Xylanase from the psychrophilic yeast Cryptococcus adeliae (original) (raw)
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Structural characterization of a xylanase from psychrophilic yeast by mass spectrometry
The complete structural characterization of the xylanase, a glycoprotein constituted of 338 amino acids, from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. The verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites and the investigation of glycosylation pattern were performed. This analysis revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures. The data obtained on both the N-and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophylic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophylic and thermophylic cells. The cysteine pairings were eventually identified as Cys173-Cys205 and Cys272-Cys278, with Cys89 showing a free thiol group. These data suggest that a common folding motif might occur within the entire xylanase family in which the second Cys is linked to the third one with the fourth and fifth joined together.
Thermostable xylanases from thermophilic fungi and bacteria: Current perspective
Bioresource Technology, 2019
Thermostable xylanases from thermophilic fungi and bacteria have a wide commercial acceptability in feed, food, paper and pulp and bioconversion of lignocellulosics with an estimated annual market of USD 500 Million. The genome wide analysis of thermophilic fungi clearly shows the presence of elaborate genetic information coding for multiple xylanases primarily coding for GH10, GH11 in addition to GH7 and GH30 xylanases. The transcriptomics and proteome profiling has given insight into the differential expression of these xylanases in some of the thermophilic fungi. Bioprospecting has resulted in identification of novel thermophilic xylanases that have been endorsed by the industrial houses for heterologous over-expression and formulations. The future use of xylanases is expected to increase exponentially for their role in biorefineries. The discovery of new and improvement of existing xylanases using molecular tools such as directed evolution is expected to be the mainstay to meet increasing demand of thermostable xylanases.
We report the crystal structure at 1.59 A Ê and the proposed amino acid sequence of an endo-1,4-b-xylanase (PVX) from the thermophilic fungus Paecilomyces varioti Bainier (PvB), stable up to 75 C. This fungus is attracting clinical attention as a pathogen causing post-surgical infections. Its xylanase, known as a skin-contact allergen, is the ®rst protein from this fungus whose three-dimensional structure has been elucidated. The crystals of PVX conform to the space group P2 1 2 1 2 1 with a 38.76 A Ê , b 54.06 A Ê and c 90.06 A Ê . The structure was solved by molecular replacement techniques using polyalanine coordinates of the Thermomyces lanuginosus xylanase (PDB code 1YNA) and a careful model building based on the amino acid sequence known for two trypsin-digested peptide fragments (17 residues), the sequence and structural alignment of family-11 xylanases and electron density maps. The ®nal re®ned model has 194 amino acid residues and 128 water molecules, with a crystallographic R-factor of 19.07 % and a free R-factor of 21.94 %. The structure belongs to an all-b fold, with two curved b-sheets, forming the cylindrical active-site cleft, and a lone a-helix, as present in other family-11 xylanases. We have carried out a quantitative comparison of the structure and sequence of the present thermophilic xylanase (PVX) with other available native structures of mesophiles and thermophiles, the ®rst such detailed analysis to be carried out on family-11 xylanases. The analysis provides a basis for the rationalisation of the idea that the``hinge'' region is made more compact in thermophiles by the addition of a disulphide bridge between Cys110 and Cys154 and a N-H Á Á ÁO hydrogen bond between Trp159 near the extremity of the lone a-helix and Trp138 on b-strand B8. This work brings out explicitly the presence of the C-H Á Á ÁO and the C-H Á Á Áp type interactions in these enzymes. A complete description of structural stability of these enzymes needs to take account of these weaker interactions.
2003
The Humicola grisea var. thermoidea is known as a good producer of hydrolytic enzymes. The H. grisea endo-1,4-xylanase gene (xyn2) was isolated and its sequence was translated into a predicted protein coding for a xylanase of 23 kDa. A structural model of H. grisea endo-1,4-xylanase (XYN2) was built by homology modeling based on the database search results of related proteins, belonging to Glycoside Hydrolase Family 11 (GH11). The inactive/active conformation transition of the XYN2 model active site is pH sensitive as revealed by independent molecular dynamics simulations at different pH. The active conformation exhibits the common structural-sheet twisted architecture of the GH11. The active site is formed by a large cleft containing the catalytic residues (E84 and E175), and is stabilized by hydrogen bond network involving the Q134, Y75, Y88, W77, and Y169. Additionally, the structural properties described by the model explain the observed thermostability of the XYN2 protein. Acco...
The gene xynC encoding xylanase C from Bacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 3538 bp DNA fragment containing xynC gene was determined, revealing an open reading frame of 3258 bp that encodes a protein of 120567 Da. A comparison of the deduced amino acid sequence of xylanase C with known /I-glycanase sequences showed that the encoded enzyme is a modular protein containing three different domains. The central region of the enzyme is the catalytic domain, which shows high homology t o family 10 xylanases. A domain homologous t o family IX cellulose-binding domains is located in the C-terminal region of xylanase C, whilst the N-terminal region of the enzyme shows homology t o thermostabilizing domains found in several thermophilic enzymes. Xylanase C showed an activity profile similar t o that of enzymes from mesophilic microorganisms. Maximum activity was found at 45OC, and the enzyme was only stable at 55 O C or lower temperatures. Xylotetraose, xylotriose, xylobiose and xylose were the main products from birchwood xylan hydrolysis, whilst the enzyme showed increasing activity on xylo-oligosaccharides of increasing length, indicating that the cloned enzyme is an endoxylanase. A deletion derivative of xylanase C, lacking the region homologous t o thermostabilizing domains, was constructed. The truncated enzyme showed a lower optimum temperature for activity than the full-length enzyme, 35 O C instead of 4 5 OC, and a reduced thermal stability that resulted in a complete inactivation of the enzyme after 2 h incubation at 55 OC.
FEMS Yeast Research, 2005
A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae a-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3 g l À1 xylanase with a maximum activity of 3130 U l À1 to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37 kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T m) was 78°C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.
The Humicola grisea var. thermoidea is known as a good producer of hydrolytic enzymes. The H. grisea endo-1,4--xylanase gene (xyn2) was isolated and its sequence was translated into a predicted protein coding for a xylanase of 23 kDa. A structural model of H. grisea endo-1,4--xylanase (XYN2) was built by homology modeling based on the database search results of related proteins, belonging to Glycoside Hydrolase Family 11 (GH11). The inactive/active conformation transition of the XYN2 model active site is pH sensitive as revealed by independent molecular dynamics simulations at different pH. The active conformation exhibits the common structural -sheet twisted architecture of the GH11. The active site is formed by a large cleft containing the catalytic residues (E84 and E175), and is stabilized by hydrogen bond network involving the Q134, Y75, Y88, W77, and Y169. Additionally, the structural properties described by the model explain the observed thermostability of the XYN2 protein. According to our results, the thermostability of XYN2 protein, compared to mesophilic xylanases, can be explained by an additional electrostatic network and extra aromatic exposed residues.
Biochemical and Biophysical Research Communications, 2019
Xylanase is an important enzyme in industrial applications, which usually require the enzyme to maintain activity in high-temperature condition. In this study, a GH10 family xylanase XynAF0 from a thermophilic composting fungus, Aspergillus fumigatus Z5, was investigated to determine its thermostable mechanism. XynAF0 showed excellent thermostability, which could maintain 50% relative activity after incubation for 1 h at 70 C. The homologous modeling structure of XynAF0 was constructed and an a-helix composed of poly-threonine has been found in the linker region between the catalytic domain and the carbohydrate-binding module domain. Both the molecular dynamics simulation and the biochemical experiments proved that the a-helix plays an important role in the thermostability of XynAF0. Introducing of this poly-threonine region to the C-terminus of another GH10 family xylanase improved its thermostability. Our results indicated that the poly-threonine a-helix at the C-terminus of the catalytic domain was important for improving the thermophilic of GH10 family xylanases, which provides a new strategy for the thermostability modification of xylanases.