Bonnin, E., Clavurier, K., Daniel, S., Kauppinen, S., Mikkelsen, J.D.M., Thibault, J.-F.
"Pectin acetylesterases from Aspergillus are able to deacetylate homogalacturonan as well as rhamnogalacturonan"
Carbohydrate Polymers, . Article in Press. (2008)
An acetylesterase was purified from Aspergillus aculeatus on the basis of its ability to deacetylate homogalacturonan. A series of well-characterized substrates was prepared and used to investigate its specificity, including pectin with various degrees of methylation and acetylation, pectic domains with various structures, and oligomers. It was then compared to a rhamnogalacturonan acetylesterase previously isolated from the same fungus. Both enzymes were active towards various acetylated pectins, and were able to release acetyl groups from acetylated homogalacturonan, oligogalacturonates as well as rhamnogalacturonan at different extent. It was thus concluded that while the two enzymes differed in their efficiency, they are both pectin acetylesterases, able to act in the "smooth" as well as in the "hairy" regions of pectin. © 2008 Elsevier Ltd. All rights reserved.
Wang, B., Wu, W., Liu, X.
"Purification and characterization of a neutral serine protease with nematicidal activity from Hirsutella rhossiliensis"
Mycopathologia, 163 (3), pp. 169-176. (2007)
Serine protease plays an important role in fungal infection to invertebrate hosts. An extracellular protease (Hnsp) was detected in liquid culture of Hirsutella rhossiliensis OWVT-1 with nematodes (Panagrellus redivivus) as the unique nitrogen source and purified to homogeneity by ammonium sulphate precipitation, anion exchange chromatography and gel filtration. Its molecular mass was about 32 kDa, and the optimal reaction pH value and temperature were pH 7 and 40°C, respectively. The Hnsp activity was stable at pH 6-8 and decreased radically at 50°C for 10 min. Hnsp was highly sensitive to inhibitor of PMSF and well decomposed the substrate N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide, suggesting that it belonged to the chymotrypsin/subtilisin of serine proteases. The N-terminal amino acid sequence of Hnsp was SVTDQQGADCGLARISHRE, which showed high homology with other serine proteases from nematophagous fungi. Ability to kill nematode and degrade its cuticle in vitro indicated that Hnsp could be involved in the infection of nematode. © 2007 Springer.
Langston, J., Blinkovsky, A., Byun, T., Terribilini, M., Ransbarger, D., Xu, F.
"Substrate specificity of Streptomyces transglutaminases"
Applied Biochemistry and Biotechnology, 136 (3), pp. 291-308. (2007)
Transglutaminase (TGase) is a multifunctional enzyme vital for many physiologic processes, such as cell differentiation, tissue regeneration, and plant pathogenicity. The acyl transfer function of the enzyme can activate primary amines and, consequently, attach them onto a peptidyl glutamine, a reaction important for various in vivo and in vitro protein crosslinking and modification processes. To understand better the structure-function relationship of the enzyme and to develop it further as an industrial biocatalyst, we studied TGase secreted by several Streptomyces species and Phytophthora cactorum. We purified the enzyme from S. lydicus, S. platensis, S. nigrescens, S. cinnamoneus, and S. hachijoensis. The pH and temperature profiles of S. lydicus, S. platensis, and S. nigrescens TGases were determined. The specificity of S. lydicus TGase toward its acyl-accepting amine substrates was characterized. Correlation of the electronic and steric features of the substrates with their reactivity supported the mechanism previously proposed for Streptomyces mobaraensis TGase. Copyright © 2007 by Humana Press Inc. All rights of any nature whatsoever reserved.
Sørensen, H.R., Jørgensen, C.T., Hansen, C.H., Jørgensen, C.I., Pedersen, S., Meyer, A.S.
