Whole-genome sequencing of Flammulina ononidis, a wood-rotting basidiomycete, was performed to identify genes associated with carbohydrate-active enzymes (CAZymes). A total of 12,586 gene structures with an average length of 2009 bp were predicted by the AUGUSTUS tool from a total 35,524,258 bp length of de novo genome assembly (49.76% GC). Orthologous analysis with other fungal species revealed that 7051 groups contained at least one F. ononidis gene. In addition, 11,252 (89.5%) of 12,586 genes for F. ononidis proteins had orthologs among the Dikarya, and F. ononidis contained 8 species-specific genes, of which 5 genes were paralogous. CAZyme prediction revealed 524 CAZyme genes, including 228 for glycoside hydrolases, 21 for polysaccharide lyases, 87 for glycosyltransferases, 61 for carbohydrate esterases, 87 with auxiliary activities, and 40 for carbohydrate-binding modules in the F. ononidis genome. This genome information including CAZyme repertoire will be useful to understand lignocellulolytic machinery of this white rot fungus F. ononidis.
Basidiomycota ; Esterases ; Flammulina* ; Fungi ; Genome ; Glycoside Hydrolases ; Glycosyltransferases ; Polysaccharide-Lyases

Heparinases can produce biologically active oligosaccharides by specifically cleaving the α-(1,4) glycosidic linkages of heparin and heparan sulphate. Heparinases are divided into heparinase and heparanase. Because heparinase is an effective biocatalyst, more and more researchers pay attention to the application of heparinase in medical field in the recent years. Combined with the related research work in our group, the application value of heparinase in the medical field was summarized, such as the determination of the structure of heparin, the preparation of low-molecular-weight heparin and ultra-low-molecular-weight heparin, tumor therapy and as a heparin antagonist. In addition, we summarized the definition, source of heparinase and its application in the medicine field. Heparinases have a great application prospect in the field of medicine.
Heparin ; Heparin Lyase ; metabolism ; Heparitin Sulfate ; Oligosaccharides ; Polysaccharide-Lyases

Heparinase II (Hep II) from Flavobacterium heparinum is an enzyme that could specifically cleave certain sequence of heparin and heparan sulfate. In this work, fermentation conditions of recombinant heparinase II (His-Hep II) producing bacteria were optimized, including initial induction time, inducer (IPTG) concentration, induction temperature and induction time. The optimum conditions were as follows: cultivating recombinant bacteria to exponential prophase under 37 degrees C, then adding IPTG to a final concentration of 0.3 g/L, finally cultivating recombinant bacteria under 20 degrees C for 10 h. The total crude enzyme activity reached 570 U/L. Based on these results, high cell density fermentation of recombinant bacteria was studied. The final OD600 could reach 98 and the total crude enzyme activity of His-Hep II increased to 9 436 U/L.
Fermentation ; Flavobacterium ; metabolism ; Microbiological Techniques ; Polysaccharide-Lyases ; biosynthesis ; Recombinant Proteins ; biosynthesis

