Isolation and properties of recombinant inulinases from <Emphasis Type="Italic">Aspergillus</Emphasis> sp.

The genes inuA and inu1, encoding two inulinases (32nd glycosyl hydrolase family) from filamentous fungi Aspergillus niger and A. awamori, were cloned into Penicillium canescens recombinant strain. Using chromatographic techniques, endoinulinase InuA (56 kDa, pI 3) and exoinulinase Inu1 (60 kDa, pI 4.3) were purified to homogeneity from the enzymatic complexes of P. canescens new transformants. The properties, such as substrate specificity, pH- and T-optima of activity, stability at different temperatures, influence of cations and anions on the catalytic activity, etc., of both recombinant inulinases were studied.     

Cloning of Penicillium canescens Endo-1,4-β-xylanase Gene and Construction of Multicopy Strains

The complete gene xylA that encodes endo-1,4--xylanase secreted byPenicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven to eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30–50% of the total secreted protein.     

Enzymological properties of endo-(1–4)-β-glucanase Eg12p of <Emphasis Type="Italic">Penicillium canescens</Emphasis> and characteristics of structural gene <Emphasis Type="Italic">egl2</Emphasis>

Gene egl2 of secreted endo-(1–4)-β-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding β-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60°C, respectively, exhibited specific activity of 33 IU, and had K _m and V _max in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 μmol/sec per mg, respectively.     

Distribution of O-glycosylhydrolases in marine fungi of the Sea of Japan and the Sea of Okhotsk: Characterization of exocellular N-acetyl-β-D-glucosaminidase of the marine fungus <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The capacity to produce exocellular enzymes was studied for 92 samples of fungi from various marine habitats in the Sea of Okhotsk (78 strains) and the Sea of Japan (14 strains). Strains producing highly active glycanases and glycosidases were found. Synthesis of O-glycosylhydrolases was stimulated by addition of laminaran to the nutrient medium. Highly purified N-acetyl-β-D-glucosaminidase was isolated from the marine fungus Penicillium canescens. The molecular weight of the enzyme determined by SDS-Na-electrophoresis was 68 kDa. The enzyme displayed maximum activity at pH 4.5 and temperature 45°C. Inactivation half-time of the enzyme at 50°C was 25 min. N-acetyl-β-D-glucosaminidase hydrolyzed both β-glucosaminide and β-galactosaminide bonds and possessed a high transglycosylating activity.     

Enzymatic properties,crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans

A high-isoelectric-point (pI), alkaline endo-1,4-β-glucanase (Egl-257) of Bacillus circulans KSM-N257 was purified to homogeneity and crystallized. The purified enzyme hydrolyzed carboxymethyl cellulose (CMC) with optima of pH 8.5 and 55 °C. The molecular mass was 43 kDa, and the pI was pH 9.3. The structural gene contained a single open reading frame of 1221 bp, corresponding to 407 amino acids (aa), including a 30-aa signal peptide (377 aa and 41,680 Da for the mature enzyme). Egl-257 hydrolyzed lichenan and showed 76.3% aa identity to a lichenase from B. circulans WL-12 belonging to glycosyl hydrolase family 8 but did not hydrolyze laminarin, curdran, and xylan at all. This indicates that Egl-257 is a true endo-1,4-β-glucanase. However, this enzyme was not active on p-nitrophenyl β-d-cellotrioside and p-nitrophenyl β-d-cellotetraoside. It was crystallized by the hanging-drop vapor-diffusion method with phosphate plus CdCl₂ as precipitant. Pyramid-like crystals were formed, and they diffracted X-rays beyond 2.2 Å resolution. It belongs to the space group P2₁2₁2₁ with unit cell parameters of a=62.5 Å, b=71.7 Å, and c=88.6 Å.     

Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Specific xyloglucanases as a new class of polysaccharide-degrading enzymes

Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanases.     

Molecular Cloning of Novel Cellulase Genes <Emphasis Type="Italic">cel</Emphasis>9A and <Emphasis Type="Italic">cel</Emphasis>12A from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> GXN151 and Synergism of Their Encoded Polypeptides

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.     

Extracellular β-D-glucosidase of the Penicillium canescens marine fungus

Extracellular β-D-glucosidase was isolated in a homogeneous state from the Penicillium canescens marine fungus. According to SDS-electrophoresis, the molecular weight of the enzyme was 64 kDa and the maximal activity was observed at pH 5.2 and 70°C. Glucosidase catalyzed the hydrolysis of β-glycosidic bonds both in glycosides and in glucose disaccharides and had transglycosylation activity. The enzyme can be used for the deglycosylation of natural glycosides and in enzymatic synthesis of new carbohydrate—containing compounds.     