"A novel GH43 α-L-arabinofuranosidase from Humicola insolens: Mode of action and synergy with GH51 α-L-arabinofuranosidases on wheat arabinoxylan"
Applied Microbiology and Biotechnology, 73 (4), pp. 850-861. (2006)
A novel α-l-arabinofuranosidase (α-AraF) belonging to glycoside hydrolase (GH) family 43 was cloned from Humicola insolens and expressed in Aspergillus oryzae. 1H-NMR analysis revealed that the novel GH43 enzyme selectively hydrolysed (1→3)-α-l-arabinofuranosyl residues of doubly substituted xylopyranosyl residues in arabinoxylan and in arabinoxylan-derived oligosaccharides. The optimal activity of the cloned enzyme was at pH 6.7 and 53°C. Two other novel α-l-arabinofuranosidases (α-AraFs), both belonging to GH family 51, were cloned from H. insolens and from the white-rot basidiomycete Meripilus giganteus. Both GH51 enzymes catalysed removal of (1→2) and (1→3)-α-l-arabinofuranosyl residues from singly substituted xylopyranosyls in arabinoxylan; the highest arabinose yields were obtained with the M. giganteus enzyme. Combinations (50:50) of the GH43 α-AraF from H. insolens and the GH51 α-AraFs from either M. giganteus or H. insolens resulted in a synergistic increase in arabinose release from water-soluble wheat arabinoxylan in extended reactions at pH 6 and 40°C. This synergistic interaction between GH43 and GH51 α-AraFs was also evident when a GH43 α-AraF from a Bifidobacterium sp. was supplemented in combination with either of the GH51 enzymes. The synergistic effect is presumed to be a result of the GH51 α-AraFs being able to catalyse the removal of single-sitting (1→2)-α-l- arabinofuranosyls that resulted after the GH43 enzyme had catalysed the removal of (1→3)-α-l-arabinofuranosyl residues on doubly substituted xylopyranosyls in the wheat arabinoxylan. © 2006 Springer-Verlag.
Rasmussen, L.E., Sørensen, H.R., Vind, J., Viksø-Nielsen, A.
"Mode of action and properties of the β-xylosidases from Talaromyces emersonii and Trichoderma reesei"
Biotechnology and Bioengineering, 94 (5), pp. 869-876. (2006)
Enzymatic hydrolysis of arabinoxylan is an important prerequisite for the utilization of hemicellulose for ethanol fermentation or for making the low calorie sweetener xylitol by catalytic hydrogenation of the generated xylose. This study focus on cloning and characterization of two industrial relevant β-xylosidases (1,4-β-D-xylan xylohydrolase, EC 188.8.131.52) from Talaromyces emersonii (βXTE) and Trichoderma reesei (βXTR) and a comparison of these in relation to hemicellulose hydrolysis using an industrial relevant substrate. Both β-xylosidases were expressed in A. oryzae and subsequently purified. During the enzymatic hydrolysis of xylobiose, the reaction product of both enzymes was found to be β-D-xylose proving that the hydrolysis is proceeding via a retaining reaction mechanism. Based on sequence similarities and glycosyl hydrolases family membership, the active site residues of βXTE and βXTR are predicted to be Asp 242 and Glu 441, and Asp 264 and Glu 464, respectively. The involvement in catalysis of these carboxyls was examined by modification using the carbodiimide-nucleophile procedure resulting in a complete inactivation of both enzymes. The degree of xylose release from vinasse, an ethanol fermentation by-product, by βXTE and βXTR was 12.1% and 7.7%, respectively. Using the β-xylosidases in combination with the multicomponent enzyme product Ultraflo L, resulted in 41.9% and 40.8% release of xylose, respectively indicating a strong synergistic effect between the exo-acting β-xylosidases and the endo-1,4-β- xylanases and α-L-arabinofuranosidase in Ultraflo L. There seems to be no measurable differences between the two β-xylosidases when used in this specific application despite the differences in specific activity and kinetic properties. © 2006 Wiley Periodicals, Inc.
Thiemann, V., Saake, B., Vollstedt, A., Schäfer, T., Puls, J., Bertoldo, C., Freudl, R., Antranikian, G.
