纤维素酶中具有壳聚糖水解酶活性成分的鉴定

在壳聚糖酶的研究过程中,目前已发现37种酶具有非专一性地降解壳聚糖的能力[1].对这些非专一性酶水解壳聚糖的机理有两种看法:一些人认为,由于这些酶大都来自商业酶制剂,未经过进一步的纯化,故有人认为其中所含的少量杂质可能是产生水解活力的原因;但也有人认为,在所有的酶制剂中都存在同一种杂质似乎是不可能的,因为这些酶来源于广泛的微生物、真菌、哺乳动物和植物等.众所周知,酶具有高度的专一性,即对所催化的反应和底物有严格的选择性,一种酶往往只能催化一种或一类反应;有如此多的不同种类的酶能非专一性地水解壳聚糖.因而探讨具有水解…     

Purification and Characterization of Neutral and Acid Sphingomyelinases from Rat Brain

Neutral and acid sphingomyelinases were copurified from a rat brain P2 fraction by extraction with 1% Triton X-100, followed by (NH4)2SO4 fractionation, acetone powdering, extraction with 1% Triton X-100, (NH4)2SO4 fractionation, Sepharose CL-6B chromatography, and chromatofocusing. The neutral sphingomyelinase was eluted with buffer containing 0.4 M NaCl after the acid sphingomyelinase had been eluted with Polybuffer at pH 5.3. The neutral sphingomyelinase exhibited specific activity of 48,300 nmol/h/mg of protein, with 254-fold purification; the corresponding value for acid sphingomyelinase was 25,300 nmol/h/mg protein, with 668-fold purification from the P2 fraction. The purified neutral sphingomyelinase had no acid sphingomyelinase activity, and vice versa. The properties of the two enzymes were examined. A single band corresponding to a molecular weight of 67,000 was obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both enzymes. The pI was estimated to be 5.5 for both on isoelectric focusing. The native molecular weights of the neutral and acid sphingomyelinases were found to be 434,000 and 284,000, respectively, on gel filtration with Sepharose CL-6B. The single band obtained for each enzyme on SDS-PAGE was identified as an antigen with antibody raised against the purified neutral sphingomyelinase. Their amino acid compositions were very similar. The neutral and acid sphingomyelinases probably consist of common polypeptides and are immunologically cross-reactive.     

Heterologous expression of thermostable acetylxylan esterase gene from Thermobifida fusca and its synergistic action with xylanase for the production of xylooligosaccharides

The axe gene which encodes an acetylxylan esterase from Thermobifida fusca NTU22, was cloned, sequenced and expressed in Escherichia coli. The gene consists of 786 base pairs and encodes a protein of 262 amino acids. The deduced amino acid sequence of the acetylxylan esterase axe exhibited a high degree of similarity with BTA-hydrolase from T. fusca DSM43793, esterase from Thermobifida alba and lipase from Streptomyces albus. The optimal pH and temperature of the purified esterase were 7.5 and 60 °C, respectively. Cooperative enzymatic treatment of oat-spelt xylan by transformant xylanase and acetylxylan esterase significantly increased the xylooligosaccharides production compared with the xylanase or acetylxylan esterase action alone. The synergy of transformant acetylxylan esterase and xylanase cannot increase the production of reducing sugars from lignocellulolytic substrate, bagasse.     

Purification and characterization of an aminopeptidase from Mycoplasma salivarium.

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Purification and characterization of a fructosyltransferase from onion bulbs and its key role in the synthesis of fructo-oligosaccharides in vivo

A fructosyltransferase that transfers the terminal (2 --> 1)-beta-linked D-fructosyl group of fructo-oligosaccharides (1(F)(1-beta-D-fructofuranosyl)(n) sucrose, n >/= 1) to HO-6 of the glucosyl residue and HO-1 of the fructosyl residue of similar saccharides (1(F)(1-beta-D-fructofuranosyl)(m) sucrose, m >/= 0) has been purified from an extract of the bulbs of onion (Allium cepa). Successive column chromatography using DEAE-Sepharose CL-6B, Toyopearl HW65, Toyopearl HW55, DEAE-Sepharose CL-6B (2nd time), Sephadex G-100, Concanavalin A Sepharose, and Toyopearl HW-65 (2nd time) were applied for protein purification. The general properties of the enzyme, were as follows: molecular masses of 66 kDa (gel filtration chromatography), and of 52 kDa and 25 kDa (SDS-PAGE); optimum pH of c. 5.68, stable at 20-40 degrees C for 15 min; stable in a range of pH 5.30-6.31 at 30 degrees C for 30 min, inhibited by Hg(2+), Ag(+), p-chloromercuribenzoic acid (p-CMB) and sodium dodecyl sulfate (SDS), activated by sodium deoxycholate, Triton X-100 and Tween-80. The amino acid sequence of the N-terminus moiety of the 52-kDa polypeptide was ADNEFPWTNDMLAWQRCGFHFRTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYXG and the amino acid sequence from the N-terminus of the 25-kDa polypeptide was ADVGYXCSTSGGAATRGTLGPFGLL VLANQDLTENTATYFYVSKGTDGALRTHFCQDET. The enzyme tentatively classified as fructan: fructan 6(G)-fructosyltransferase (6G-FFT). The enzyme is proposed to play an important role in the synthesis of inulin and inulinneo-series fructo-oligosaccharides in onion bulbs.     