In order to enhance the alkaline polygalacturonate lyase (PGL) productivity by Pichia pastoris, we developed a constant cell concentration culture strategy by methanol feeding (called as CCCM culture) used in the continuous cultures. We controlled reasonable cell concentrations in the bioprocess by different strategies of methanol feeding. Using this CCCM culture with DCW 75 g/L, we significantly enhanced the PGL productivity (Qv) and the average specific enzyme production rate (Qx) of PGL to 6.11 U/(mL x h) and 81.5 U/(g x h), increased by 42.1% and 191.2% than the fed-batch culture with high cell density, respectively. The final PGL activity was 441.9 U/mL. Moreover, the extracellular protease concentration is 1.9 mg/L and the cell viability is more than 94% after 120 hour induction. The results show that this new strategy is advantageous in reducing proteolytic degradation and enhancing cell viability.
Cell Count ; Culture Media ; Culture Techniques ; methods ; Fermentation ; Pichia ; genetics ; metabolism ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Heparinase III (HepIII) is a 73-kDa polysaccharide lyase (PL) that degrades the heparan sulfate (HS) polysaccharides at sulfate-rare regions, which are important co-factors for a vast array of functional distinct proteins including the well-characterized antithrombin and the FGF/FGFR signal transduction system. It functions in cleaving metazoan heparan sulfate (HS) and providing carbon, nitrogen and sulfate sources for host microorganisms. It has long been used to deduce the structure of HS and heparin motifs; however, the structure of its own is unknown. Here we report the crystal structure of the HepIII from Bacteroides thetaiotaomicron at a resolution of 1.6 Å. The overall architecture of HepIII belongs to the (α/α)₅ toroid subclass with an N-terminal toroid-like domain and a C-terminal β-sandwich domain. Analysis of this high-resolution structure allows us to identify a potential HS substrate binding site in a tunnel between the two domains. A tetrasaccharide substrate bound model suggests an elimination mechanism in the HS degradation. Asn260 and His464 neutralize the carboxylic group, whereas Tyr314 serves both as a general base in C-5 proton abstraction, and a general acid in a proton donation to reconstitute the terminal hydroxyl group, respectively. The structure of HepIII and the proposed reaction model provide a molecular basis for its potential practical utilization and the mechanism of its eliminative degradation for HS polysaccarides.
Amino Acid Sequence ; Bacteroides ; enzymology ; Catalytic Domain ; Crystallography, X-Ray ; Heparitin Sulfate ; metabolism ; Kinetics ; Models, Molecular ; Molecular Sequence Data ; Polysaccharide-Lyases ; chemistry ; metabolism ; Substrate Specificity

Marine can be considered as a rather unexplored source of biological material. Production of algal oligosaccharides by using valuable enzymes from marine origin has become an important way to utilize marine resources. As one of algal tool enzymes, the use of alginate lyases has been focused mainly on development and application of alginate oligosaccharides with bioactive function in recent years. In this paper, we reviewed the research of alginate lyases over the past decade in several aspects, including their origin, diversity, substrate specification, mode of action, structure and catalysis mechanism, assay of enzyme activity, enzyme characterization, as well as our own experience on this subject. At the end of the review, the application prospects of alginate lyases are presented.
Alginates ; metabolism ; Glucuronic Acid ; metabolism ; Hexuronic Acids ; metabolism ; Marine Biology ; methods ; Oligosaccharides ; metabolism ; Phaeophyta ; enzymology ; Polysaccharide-Lyases ; classification ; isolation & purification ; metabolism ; Substrate Specificity

The structure gene pelA from Thermotoga maritima MSB8 encoding pectate lyase was amplified and ligated into pHsh, resulting pHsh-pelA. Through structural optimization on pHsh-pelA, the ultimate plasmid, pHsh-pelC, which possessed the most appropriate structure and free energy of mRNA, was obtained. Pectate lyase C (PelC) was obtained after expressing pHsh-pelC in Escherichia coli JM109. The optimum activity of PelC was determined at pH 8.5 at 90 degrees C, with a half-life for almost 2 h at 95 degrees C. PelC was stable at the pH range of 8.2-9.8, and was dependent on Ca2+ for activity and stability. The enzyme kept stable for a long time and possessed a high level of activity at 60 degrees C. The kinetic assay using polygalacturonic acid (PGA) as substrate gave K(m) and V(max) of 0.11 mmol/L and 327 U per mg of protein. SDS-PAGE analysis showed that the molecular mass of the expressed recombinant PelC was about 43 kD, which was exactly the size predicted. The expression vector system of the heat shock plasmid pHsh owned such advantages as high expression level and cheap induction. Moreover, the superior stability of the recombinant enzyme laid the base for large-scale fermentation application.
Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Hot Temperature ; Polysaccharide-Lyases ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Recombination, Genetic ; Thermotoga maritima ; enzymology

