Production,purification and characterization of <Emphasis Type="Italic">Bacillus</Emphasis> sp. GRE7 xylanase and its application in eucalyptus Kraft pulp biobleaching

Production of extracellular xylanase from Bacillus sp. GRE7 using a bench-top bioreactor and solid-state fermentation (SSF) was attempted. SSF using wheat bran as substrate and submerged cultivation using oat-spelt xylan as substrate resulted in an enzyme productivity of 3,950 IU g⁻¹ bran and 180 IU ml⁻¹, respectively. The purified enzyme had an apparent molecular weight of 42 kDa and showed optimum activity at 70°C and pH 7. The enzyme was stable at 60–80°C at pH 7 and pH 5–11 at 37°C. Metal ions Mn²⁺ and Co²⁺ increased activity by twofold, while Cu²⁺ and Fe²⁺ reduced activity by fivefold as compared to the control. At 60°C and pH 6, the K _m for oat-spelt xylan was 2.23 mg ml⁻¹ and V _max was 296.8 IU mg⁻¹ protein. In the enzymatic prebleaching of eucalyptus Kraft pulp, the release of chromophores, formation of reducing sugars and brightness was higher while the Kappa number was lower than the control with increased enzyme dosage at 30% reduction of the original chlorine dioxide usage. The thermostability, alkali-tolerance, negligible presence of cellulolytic activity, ability to improve brightness and capacity to reduce chlorine dioxide usage demonstrates the high potential of the enzyme for application in the biobleaching of Kraft pulp.     

Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum

A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB. The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74 511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49 883 Da. XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-11 xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain. XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter. These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication. XynA was purified from a recombinant Escherichia coli strain and characterized. The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein. Immunological and zymogram analyses suggested that XynA and XynB are components of the C. thermocellum F1 cellulosome. Received: 21 September 1998 / Received revision: 30 October 1998 / Accepted: 29 November 1998     

Identification and characterization of a novel xylanase derived from a rice straw degrading enrichment culture

A metagenomic library containing ca. 3.06 × 10⁸ bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan. However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K _m and V _max of Umxyn10A toward oat spelt xylan were 3.2 mg ml⁻¹ and 0.22 mmol min⁻¹ mg⁻¹ and were 2.7 mg ml⁻¹ and 1.0 mmol min⁻¹ mg⁻¹ against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme. The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan.     

Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping. Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998     

Debranching of arabinoxylan: properties of the thermoactive recombinant α-L-arabinofuranosidase fromClostridium stercorarium (ArfB)

 The gene arfB encoding α-L-arabino-furanosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment. The recombinant gene product ArfB was purified by fast-performance liquid chromatography. It has a tetrameric structure with a monomeric relative molecular mass of 52 00. The optima for temperature and pH are 70 °C and 5.0 respectively. The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents. It hydrolyzes aryl and alkyl α-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced by recombinant endoxylanase XynA from the same organism. The identity of the N-terminal amino acid sequences indicates that ArfB corresponds to the major α-arabinosidase activity present in the culture supernatant of C. stercorarium. Received: 30 September 1994/Received revision: 24 November 1994/Accepted: 16 December 1994     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

Purification, characterization and immobilization of an NADPH-dependent enzyme involved in the chiral specific reduction of the keto ester M, an intermediate in the synthesis of an anti-asthma drug, Montelukast, from Microbacterium campoquemadoensis (MB5614)

(S)(E)-2-{3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]-phenyl]-3-hydroxypropyl} benzoic acid methyl ester,␣a key intermediate in the synthesis of the anti-asthma drug, Montelukast, was prepared from the corresponding ketone (keto ester M) by microbial transformation. The biotransforming organism, Microbacterium campoquemadoensis (MB5614), was discovered as a result of an extensive screening program and was used for the isolation and purification of the responsible enzyme. The enzyme is a soluble cytoplasmic protein which was purified as a complex with a low-molecular-mass molecule that had a visible-light absorption maximum at 460 nm. The purified enzyme has an apparent molecular mass of 60 kDa, when denatured, and is isolated in the native state as an oligomer. The isolated enzyme requires NADPH for its activity and reduces the keto ester M to the desired (S)-hydroxy ester with an enantiomeric excess greater than 95% at the optimum temperature of 30 °C and pH 8. The enzyme was immobilized on oxirane-activated acrylamide beads with some loss of activity, but it was fully active in a two-phase (water/hexane 25:75) solvent system, both as a free solution and in an immobilized form. Received: 31 October 1997 / Received revision: 8 January 1998 / Accepted: 24 January 1998     

Purification and Characterization of a Low-Molecular-Weight Xylanase Produced by Acrophialophora nainiana