Cloning and Characterization of Xylanase A from the Strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. BP-7: Comparison with Alkaline pI-Low Molecular Weight Xylanases of Family 11

The xynA gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-7 has been cloned and expressed in Escherichia coli. Recombinant xylanase A showed high activity on xylans from hardwoods and cereals, and exhibited maximum activity at pH 6 and 60°C. The enzyme remained stable after incubation at 50°C and pH 7 for 3 h, and it was strongly inhibited by Mn²⁺, Fe³⁺, Pb²⁺, and Hg²⁺. Analysis of xylanase A in zymograms showed an apparent molecular size of 24 kDa and a pI of above 9. The amino acid sequence of xylanase A, as deduced from xynA gene, shows homology to alkaline pI-low molecular weight xylanases of family 11 such as XynA from Bacillus subtilis. Analysis of codon usage in xynA from Bacillus sp. BP-7 shows that the G+C content at the first and second codon positions is notably different from the mean values found for glycosyl hydrolase genes from Bacillus subtilis.     

Cloning,Isolation, and Properties of a New Homologous Exoarabinase from the <Emphasis Type="Italic">Penicillium canescens</Emphasis> Fungus

A novel exo-arabinase (GH93, exo-ABN) enzyme produced by the ascomycete Penicillium canescens has been studied. Cloning of the abn1 gene coding for exo-ABN into the recipient P. canescens strain RN3-11-7 yielded recombinant producing strains characterized by a high yield of extracellular exo- ABN production (20–30% of the total amount of extracellular protein). Chromatographic purification yielded a homogenous exo-ABN with a molecular weight of 47 kDa, as shown by SDS-PAGE. The enzyme showed high specific activity towards linear arabinan (117 U/mg) and low specific activity towards branched arabinan and arabinoxylan (4–5 U/mg) and para-nitrophenyl-α-L-arabinofuranoside (0.3 U/mg), whereas arabinogalactan and para-nitrophenyl-α-L-arabinopyranoside, the substrates that contained the pyranose form of arabinose, were not hydrolyzed. Arabinohexaose was the major product of linear arabinan hydrolysis. Exo-ABN had a pH optimum at 5.0 and a temperature optimum at 60°C. The enzyme was stable in a broad pH range (4.0–7.0) and upon heating to 50°C during 180 min. Extensive hydrolysis of linear and branched arabinans by exo- and endo-arabinase mixtures, arabinofuranosidase, and arabinofuran-arabinoxylan hydrolase has been performed. The degree of substrate conversion amounted to 67 and 83% of the maximal possible value, respectively.     

Cloning of the gene <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis> chitinase <Emphasis Type="Italic">Lpchi1</Emphasis> and identification of its potential role in the biocontrol of root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

The complete genome sequence of Bacillus subtilis reveals that sequences encoding several hemicellulases are co-localised with a gene (xynD) encoding a putative family 43 glycoside hydrolase that has not yet been characterised. In this work, xynD has been isolated from genomic DNA of B. subtilis subsp. subtilis ATCC 6051 and cloned for cytoplasmatic expression in Escherichia coli. Recombinant XynD (rXynD) was purified using ion-exchange chromatography and gel permeation chromatography. The enzyme had a molecular mass of approximately 52 kDa, a pI above 9.0 and releases α-l-arabinose from arabinoxylo-oligosaccharides as well as arabinoxylan polymers with varying degree of substitution. Using para-nitrophenyl-α-l-arabinofuranoside as substrate, maximum activity was observed at pH 5.6 and 45°C. The enzyme retained its activity over a large pH range, while activity was lost after pre-incubation above 50°C. Gas–liquid chromatography and proton nuclear magnetic resonance spectrometry analysis indicated that rXynD specifically releases arabinofuranosyl groups from mono-substituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. As rXynD did not display endoxylanase, xylosidase or arabinanase activity and was inactive on arabinan, we conclude that this enzyme is best described as an arabinoxylan arabinofuranohydrolase.     

Cloning of the <Emphasis Type="Italic">Penicillium canescens</Emphasis> endo-1,4-β-glucanase gene <Emphasis Type="Italic">egl3</Emphasis> and the characterization of the recombinant enzyme

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-β-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for β-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54°C, respectively. The K _m and V _m values for CMC hydrolysis were determined; they amounted to 17.1 g/l and 0.31 μM/(mg s), respectively.     