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.     

Purification and characterization of a novel chitinase from Burkholderia cepacia strain KH2 isolated from the bed log of Lentinus edodes,Shiitake mushroom

One of the chitinases secreted in the culture filtrate of a gram-negative bacteria, Burkholderia cepacia strain KH2, which was isolated from the bed log of Lentinus edodes, Shiitake mushrooms, was purified by DEAE Sepharose CL-6B chromatography, followed by Sephacryl S-100 HR gel filtration. The purified enzyme was homogenous, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with an estimated molecular weight of 34,000 and an isoelectric point (pI) of 5.9. The enzyme was stable at pH values of 4.0-6.0, and at temperatures up to 50 degrees C; the optimum pH and temperature were 4.5 and 50 degrees C, respectively. The enzyme exhibited higher activities toward chitosan 7B, a 62% deacetylated chitosan, than toward the highly deacetylated chitosan substrates. The enzyme was observed to drastically hydrolyze partially deacetylated chitin substrates, with the subsequent formation of N-acetylchitooligosaccharides [(GlcNAc) (n), n=2-7]. Separation and quantification of the hydrolysis products of (GlcNAc) (n), n52-6, by HPLC showed the splitting into (GlcNAc)(n), n=3-6. Activity toward N-acetylchitobiose was not detected. Oligomers with a higher number of units than the starting substrate were also detected, which indicate transglycosylation activity.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of an inducible cysteine proteinase inhibitor from submandibular glands of isoproterenol-treated rats

A low-molecular-weight protein, induced in rats following prolonged isoproterenol treatment, has been purified from rat submandibular glands by chromatography on columns of Sepharose CL-6B, DEAE-Sepharose CL-6B, and Sephadex G-75. The purified protein is homogeneous based on gel electrophoresis and Ouchterlony double diffusion. The molecular weight of the purified protein was 14,000 and 15,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Superose 12 column, respectively. This protein contains 31% glutamic acid/glutamine and aspartic acid/asparagine, 3.6% cysteine, and 2.5% proline. This protein is shown to be an inhibitor of several cysteine proteinases, papain and ficin being inhibited very strongly in approximately 1:1 molar ratio of enzyme to inhibitor. The protein is not detected in normal rat tissues but is induced in submandibular and sublingual glands even after 1 day of isoproterenol treatment of rats as early as 7 days after birth. Based on cysteine proteinase inhibitor activity, molecular size, and chemical composition this protein appears to belong to the cystatin superfamily.     

Purification and characterization of an acidic amino acid specific endopeptidase of Streptomyces griseus obtained from a commercial preparation (Pronase)

A protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20,000 by gel filtration on Sepharose 6B using 6 M guanidine hydrochloride as an eluent, and 22,000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-chloromercuribenzoate (PCMB) or EDTA.     

trans-2, cis-4-Heptadienoic Acid,One of Metabolites of Sporobolomyces odorus

Water-soluble phospholipase B was purified to homogeneity from Torulaspora delbrueckii cell washings. The washings were concentrated by ultrafiltration, and then a fraction with phospholipase B activity was precipitated with ammonium sulfate, and purified by sequential column chromatographies on Octyl-Sepharose CL-4B, DEAE-Sephacel, and Sepharose 6B. The molecular weight of the enzyme was estimated to be 170,000~200,000 by SDS-polyacrylamide gel electrophoresis and by gel filtration with a Sephadex G-200 column. The isoelectric point of the enzyme was 4.0. The purified enzyme had two pH optima at pH 2.5 and pH 7.5. The activity at acidic pH was greatly stimulated by the divalent metal ions tested, but the activity at alkaline pH was stimulated mainly by Ca²⁺ and Fe²⁺. The purified enzyme had both lysophospholipase activity and phospholipase B activity in a ratio of 37:1 at acidic pH and 73:1 at alkaline pH. The amino acid composition of the enzyme was characterized by high contents of Asp, Ser, Leu, and Gly.     

Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes

The (H+,K+)ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of approximately 94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-ATPase and K+-pNPPase activities.     

Improved isolation method and some properties of soybean gamma-conglycinin

Soybean gamma-conglycinin was isolated by isoelectric precipitation and ammonium sulphate fractionation. The crude protein was purified by ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sepharose CL-6B. The purified gamma-conglycinin was homogeneous on two kinds of gel electrophoresis and an ultracentrifugal analysis. A subunit band, distinguishable from other subunit bands of beta-conglycinin and glycinin, was detected by sodium dodecyl sulphate electrophoresis. Amino acid composition was similar to those of the other storage proteins of soybean. Some physical properties were also studied.     