A low-molecular-weight xylanase activity (XynI) was isolated from the fungus Acrophialophora nainiana after growth in a solid medium containing wheat bran. XynI was purified to apparent homogeneity by ultrafiltration and gel filtration chromatography. The purified enzyme had a molecular weight value of approx. 17 kDa, as determined by SDS-PAGE. This enzyme was most active at 50°C and pH 6.0. At 50°C the half-life was 150 min. The apparent K _m value for birchwood xylan was much lower than the K _m value for oat spelt xylan. XynI was activated by L-cysteine, DTE, β-mercaptoethanol, and L-tryptophan. XynI did not show significant sequence homology with other xylanases. The analysis of hydrolysis products of xylans and wood pulps showed that XynI was able to release xylooligomers ranging from X2 to X3 and X2 to X6, respectively. The enzyme was not active against acetylated xylan. A small amount of xylose was released from deacetylated, birchwood, and oat spelt xylans. The results obtained with enzymatic treatment of Kraft pulp indicated a reduction in the amount of chlorine compounds required for the process and enhanced brightness gain. Received: 6 May 1998 / Accepted: 29 July 1998     

A novel cold-active xylanase from the cellulolytic myxobacterium Sorangium cellulosum So9733-1: gene cloning, expression, and enzymatic characterization

The cellulolytic myxobacterium Sorangium cellulosum is able to efficiently degrade many kinds of polysaccharides, but none of the enzymes involved have been characterized. In this paper, a xylanase gene (xynA) was cloned from S. cellulosum So9733-1 using thermal asymmetric interlaced PCR. The gene is composed of 1,209 bp and has only 52.27% G + C content, which is much lower than that of most myxobacterial DNA reported (67–72%). Gene xynA encodes a 402 amino acid protein that contains a single catalytic domain belonging to the glycoside hydrolase family 10. The novel xylanase gene, xynA, was expressed in Escherichia coli BL21 (DE3) and the recombinant protein (r-XynA) was purified by Ni-affinity chromatography. The r-XynA had the optimum temperature of 30–35°C and exhibited 33.3% activity at 5°C and 13.7% activity at 0°C. Approximately 80% activity was lost after 20-min pre-incubation at 50°C. These results indicate that r-XynA is a cold-active xylanase with low thermostability. At 30°C, the K _m values of r-XynA on beechwood xylan, birchwood xylan, and oat spelt xylan were 25.77 ± 4.16, 26.52 ± 4.78, and 38.13 ± 5.35 mg/mL, respectively. The purified r-XynA displayed optimum activity at pH 7.0. The activity of r-XynA was enhanced by the presence of Ca²⁺. The r-XynA hydrolyzed beechwood xylan, birchwood xylan, and xylooligosaccharides (xylotriose, xylotetraose, and xylopentose) to produce primarily xylose and xylobiose. To our knowledge, this is the first report on the characterization of a xylanase from S. cellulosum.     

Purification of the alkaliphilic xylanases from Myceliophthora sp. IMI 387099 using cellulose-binding domain as an affinity tag

Ten xylanase isoforms produced by Myceliophthora sp. were characterized for their ability to bind to avicel. Three of the xylanases showing differential affinity for avicel were purified by column chromatography. The purified xylanase Xyl IIa, IIb and IIc showed molecular mass of 47, 41 and 30 kDa and pI of ∼3.5, 4.8 and 5.2, respectively. Xyl IIa was optimally active at pH 8.0 and temperature 70 °C, while Xyl IIb and IIc were optimally active at pH 9.0 and 60 °C and 7.0 and 80 °C, respectively. Xyl IIa and Xyl IIb showed higher stability under alkaline conditions (pH 9.0) and retained 80% of the original activity upto 1 h and 3 h respectively, at 50 °C. All three purified iso-xylanases showed enhanced activities in presence of Na⁺, Mg²⁺, Mn²⁺ and K⁺ ions, whereas, Zn²⁺ and Cu²⁺ showed negative effect on Xyl IIa. The activity of Xyl IIa increased in presence of reducing agents DTT and mercaptoethanol, however, SDS showed inhibitory effect. Kinetic studies showed that Xyl IIb and IIc degrade rye arabinoxylan, much more efficiently than oat spelt xylan, whereas, Xyl IIa showed much higher Kcat/Km value for birch wood xylan as compared to oat spelt xylan. The purified xylanases were apparently classified in family 10.     

