l of NaCl. The results indicate that the enzyme may lead to the accumulation of proline from dipeptides and oligopeptides during the ripening of cheese.     

Pectin lyase and polygalacturonase of Aspergillus niger pathogenic for yam tubers (Diascorea rotundata)

Polygalacturonase and pectin lyase of Aspergillus niger partially purified by ethanol, ammonium sulphate precipitation, DEAE-cellulose and Sephadex G-150 column chromatography were characterized. Polygalacturonase gave optimum activity at pH 4–5, and at 35°C. It was stable at pH 3–7 and at 20–50°C. The molecular weight was 38020. For pectin lyase optimum activity occurred at pH 5 and 45°C. The enzyme was stable at pH 3–4 and at 40–50°C. The molecular weight was 30 900. Yam tissue was optimally macerated at pH 4–5 by the enzymes. At pH 4.5, potassium sorbate (0.6 mg/ml), benzoic acid (0.8 mg/ml) and sodium benzoate (1.0 mg/ml) caused complete inhibition of polygalacturonase activity. With pectin lyase, this effect was achieved with potassium sorbate and benzoic acid each at 0.9 mg/ml, but not with sodium benzoate.     

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.