"Heterologous expression and characterization of a novel branching enzyme from the thermoalkaliphilic anaerobic bacterium Anaerobranca gottschalkii"
Applied Microbiology and Biotechnology, 72 (1), pp. 60-71. (2006)
The gene encoding the branching enzyme (BE) from the thermoalkaliphilic, anaerobic bacterium Anaerobranca gottschalkii was fused with a twin arginine translocation protein secretory-pathway-dependent signal sequence from Escherichia coli and expressed in Staphylococcus carnosus. The secreted BE was purified using hydrophobic interaction and gel filtration chromatography. The monomeric enzyme (72 kDa) shows maximal activity at 50°C and pH 7.0. With amylose the BE displays high transglycosylation and extremely low hydrolytic activity. The conversion of amylose and linear dextrins was analysed by applying high-performance anion exchange chromatography and quantitative size-exclusion chromatography. Amylose (104-4×107 g/mol) was converted to a major extent to products displaying molecular masses of 10 4-4×105 g/mol, indicating that the enzyme could be applicable for the production of starch or dextrins with narrow molecular mass distributions. The majority of the transferred oligosaccharides, determined after enzymatic hydrolysis of the newly synthesized α-1,6 linkages, ranged between 103 and 104 g/mol, which corresponds to a degree of polymerisation (DP) of 6-60. The minimal donor chain length is DP 16. Furthermore, the obtained results support the hypotheses of a random endocleavage mechanism of BE and the occurrence of interchain branching. © Springer-Verlag 2006.
Langston, J., Sheehy, N., Xu, F.
"Substrate specificity of Aspergillus oryzae family 3 β-glucosidase"
Biochimica et Biophysica Acta - Proteins and Proteomics, 1764 (5), pp. 972-978. (2006)
Among glycoside hydrolases, β-glucosidase plays a unique role in many physiological and biocatalytical processes that involve the β-linked O-glycosyl bond of various oligomeric saccharides or glycosides. Structurally, the enzyme can be grouped into glycoside hydrolase family 1 and 3. Although the basic ("retaining, double-displacement") mechanism for the catalysis of family 3 β-glucosidase has been established, in-depth understanding of its structure-function relationship, particularly the substrate specificity that is of great interest for developing the enzyme as a versatile biocatalyst, remains limited. To further probe the active site, we carried out a comparative study on a family 3 β-glucosidase from Aspergillus oryzae with substrates and competitive inhibitors of different structures, in attempt to evaluate the site-specific spatial and chemical interactions between a pyranosyl substrate and the enzyme. Our results showed the enzyme having a strict stereochemical requirement (to accommodate β-d-glucopyranose) for its "- 1" active subsite, in contrast to its family 1 counterpart. © 2006 Elsevier B.V. All rights reserved.
Viksø-Nielsen, A., Andersen, C., Hoff, T., Pedersen, S.
"Development of new α-amylases for raw starch hydrolysis"
Biocatalysis and Biotransformation, 24 (1-2), pp. 121-127. (2006)
This paper describes the discovery of a new 4 domain α-amylase from Anoxybacillus contaminans which very efficiently hydrolyses raw starch granules. Compared to traditional starch liquefying α-amylases, this new 4 domain α-amylase contains a starch binding domain. The presence of this starch-binding domain enables the enzyme to efficiently hydrolyse starch at a temperature below the gelatinisation temperature. At a reaction temperature of 60°C and in combination with a glucoamylase from Aspergillus niger it was possible to liquefy 99% of the starch obtaining a DX value of 95%. Furthermore, we describe how the current HFCS process can be turned into a low temperature simultaneous liquefaction and saccharification process by using this new 4 domain α-amylase in combination with a glucoamylase. © 2006 Taylor & Francis.
Yang, J., Huang, X., Tian, B., Sun, H., Duan, J., Wu, W., Zhang, K.
"Characterization of an extracellular serine protease gene from the nematophagous fungus Lecanicillium psalliotae"
Biotechnology Letters, 27 (17), pp. 1329-1334. (2005)
The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3′ and 5′ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi. © Springer 2005.