d-Xylose isomerases from a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii : isolation, characterization and immobilisation to solid supports

d-Xylose/d-glucose isomerases from two strains, a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii are described herein. The enzymes were purified to apparent homogeneity. Both of these d-xylose isomerases are homotetramers with relative subunit molecular masses of 45 000 and 53 000, respectively, as estimated by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The native molecular masses determined on Superose 12 gel chromatography are 181 kDa for the enzyme from Paenibacillus sp. and 199 kDa for that from A. ruhlandii. The activity of both enzymes shows a requirement for divalent metal ions; the d-xylose isomerase from Paenibacillus sp. has the highest activity with Mn²⁺, while the enzyme from A. ruhlandii prefers Mg²⁺. Both enzymes also accept Co²⁺ with a somewhat lower efficiency, while Cu²⁺ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two non-identical binding sites per subunit. d-Glucose is converted to d-fructose at a rate that is two- to three-fold slower than for the d-xylose isomerisation. d-Xylitol and d-lyxose are competitive inhibitors of both enzymes. Both enzymes have a pH optimum between 6.5 and 7.0, and they are active up to 60 °C. The enzyme from Paenibacillus sp. retained 50% of its activity after 4 days at 55 °C, whereas that from A. ruhlandii still retained 50% of its activity after 6 days at 55 °C. Polyacrylamide entrapment and immobilisation to both controlled pore glass and cyanogen-bromide-activated Sepharose were achieved for both enzymes with high efficiency. Received: 14 May 1998 / Received last revision: 29 July 1998 / Accepted: 29 July 1998     

Analysis of the gene for β-fructosidase (invertase, inulinase) of the hyperthermophilic bacterium Thermotoga maritima, and characterisation of the enzyme expressed in Escherichia coli

This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases. On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis of sucrose and inulin with k _cat/K _m values (at 75 °C, pH 5.5) of about 4.1 × 10⁴M⁻¹s⁻¹ and 3.1 × 10⁴M⁻¹s⁻¹ respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation. During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability recommend it for use in biotechnology. Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998     

Biochemical properties of xylanases from a thermophilic fungus,Melanocarpus albomyces, and their action on plant cell walls

Melanocarpus albomyces, a thermophilic fungus isolated from compost by enrichment culture in a liquid medium containing sugarcane bagasse, produced cellulase-free xylanase in culture medium. The fungus was unusual in that xylanase activity was inducible not only by hemicellulosic material but also by the monomeric pentosan unit of xylan but not by glucose. Concentration of bagasse-grown culture filtrate protein followed by size-exclusion and anion-exchange chromatography separated four xylanase activities. Under identical conditions of protein purification, xylanase I was absent in the xylose-grown culture filtrate. Two xylanase activities, a minor xylanase IA and a major xylanase IIIA, were purified to apparent homogeneity from bagasse-grown cultures. Both xylanases were specific forβ-1,4 xylose-rich polymer, optimally active, respectively, at pH 6.6 and 5.6, and at 65°C. The xylanases were stable between pH 5 to 10 at 50°C for 24 h. Xylanases released xylobiose, xylotriose and higher oligomers from xylans from different sources. Xylanase IA had a M_r of 38 kDa and contained 7% carbohydrate whereas xylanase IIIA had a M_r of 24 kDa and no detectable carbohydrate. The K_m for larchwood xylan (mg ml⁻¹) and V_max (μmol xylose min⁻¹ mg⁻¹ protein) of xylanase IA were 0.33 and 311, and of xylanase IIIA 1.69 and 500, respectively. Xylanases IA, II and IIIA showed no synergism in the hydrolysis of larchwood glucuronoxylan or oat spelt and sugarcane bagasse arabinoxylans. They had different reactivity on untreated and delignified bagasse. The xylanases were more reactive than cellulase on delignified bagasse. Simultaneous treatment of delignified bagasse by xylanase and cellulase released more sugar than individual enzyme treatments. By contrast, the primary cell walls of a plant, particularly from the region of elongation, were more susceptible to the action of cellulase than xylanase. The effects of xylanase and cellulase on plant cell walls were consistent with the view that hemicellulose surrounds cellulose in plant cell walls.     

High-level expression of an endoxylanase gene from Bacillus sp. in Bacillus subtilis DB104 for the production of xylobiose from xylan

To produce xylobiose from xylan, high-level expression of an endoxylanase gene from Bacillus sp. was carried out in Bacillus subtilis DB104. A 1.62-kb SmaI DNA fragment, coding for an endoxylanase of Bacillus sp., was ligated into the Escherichia coli/B. subtilis shuttle vector pJH27Δ88, producing pJHKJ4, which was subsequently transformed into B. subtilis DB104. A maximum endoxylanase activity of 105 U/ml was obtained from the supernatant of B. subtilis DB104 harboring pJHKJ4. The endoxylanase was purified to homogeneity by ion-exchange chromatography and the production profile of xylooligosaccharides from xylan by the endoxylanase was examined by HPLC with a carbohydrate analysis column. Xylobiose was the major product from xylan at 40 °C and its proportion in the xylan hydrolyzates increased with the reaction time; at 12 h, over 60% of the reaction products was xylobiose. These results suggest that xylobiose, which has a stimulatory effect on the selective growth of the intestinal bacterium Bifidobacterium, can be mass-produced effectively by the endoxylanase of Bacillus sp. cloned in B. subtilis. Received: 2 January 1998 / Received revision: 4 March 1998 / Accepted: 4 March 1998     