Purification and Properties of Soluble Chlorophyllase from Tea Leaf Sprouts

Soluble chlorophyllase (chlorophyll-chlorophyllido-hydrolase,EC 3.1.1.14 [EC] ) was purified 650-fold from tea leaf sprouts byammonium sulfate fractionation and gel filtration through SephadexG-200 and Sepharose CL-6B. The purified enzyme showed two bandson polyacrylamide gel electrophoresis and the specific activitywas 2.6 µmol chlorophyll a hydrolyzed min^–1 mg^–1of protein. The molecular weights determined by Sepharose CL-6Bwere 910,000 and 350,000, indicating high molecular aggregates.The subunit molecular weight estimated by sodium lauryl sulfate-polyacrylamidegel electrophoresis was 38,000. The isoelectric point was 3.9.The optimum pH was 5.5 in acetate buffer and the Km value forchlorophyll a was 10 µM. This enzyme did not require athiol compound nor metal ion such as Mg²⁺. (Received January 26, 1981; Accepted April 3, 1981)     

Purification and some properties of a novel chitosanase from Bacillus subtilis KH1

One of at least two chitosanases secreted in the culture filtrate of Bacillus subtilis KH1 was purified by two sequential DEAE Sepharose CL-6B chromatographies, followed by Sephacryl S-100 HR gel chromatography. The purified enzyme was homogenous as judged by SDS-PAGE. It showed an estimated molecular weight and pI of 28,000 and 8.3, respectively. The enzyme drastically reduced the viscosity of highly deacetylated chitosan substrates, with the subsequent formation of chitooligosaccharides [(GlcN)(n), n=2-6]. No activity toward carboxymethylcellulose (CMC), chitobiose (GlcN)(2), or chitotriose (GlcN)(3) was detected. Separation and quantification of products of hydrolysis of 10% (w/v) solutions of chitooligosaccharides, (GlcN)(n), n=2-6, by HPLC showed the splitting of (GlcN) (n), n=4-6, in an endo-splitting manner. Oligomers comprising higher units than the starting substrate were also detected, indicating transglycosylation activity. The amino terminal sequence of this enzyme (A-G-L-N-K-D-Q-K-R-R) is identical to that of the chitosanase derived from Bacillus pumilus BN262 and to the deduced amino terminal sequences of Bacillus subtilis 168 and Bacillus amyloliquefaciens UTK chitosanases.     

Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Purification of a membrane-bound neutral endopeptidase from rat kidney and its activation by polyamines

An endopeptidase which cleaves succinyl trialanine p-nitroanilide (Suc(Ala)3-pNA) into succinyl dialanine and alanine p-nitroanilide (Ala-pNA) was solubilized from a microsomal membrane fraction of rat kidney with Nonidet P-40 following treatment with 1 M KCl and Brij 35. The solubilized enzyme was purified to homogeneity by DEAE-Sephadex chromatography, Sepharose CL-6B gel filtration and sucrose gradient centrifugation. The final enzyme preparation had a specific activity of 1.69 mumol/min/mg protein, representing about 140-fold purification over the starting membrane. The enzyme hydrolyzes Suc(Ala)3-pNA with a Km value of 0.28 mM and a Vmax value of 1.3 mumol/min. The molecular weight of the undenatured enzyme was estimated to be 360,000 by gel filtration on a Sepharose CL-6B column and that of the denatured enzyme to be 92,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, revealing the presence of a single polypeptide chain. The enzyme was markedly activated by polyamines, producing increases in the values of both Km and Vmax. Comparatively less activation was found in the presence of some monovalent cations and Ca2+. The activation by polyamines was inversely proportional to the concentration of monovalent cations, but Ca2+ and polyamines seemed to stimulate additively.     

Regulation and Characterization of Xylanolytic Enzymes of Thermoanaerobacterium saccharolyticum B6A-RI

During growth on xylan and xylose Thermoanaerobacterium saccharolyticum B6A-RI produced endoxylanase, β-xylosidase, arabinofuranosidase, and acetyl esterase, and the first three activities appeared to be produced coordinately. During nonlimiting growth on xylan, these enzyme activities were predominantly cell associated; however, during growth on limiting concentrations of xylan, the majority of endoxylanase activity was extracellular rather than cell associated. Endoxylanase, β-xylosidase, and arabinofuranosidase activities were induced by xylan, xylose, and arabinose, respectively. Acetyl esterase activity was constitutive, and endoxylanase activity was catabolite repressed by glucose. Extracellular endoxylanase existed as a high-molecular-weight complex (molecular weight, more than 10⁶). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms, the crude endoxylanase complex was composed of at least six activity bands. Endoxylanase was purified by gel filtration with Sephacryl S-300 and affinity chromatography with xylan coupled to Sepharose CL-4B preequilibrated to 45°C with 50 mM sodium acetate buffer (pH 4.0) and eluted with 0.1% soluble xylan. A single area of endoxylanase activity was identified on the zymogram; when this activity was analyzed by SDS-PAGE, it was composed of a major protein with a molecular weight of approximately 160,000 and a minor protein with a molecular weight of approximately 130,000. The endoxylanase activity stained with Schiff's reagent, indicative of glycoproteins, displayed a specific activity of 41 U/mg of protein on xylan, and had pH and temperature optima of 6.0 and 70°C, respectively.