Bermek, H., Yazici, H., Öztürk, H., Tamerler, C., Jung, H., Li, K., Brown, K.M., Ding, H., Xu, F."Purification and characterization of manganese peroxidase from wood degrading fungus Trichophyton rubrum LSK-27"
Enzyme and Microbial Technology, 35 (1), pp. 87-92. (2004)
A manganese peroxidase (MnP) from a wood-degrading fungus Trichophyton rubrum LSK-27 was purified to homogeneity by anion-exchange chromatography followed by gel filtration. Molecular mass of the purified enzyme was determined to be about 42 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Spectrophotometric analysis of the enzyme revealed one Soret maximum at 407 nm, and two visible peaks at 502 and 644 nm, which are consistent with photometric spectra of other MnPs. Mass spectrometric analysis of the digested protein revealed that it had a very high homology to a unique peroxidase (a hybrid of MnP and lignin peroxidase) from Bjerkandera sp. B33/3. Bjerkandera MnP was able to oxidize veratryl alcohol, whereas T. rubrum LSK-27 MnP could not. T. rubrum LSK-27 MnP had the highest pI of 8.2 among MnPs reported so far. The enzyme was stable at rather high temperatures, and when compared with other MnPs, this MnP was more stable in the presence of high concentrations of H2O2. © 2004 Elsevier Inc. All rights reserved.
Bertoldo, C., Armbrecht, M., Becker, F., Schäfer, T., Antranikian, G., Liebl, W.
"Cloning, sequencing, and characterization of thermoalkalistable type I pullulanase from Anaerobranca gottschalkii"
Applied and Environmental Microbiology, 70 (6), pp. 3407-3416. (2004)
The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.
M.L. Shinohara; M.Ihara; M.Abo; M.Hashida; S.Takagi; T.C.Beck.
"A novel thermostable branching enzyme from an extremely thermophilic bacterial species, Rhodothermus obamensis".
Appl Microbiol Biotechnology, 57, 653-659 (2001)
A branching enzyme (EC 184.108.40.206) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular weight of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the E. coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E.coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0 - 6.5 and 65 °C. The enzyme was stable after 30 min at 80 °C, and retained 50 % of activity at 80 °C after 16 hours. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.
F. Xu; E.J. Golightly; C.C. Fuglsang; P. Schneider P, K.R. Duke. L. Lam; S. Christensen; K.M. Brown; C.T. Jorgensen; S.H. Bro wn.
"A novel carbohydrate : acceptor oxidoreductase from Microdochium nivale".
Eur. J. Biochem., 268, 1136-1142 (2001)
A Microdochium nivale carbohydrate:acceptor oxidoreductase was purified, cloned, heterologously expressed, and characterized. The gene encoding the protein showed one intron, and the ORF showed a sequence with low homology (less than or equal to 25% identity or 65% similarity) to other known flavin-containing carbohydrate oxidases. The maturation of the protein required the cleavage of a tetrameric propeptide in addition to an 18 amino-acid signal peptide. The enzyme was found to have a relative molecular mass of 55 000 Da, an isoelectric point of 9, and one FAD per protein. It could oxidize mono-, oligo-, or polymeric saccharides, and transfer their electrons to O-2 or other accepters. When D-glucose served as electron-donating substrate, an activity of 2 s(-1) was observed at pH 5.5 and 23 degreesC. Among various oligosaccharides, the enzyme preferred tetrameric dextrins, indicating a favorable interaction of four linked glucose units with the substrate pocket. The unique structure and ability of oxidizing oligo/polymeric saccharides suggest a promising prospect of this enzyme for various industrial/medicinal applications.
F. Xu; E.J. Golightly; P. Schneider; R.M. Berka; K.M. Brown; J.A. Johnstone; D.H. Baker; C.C. Fuglsang; S.H. Brown; A.V. Klotz.
"Expression and characterization of a recombinant Fusarium spp. galactose oxidase."
Appl. Biochem. Biotechnol. 88, 23-32 (2000)
The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger á-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after Cu saturation. Based on peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnovers per second toward D-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of D-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and cyclic hemiacetal O sites. The recombinant oxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.
R.M. Berka; P. Schneider; E.J. Golightly; S.H. Brown; M. Madden, K.M. Brown, T. Halkier, K. Mondorf; F. Xu.
"Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme produced in Aspergillus oryzae."
Appl. Environ. Microbiol. 63, 3151-3157 (1997)
A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion- exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase shows an absorbance spectrum that is typical of blue copper oxidases, with maxima at 276 and 589 nm, and contains 3.9 copper atoms per subunit. With syringaldazine as a substrate, MtL has optimal activity at pH 6.5 and retains nearly 100% of its activity when incubated at 60 degrees C for 20 min. This is the first report of the cloning and heterologous expression of a thermostable laccase.