A cel44C-man26A gene of endophytic Paenibacillus polymyxa GS01 has multi-glycosyl hydrolases in two catalytic domains

A bacterial strain Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). The cel44C-man26A gene was cloned from this endophytic strain. This 4,056-bp gene encodes for a 1,352-aa protein which, based on BLAST search homologies, contains a glycosyl hydrolase family 44 (GH44) catalytic domain, a fibronectin domain type 3, a glycosyl hydrolase family 26 (GH26) catalytic domain, and a cellulose-binding module type 3. The multifunctional enzyme domain GH44 possesses cellulase, xylanase, and lichenase activities, while the enzyme domain GH26 possesses mannanase activity. The Cel44C enzyme expressed in and purified from Escherichia coli has an optimum pH of 7.0 for cellulase and lichenase activities, but is at an optimum pH of 5.0 for xylanase and mannanase activities. The optimum temperature for enzymatic activity was 50°C for all substrates. No detectable enzymatic activity was detected for the Cel44C-Man26A mutants E91A and E222A. These results suggest that the amino acid residues Glu₉₁ and Glu₂₂₂ may play an important role in the glycosyl hydrolases activity of Cel44C-Man26A.     

A β-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae

 A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding. Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999     

Production of enzyme preparations on the basis of Penicillum canescens recombinant strains with a high ability for the hydrolysis of plant materials

An enzyme preparation has been produced on the basis of Penicillium canescens strains with the activity of cellibiohydrolase I, II; endo-1,4-β-gluconase of Penicillium verruculosum; and β-glucosidase of Aspergillus niger. It was shown that for the most effective hydrolysis of aspen wood pulp the optimal ratio of cellobiohydrolase and endo-1,4-β-gluconase in enzyme preparations was 8: 2 (by protein). It was also established that the homologous xylanase secreted by the Penicillium canescens fungus is a required component for the enzyme complex for hydrolysis of the hemicellulose matrix of aspen wood.     

Characterization of two novel family 12 xyloglucanases from the thermophilic Rhizomucor miehei

Two novel glycoside hydrolase (GH) family 12 xyloglucanase genes (designated RmXEG12A and RmXEG12B) were cloned from the thermophilic fungus Rhizomucor miehei. Both genes contained open reading frames of 729 bp encoding 242 amino acids. Their deduced amino acid sequences shared 68 % identity with each other and less than 60 % with other xyloglucanases. The two genes, without the sequences for the signal peptides, were cloned and successfully expressed in Escherichia coli as active xyloglucanases, designated RmXEG12A and RmXEG12B, with similar molecular masses—25.6 and 25.9 kDa, respectively. RmXEG12A showed optimal activity at pH?6.5 and 65 °C, RmXEG12B at pH?5.0 and 60 °C. Both recombinant xyloglucanases displayed very high specific activities, 6,681.4 and 3,092.2 U?mg^?1, respectively, toward tamarind xyloglucan, but no activity toward carboxymethylcellulose, Avicel, or p-nitrophenyl derivatives. The main products of tamarind xyloglucan hydrolysis by the two xyloglucanases were XXXG, XXLG/XLXG, and XLLG (where G is an unsubstituted β-d-Glc residue, X is a xylosylated β-d-Glc residue, and L is a β-d-Glc residue substituted by xylosyl-galactose).     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.     

Comparison of chitinolytic enzymes from an alkaline, hypersaline lake and an estuary

We examined the genetic and physiological characteristics of chitin degrading enzymes expressed by fosmids cloned from two strains of chitinolytic gammaproteobacteria isolated from alkaline, hypersaline Mono Lake, California; and from a metagenomic library derived from an estuarine bacterial community (Dean Creek, Sapelo Island, GA, USA). The Mono Lake chitinolytic enzymes presented unique adaptations in terms of halo- and alkalitolerance. The sequence from one of the Mono Lake isolates (strain 12A) was a conventional family 18 glycosyl hydrolase; however, the expressed protein had a novel secondary activity peak at pH 10. We obtained a novel family 20 glycosyl hydrolase sequence from Mono Lake strain AI21. The activity of the expressed protein had a pH optimum of 10, several pH units higher than any other enzyme currently assigned to this family, and the enzyme retained 80% of its activity at pH 11. The enzyme was also halotolerant, retaining activity in salt solutions of up to 225 g l(-1). Sequence analysis indicated a molecular weight of approximately 90 kDa for the protein, and that it contained two active sites. Culture supernatant contained two chitinolytic proteins, 45 and 31 kDa, suggesting possible post-expression modification of the gene product. In contrast, the sequence found in the estuarine metagenomic library and the functional characteristics of the protein expressed from it were those of a conventional family 18 glycosyl hydrolase.