Purification,characterization and regulation of the synthesis of an Aspergillus nidulans acidic xylanase

An acidic xylanase from a culture filtrate of Aspergillus nidulans grown on oat-spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a molecular mass of 34,000 Da and had an isoelectric point of approximately 3.4. The enzyme was a non-debranching endoxylanase highly specific for xylans. The xylanase showed an optimal activity at pH 6.0 and 56° C and had a Michaelis constant K_m of 0.97 mg oat-spelt xylan (soluble fraction) ml and a maximed reaction velocity (Vmax) of 1,091 mol min^–1 (mg^–1protein)^–1. Using polyclonal antibodies raised against the purified enzyme, the regulation of its synthesis has been studied. The xylanase production is repressed by glucose and induced by oat-spelt xylan, arabinoxylan, 4-O-methylglucurono-xylan, birchwood xylan and xylose.     

Thermostable β-xylosidase from Thermomonospora curvata

Thermomonospora curvata produced a thermostable β-xylosidase during growth on birch xylan. The enzyme, extracted by sonication of early stationary phase mycelia, was purified by isoelectric focusing and size exclusion HPLC. The isoelectric point was pH 4.8. The molecular weight was estimated to be 102 000 by size exclusion HPLC and 112 000 by SDS-PAGE. Maximal activity occurred at pH 6–7 and 60–68°C. K _m values for xylobiose and p-nitrophenyl-β -D-xylopyranoside were 4.0 M and 0.6 M respectively. The enzyme was sensitive to low levels of Hg²⁺ (50% inhibition at 0.2 μM), but was stimulated by Co²⁺ and Pb²⁺. Addition of the xylosidase to a xylanase reaction mixture increased the liberation of xylose equivalents from xylan and decreased the proportion of xylobiose in the hydrolysate. Received 14 April 1997/ Accepted in revised form 21 October 1997     

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation. Received: 25 September 1998 / Received revision: 15 December 1998 / Accepted: 21 December 1998     

Cloning and characterization of a heat-stable CMP-<Emphasis Type="Italic">N</Emphasis>-acylneuraminic acid synthetase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

In this study, we report the cloning, recombinant expression, and biochemical characterization of a heat-stable CMP-N-acylneuraminic acid (NeuAc) synthetase from Clostridium thermocellum ATCC 27405. A high throughput electrospray ionization mass spectrometry (ESI-MS)-based assay demonstrates that the enzyme has an absolute requirement for a divalent cation for activity and reaches maximum activity in the presence of 10 mM Mn²⁺. The enzyme is active at pH 8–13 in Tris–HCl buffer and at 37–60 °C, and maximum activity is observed at pH 9.5 and 50 °C in the presence of 0.2 mM dithiothreitol. In addition to NeuAc, the enzyme also accepts the analog N-glycolylneuraminic acid (NeuGc) as a substrate. The apparent Michaelis constants for cytidine triphosphate and NeuAc or NeuGc are 240 ± 20, 130 ± 10, and 160 ± 10 μM, respectively, with corresponding turnover numbers of 3.33, 2.25, and 1.66 s⁻¹, respectively. An initial velocity study of the enzymatic reaction indicates an ordered bi–bi catalytic mechanism. In addition to demonstration of a thermostable and substrate-tolerant enzyme, confirmation of the biochemical function of a gene for CMP-NeuAc synthetase in C. thermocellum also opens the question of the biological function of CMP-NeuAc in such nonpathogenic microorganisms.     

Characterisation of a starch-hydrolysing enzyme of Aspergillus niger

A UV-induced mutant strain of Aspergillus niger (CFTRI-1105-U9) overproduced a starch-hydrolysing enzyme with properties characteristically different from the known amylases of the fungus. The purified enzyme of 4.0 pI had an apparent molecular mass of 125 kDa and it dextrinised starch and then saccharified the dextrins. Patterns of the enzyme activity on starch, resulting in glucose at 60 °C and glucose, maltose and maltodextrins at 70 °C as primary products, suggested significant applications for the enzyme in starch-processing industries. Received: 29 October 1998 / Received revision: 11 January 1999 / Accepted: 19 January 1999     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998