Hyaluronidase is a goat testicular protein that hydrolyzes hyaluronic acid, a structural component of the intercellular matrix. It is commonly used as a spreading factor to improve the diffusion of drugs, including local anesthetics and chemotherapeutics. We experienced a 55-yr-old female with generalized urticaria that developed within 1 hr after the epidural injection of hyaluronidase. She had a history of allergic rhinitis, and had suffered from post-herpetic neuralgia and a herniated disc for several years. To relieve her pain, she had been given epidural injections consisting of mepivacaine hydrochloride, triamcinolone acetonide, and morphine sulfate biweekly for one year. Hyaluronidase had been administered several times with these drugs before this episode of generalized urticaria. Skin prick testing showed a positive response to 1,500 IU/mL of hyaluronidase extract, as compared to histamine. The patient's serum hyaluronidase-specific IgE level, determined using an enzyme-linked immunosorbent assay (ELISA), was markedly elevated, as compared to unexposed healthy controls. An IgE immunoblot analysis using hyaluronidase extract and the patient's serum showed IgE binding components at 31 and 21 kDa, whereas no corresponding IgE binding component was found in healthy controls. An ELISA inhibition test showed significant, dose-dependent inhibition with the serial addition of hyaluronidase extract. This is the first case of an IgE-medicated allergic reaction to goat (Naemorhedus goral raddenus) hyaluronidase, demonstrated by skin testing and a specific IgE and immunoblot assay.
Anesthetics, Local ; Diffusion ; Enzyme-Linked Immunosorbent Assay ; Female ; Goats ; Histamine ; Humans ; Hyaluronic Acid ; Hyaluronoglucosaminidase ; Hypersensitivity ; Hypersensitivity, Immediate ; Immunoglobulin E ; Injections, Epidural ; Intervertebral Disc Displacement ; Mepivacaine ; Morphine ; Neuralgia ; Polysaccharide-Lyases ; Rhinitis ; Rhinitis, Allergic, Perennial ; Skin ; Skin Tests ; Triamcinolone Acetonide ; Urticaria

Hyaluronidase is a goat testicular protein that hydrolyzes hyaluronic acid, a structural component of the intercellular matrix. It is commonly used as a spreading factor to improve the diffusion of drugs, including local anesthetics and chemotherapeutics. We experienced a 55-yr-old female with generalized urticaria that developed within 1 hr after the epidural injection of hyaluronidase. She had a history of allergic rhinitis, and had suffered from post-herpetic neuralgia and a herniated disc for several years. To relieve her pain, she had been given epidural injections consisting of mepivacaine hydrochloride, triamcinolone acetonide, and morphine sulfate biweekly for one year. Hyaluronidase had been administered several times with these drugs before this episode of generalized urticaria. Skin prick testing showed a positive response to 1,500 IU/mL of hyaluronidase extract, as compared to histamine. The patient's serum hyaluronidase-specific IgE level, determined using an enzyme-linked immunosorbent assay (ELISA), was markedly elevated, as compared to unexposed healthy controls. An IgE immunoblot analysis using hyaluronidase extract and the patient's serum showed IgE binding components at 31 and 21 kDa, whereas no corresponding IgE binding component was found in healthy controls. An ELISA inhibition test showed significant, dose-dependent inhibition with the serial addition of hyaluronidase extract. This is the first case of an IgE-medicated allergic reaction to goat (Naemorhedus goral raddenus) hyaluronidase, demonstrated by skin testing and a specific IgE and immunoblot assay.
Anesthetics, Local ; Diffusion ; Enzyme-Linked Immunosorbent Assay ; Female ; Goats ; Histamine ; Humans ; Hyaluronic Acid ; Hyaluronoglucosaminidase ; Hypersensitivity ; Hypersensitivity, Immediate ; Immunoglobulin E ; Injections, Epidural ; Intervertebral Disc Displacement ; Mepivacaine ; Morphine ; Neuralgia ; Polysaccharide-Lyases ; Rhinitis ; Rhinitis, Allergic, Perennial ; Skin ; Skin Tests ; Triamcinolone Acetonide ; Urticaria

In this study, the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique. The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA. By using electric shocks, we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115. The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis revealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.
Aspergillus niger ; enzymology ; genetics ; Electroporation ; Pichia ; genetics ; metabolism ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction