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

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

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

Chondroitinase has been used as an important tool in the study of the structure, function and distribution of glycosaminoglycans for many years. Recently, the enzyme has been reported to be a potential enzyme for chemonucleolysis, an established treatment for intervertebral disc protrasion. In this paper, a chondroitinase had been purified from the culture supernatant of Aeromonas sobria YH311 using a simple purification procedure of ammonium sulfate precipitation, QAE-Sephadex A50 ion exchange chromatography and Sephadex G-150 gel filtration. The immobilization of purified chondroitinase using sodium alginate or cellulose as carriers has also been studied. The chondroitinase obtained from Aeromonas sobria YH311 was purified 55-fold to 95.3% pure, the specific activity of the purified enzyme was 31.86u/mg and the yield was 37%. The molecular weight of chondroitinase from Aeromonas sobria YH311 was determined by SDS-PAGE to be 80kD, which was almost the same as those chondroitinase AC from Arthrobacter aurescens, Aeromonas liquefaciens and Flavobacterium heparinum. But its isoelectric point was 4.3 - 4.6, which was far lower than the microbial chondroitinase AC. After the immobilization on sodium alginate or cellulose, the properties of chondroitinase changed greatly. The optimum temperature and pH of the free enzyme were 50 degrees C and 7.0 respectively, and about 10% activity remained after heat treatment at 80 degrees C for 20 minutes, and 47% activity remained after two weeks storage at 4 degrees C. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40 degrees C and 7.0 respectively, about 50% activity remained after 80 degrees C heat treatment for 120 minutes and 50% remained after 30 days storage at 4 degrees C. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70 degrees C and 6.0 respectively, and more than 70% activity remained after heat treatment at 80 degrees C and 30 days storage at 4 degrees C. The yield of the immobilization was very low, with 18.56% for alginate and 18.86% for cellulose.
Aeromonas ; enzymology ; Chondroitinases and Chondroitin Lyases ; isolation & purification ; metabolism ; Enzyme Stability ; Enzymes, Immobilized ; metabolism ; Temperature

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

In order to increase the production of alkaline polygalacturonate lyase (PGL) by recombinant Pichia pastoris GS115, the effect of cell and methanol concentration on the PGL production was carefully investigated by single factor experiment. The optimum conditions were listed as follows: the cell concentration 122 g/L, the methanol concentration 20 g/L, and the ratio of methanol and cell concentration 0.16-0.20 g/g (methanol/cell). With the glycerol and methanol feeding strategies, the ratio of methanol and cell concentration could be controlled at the range of 0.171 to 0.195 g/g. And the highest PGL activity (430 u/mL) and highest PGL productivity (4.34 u/mL/h) were achieved.
Alkalies ; metabolism ; Fermentation ; Genetic Vectors ; Methanol ; chemistry ; Pichia ; enzymology ; genetics ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic

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

Pectin lyases from Aspergillus oryzae and Aspergillus niger are usually used for the production of traditional fermented foods, but these fungi produce less pectinases under natural conditions. The cDNA coding mature Pell (without signal peptide) was amplified from Aspergillus oryzae by RT-PCR. Pell cDNA was cloned into pET-28a ( + ) expression vector, then was transformed into E. coli Turner (DE3) plac I cells to express Pell with 6-His tag. For improving the efficiency of Pell expression in E. coli, the conditions of expressing the Pell in E. coli were optimized. E. coli Turner (DE3) plac I cells with pET-28a ( + )-pell was first cultivated at 37 degrees C, 220 r/min until OD600 reached about 0.8. Then, cultivation broth was added with 0.05-0.1 mmol/L IPTG and continuously incubated at 15 degrees C, at 170 r/min for 60 h for expressing of Pell. The recombinant expressed Pell activity could reach 400 u/mL medium, which is 4000-fold of Pell produced naturally by A. oryzae and superior than known recombinant amount of pectin lyases expressed in different fungi expression systems.
Amino Acid Sequence ; Aspergillus oryzae ; enzymology ; genetics ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Polysaccharide-Lyases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism