Exo-type Enzyme Research Articles

Cloning, sequencing, and expression of the gene encoding an intracellular beta-D-xylosidase from Streptomyces thermoviolaceus OPC-520.

The intracellular beta-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50 degrees C in a minimal medium containing xylan or xylooligosaccharides. The 82-kDa protein with beta-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with beta-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-beta-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylooligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0-7.0 and temperature range 40-50 degrees C.

Crystal structure of prolyl aminopeptidase from Serratia marcescens.

Prolyl aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.

Extracellular dextran-induced p-nitrophenyl-alpha-D-glucoside-hydrolyzing enzyme of Bacillus circulans KA-304: a producer of Schizophyllum commune-lytic enzyme.

p-NP-alpha-D-Glucoside-hydrolyzing activity in the culture filtrate of Bacillus circulans KA-304, a producer of Schizophyllum commune cell-wall lytic enzyme, increased remarkably when the bacterium was grown on dextran as a carbon source. It was suggested that the increase of the activity was caused by increases of two major species, alpha-D-glucosidase I and alpha-D-glucosidase II. alpha-D-Glucosidase I, which showed a certain reactivity toward dextran, was isolated from the filtrate (MW 70 kDa, 35-fold, 10% recovery). The enzyme was stable around pH 6.5-7.5 and showed its highest activity at pH 6.5. The enzyme preparation inactivated with p-chloromerucuribenzoic acid recovered its activity by incubating with ditiothereitol. Its substrate specificity suggested that the enzyme was an exo-type enzyme with certain affinity toward alpha-1,6-glucosidic linkage.

Mode of Action of Exo- and Endo-Type Cellulases from Irpex lacteus in the Hydrolysis of Cellulose with Different Crystallinities

The mode of action of two highly purified cellulases of exo- and endo-types from Irpex lacteus was investigated by using pure cellulosic substrates with different crystallinities derived from cellulose I. Exo-type cellulase saccharified all celluloses more effectively than endo-type enzyme, and the saccharification activities of both cellulases similarly increased with decreasing crystallinity of cellulose. The DP-lowering activity of exo-type cellulase remained similar for celluloses with higher crystallinity, while this cellulase showed a degradation mode resembling that of the endo-type enzyme for the substrates with lower crystallinity. Compared with exo-type cellulase, endo-type cellulase remarkably decreased the DP of cellulose with higher crystallinity, while this activity was abated for cellulose with lower crystallinity. Thus, the effects of both cellulases became similar in the degradation of amorphous substrates such as H3PO4-treated cellulose. Endo-type cellulase produced several kinds of cellooligosaccharide from all kinds of cellulose used, while the product of the exo-type enzyme was only cellobiose from crystalline cellulose such as cotton and cotton linter even after a 12-h incubation period. The results indicate that each cellulase shows a typical mode of action (exo or endo) for crystalline cellulose, but that their characteristic modes of attack may change with decreasing crystallinity of cellulose.

Xylan structure, microbial xylanases, and their mode of action

Xylans, the major portion of the hemicellulose of plant cell walls and grasses, are heteropolymers consisting principally of xylose and arabinose. Microbial xylanases with different multiplicities and properties are reported. Most studies on the mode of action of these xylanases have been carried out with fungi and there is very little information available on bacterial xylanases. Fungal xylanases have three or more substrate binding sites: for exampleAspergillus niger, Ceratocytis paradoxa, Cryptococcus albidus andChainia sp. endoxylanases have four to seven subsites with the catalytic site located at the centre of these sub-sites. The analysis of these sub-sites is either by kinetic or end-product analysis studies. Kinetic studies are used for exo-type enzymes while the end-product analysis studies are more convenient for endo-type enzymes. This review covers microbial xylanases with special emphasis on studies of sub-site mapping. The industrial applications of the microbial xylanases are also discussed.

New substrate specificity of modified porcine pancreatic α-amylase

Conversion of the substrate specificity of porcine pancreatic α-amylase (PPA) was studied using chemical modification of His residues. Diethyl pyrocarbonate modified His residues in PPA and the activity of the modified PPA for the hydrolysis of the α- d-(1,4) glucoside bond in starch or oligosaccharides decreased to less than 1% of that of the native enzyme. However, the activity for the hydrolysis of the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides was increased by chemical modification. When the modified PPA was incubated with a proteinaceous α-amylase inhibitor ( M r 60,000) purified from white kidney bean ( Phaseolus vulgaris), it bound to the inhibitor. As a result, the remaining less than 1% hydrolytic activity of the modified PPA for starch disappeared completely but that for p-nitrophenyl oligosaccharides remained unaltered. The hydrolytic activity of the native PPA for the α- d-(1,4)glucoside bond in oligosaccharides was stronger than that between p-nitrophenyl and oligosaccharides in p-nitrophenyl oligosaccharides. Therefore, when p-nitrophenyl oligosaccharides (three to five glucose residues) were used as substrates for the native PPA, the α- d-(1,4) glucoside bonds in the oligosaccharides were hydrolyzed. However, the modified PPA-inhibitor complex hydrolyzed only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides. The above results reveal that, by chemical modification with diethyl pyrocarbonate and biochemical modification with an amylase inhibitor, amylase can be converted to a new exo-type enzyme which hydrolyzes only the bond between p-nitrophenol and oligosaccharides in p-nitrophenyl oligosaccharides.

The application of recombinant-DNA technology to cellulases and lignocellulosic wastes

The efficient utilization of different lignocellulosic wastes will require an understanding of the roles of the different enzymes involved in enzymatic hydrolysis. Towards this goal, we have isolated and characterized the genes coding for four major cellulases, CBH I, CBH II, EG I and EG III, produced by the cellulolytic fungus Trichoderma reesei . It seems that T. reesei produces at least two classes of cellulose and in each class both endo- and exo-type enzyme are found. Full-length cDNAs coding for CBH I, CBH II, EG I and EG III have been expressed in the yeast Saccharomyces cerevisiae , and the cellulases are secreted by these strains. The recombinant cellulases produced by the yeast all show activity towards cellulosic substrates. The information obtained from the cloned genes makes possible the construction of new cellulolytic organisms and may also make possible the development of improved enzymes.

Purification and Properties ofβ-N-Acetyl-D-glucosaminidase from Alimentary Canal of the Silkworm,Bombyx mori

β-N-Acetyl-d-glucosaminidase (EC 3.2.1.30) was purified from the alimentary canal of the silkworm, Bombyx mori, by ammonium sulfate fractionation and chromatography with hydroxylapatite, DEAE Bio-Gel A, chromatofocusing, and Sephacryl S-200. The purified enzyme was a single band on disc-PAGE. The molecular weight was 119,000 by gel filtration and 125,000 by SDS-PAGE. The enzyme was separated into two peptides whose apparent molecular weights were 67,500 and 57,500 by SDS-PAGE. The pi was 4.86 by chromatofocusing. The optimum pH was 5.5 to 6.0 and the optimum temperature, 45°C, using pNp-β-GlcNAc as the substrate. The enzyme was stable from pH 5.5 to 8.5 and below 30°C. It was strongly inhibited by HgCl2. Small N-acetylchitooligomers were as good substrates as pNp-β-GlcNAc, and the enzyme cleaved colloidal chitin to GlcNAc, even though the relative velocity was slow. Smaller N-acetylchitooligomers were preferred substrates with Km 0.787 to 0.056mm, A:cat 1013 to 52sec–1, and kcat/Km 1690 to 754 mm–1 sec–1. The enzyme precipitated in as band with moulting fluid chitobiase antiserum, but not with haemolymph exo-β-N-acetylglucosaminidase antiserum. The results suggest that this enzyme is an exo-type enzyme which is involved in the degradation of chitin to GlcNAc.

Urocanic Acid Production by Sporogenous Bacteria

As the results of a screening of several type cultures of bacteria, Bacillus subtilis var. thermophilus was revealed to- be the most powerful strain for urocanic acid production. The accumulation of urocanic acid by this bacteria is caused by deamination of L-histidine, and is particularly accelerated in the presence of a component (X-factor) in meat extract. In the decomposition of urocanic acid the optimal pH of urocanase activity is markedly inhibited by the deviation of pH of the culture medium. The histidase of this bacteria is supposed to be a new exo-type enzyme.