CN103923899A - Glycosaminoglycan lyase and encoding gene and application thereof - Google Patents

Glycosaminoglycan lyase and encoding gene and application thereof Download PDF

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CN103923899A
CN103923899A CN201410160095.3A CN201410160095A CN103923899A CN 103923899 A CN103923899 A CN 103923899A CN 201410160095 A CN201410160095 A CN 201410160095A CN 103923899 A CN103923899 A CN 103923899A
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glycosaminoglycan
glycosaminoglycan lyase
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李福川
韩文君
王文爽
赵梅
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Shandong University
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Abstract

The invention relates to glycosaminoglycan lyase and an encoding gene and an application thereof. The amino acid sequence of the glycosaminoglycan lyase is as shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene of the glycosaminoglycan lyase is as shown in SEQ ID NO.1; the specific activity of the glycosaminoglycan lyase prepared in the invention to hyaluronic acid is 110,000 U/Mg, and the specific activity to chondroitin sulfate is 45, 000 U/Mg; the enzyme activity is tens to hundreds of times as large as that of the existing commercial glycosaminoglycan lyase (such as CSaseABC, CSaseAC, I and II and the like), and the glycosaminoglycan lyase can be used in such fields as medicine and cosmetics and the like, thereby having a broad application prospect.

Description

A kind of glycosaminoglycan lyase and encoding gene and application
Technical field
The present invention relates to a kind of glycosaminoglycan lyase and encoding gene and application, belong to gene engineering technology field.
Background technology
Glycosaminoglycan (Proteoglycans, GAG) is called again mucopolysaccharide, is the straight-chain polysaccharide that repetition disaccharide unit forms.Mainly comprise hyaluronic acid (Hyaluronic Acid, HA), heparin/Suleparoid (Heparin/Heparan Sulfate, Hep/HS), chondroitin sulfate/dermatan sulfate (Chondroitin Sulfate/Dermatan Sulfate, CS/DS), keratan sulfate (Keratan Sulfate, KS).Except the disaccharide unit of keratan sulfate is comprised of neutral D-semi-lactosi and N-acetyl-glucosamine, the disaccharide unit of other glycosaminoglycan is all comprised of hexuronic acid (D-Glucose aldehydic acid/L-iduronic acid) and hexosamine (N-acetyl ammonia glucosamine/N-acetylgalactosamine).Wherein hyaluronic acid structure is relatively simple, and the disaccharide unit being comprised of D-Glucose aldehydic acid and N-acetyl-glucosamine is formed by connecting through β-Isosorbide-5-Nitrae-glycosidic link.Other glycosaminoglycan is because the effect of various modifying enzymes makes the sugar chain complex that becomes; be mainly manifested in the sulfation of different sites hydroxyl (OH) in sugar chain and amino (NH2); D-Glucose aldehydic acid changes L-iduronic acid under the effect of C5-epimerase, the acetylize of hexosamine two bit aminos etc.This high complexity of glycosaminoglycan sugar chain structure is not random generation, but there is Space-time speciality highly, that the synthetic relevant enzyme of various sugar chains is due to the different developmental phases expression regulation level of different cell tissue and organ, different structures makes it have different functions, and the complicacy of structure is given the diversity of its function.
Glycosaminoglycan is extensively present in zooblast cell surface and cell matrix, increment and the differentiation of cell by the special interaction with various albumen, have been participated in, intercellular identification, cell shifts, tissue morphology occurs, the various physiology such as canceration and pathologic process (Knudson and Knudson1993, Perrimon and Bernfield2000, Karbownik and Nowak2013) a series of important biological function that glycosaminoglycan has becomes important bioactive molecules, in medicine and functional food, be used widely, as heparin, chondroitin sulfate, hyaluronic acid (Noble2002, Jiang, Liang et al.2007) etc.But due to the unhomogeneity of glycosaminoglycan structure, particularly the high complexity of sulfated glycosaminoglycans structure, makes the same glycosaminoglycan of different sources and different batches in activity, have very big-difference (Sugahara, Mikami et al.2003).Large quantity research in recent years shows, the biological function of glycosaminoglycan is by having the functional zone of special construction in polysaccharide chain and the special interaction of specific protein realizes, in same polysaccharide chain, there are the functional zone of different structure, can exercise different functions from different protein-interactings, therefore can pass through selectivity Partial digestion glycosaminoglycan polysaccharide chain, the specific function district oligosaccharides of preparation structure homogeneous or relatively homogeneous is for medicine and functional food, this series products not only structure and activity is determined, but also because molecular weight minor benefit has improved utilization ratio greatly in absorption.
Glycosaminoglycan degrading enzyme is the important tool enzyme of research glycosaminoglycan structure activity relationship, mainly comprises microbe-derived lyase system and the lytic enzyme system of animal-origin.According to the difference of substrate, can be divided into Unidasa, chondroitin sulfate/dermatan sulfate degrading enzyme, heparin/Suleparoid degrading enzyme, the large class of keratan sulfate degrading enzyme four, can simultaneously degrade non-sulfuric acid and N-acetylgalactosamine 4 (CS-A) or 6(CS-C) the low degree of sulfation chondroitin sulfate section of position sulfovinic acid of most of Unidasa wherein, but degradation speed is far below degraded hyaluronic acid.As the hyaluronic acid lytic enzyme from sheep and bull testis (Zaneveld, Polakoski et al.1973), can be under acid PH by hyaluronic acid degradable be tetrose and six sugar.From the hyaluronate lyase of streptomycete the hyaluronic acid of can only degrading, end product is tetrose and six sugar (Ohya and Kaneko1970) of non-reducing end band unsaturated double-bond.Do not find in animal body single-minded chondroitin sulfate/dermatan sulfate degrading enzyme family always, more and more studies show that the degraded of chondroitin sulfate/sulfuric acid skin element in mammalian body is mainly that some enzymes by hyaluronic acid enzyme family complete, Unidasa family member HYAL4 is accredited as a chondroitin sulfate lytic enzyme (Yamada, Sugahara et al.2011) recently.Microbe-derived chondroitinase (CSase), as CSase ABC and CSaseAC, all to show certain hyaluronic acid degradation active, but CSase B is height specificity dermatan sulfate degrading enzyme, to chondroitin sulfate and hyaluronic acid all without degrading activity.Different substrate selectives and the product diversity of utilizing various glycosaminoglycan degrading enzymes to have, can form structure and function research to baroque glycosaminoglycan, in conjunction with various compartment analysis means, the specific function district of being combined with target protein in polysaccharide chain is identified, and prepared by final enzyme process medicinal for high reactivity and function oligosaccharides.
Glycosaminoglycan has formed cell protective barrier together with extraneous as the chief component composition of extracellular matrix; protection body cell is avoided damage; but in disease treatment; fine and close extracellular matrix has suppressed the permeability of medicine in tissue and effectively diffusion; onset time and the curative effect of medicine have been reduced; especially to some macromolecular drugs, as protein medicaments such as Regular Insulin and antibody.For many years, Unidasa is widely used in clinical, the commercialization Unidasa of You Sanzhong FDA Food and Drug Administration (FDA) approval at present can utilize, respectively the Amphadase in bull testis source, the Vitrase in sheep testicle source, and the just people of the listing Unidasa Hylenex that recombinates recently.The main application of Unidasa comprises clinically: as auxiliary agent, promote absorption and the diffusion (Schuller, Castonguay et al.1991) of other medicines; For the subcutaneous perfusion of liquid; For hyaluronic acid, filling the unexpected hyaluronic degraded of Cosmetics Surgery eliminates; Be used for the treatment of to glycosaminoglycan metabolism disorder and accumulate relevant refractory skin, as (PCT publications WO00/38732 such as diabetic scleroderma neonatorum, scleroderma, keloids; WO01/45743; And German Patent No.19963538) (Lee, Grummer et al.2010).In addition, a large amount of report proofs, chondrosulphatase CSase ABC can promote neuron axon regeneration (Moon in nerve injury by degraded chondroitin sulfate, Asher et al.2001, Bradbury, Moon et al.2002, Kwok, Afshari et al.2008, Bradbury and Carter2011).
In sum, glycosaminoglycan degrading enzyme is not only glycosaminoglycan structure function and is studied requisite instrument, and has important using value in the active oligosaccharides preparation of glycosaminoglycan and disease treatment.But there is at present the high vigor osamine Polyose degradation enzyme of using value seldom, therefore find and identify that new and effective stable glycosaminoglycan degrading enzyme is significant.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of glycosaminoglycan lyase and encoding gene and application are provided.
A glycosaminoglycan lyase, aminoacid sequence is as (a) or (b):
(a) aminoacid sequence is as shown in SEQ ID NO.2;
(b) aminoacid sequence in (a) through replacement, lack or add one or several amino acid and have glycosaminoglycan lyase activity by (a) derivative protein.
Preferred according to the present invention, described aminoacid sequence (b) is as shown in SEQ ID NO.3 or SEQ ID NO.4.
An encoding gene for glycosaminoglycan lyase, nucleotide sequence is as (i) or (ii):
(i) nucleotide sequence is as shown in SEQ ID NO.1;
(ii) the protein DNA molecule that there is glycosaminoglycan lyase activity with the DNA sequence dna hybridization (i) limiting and coding under stringent condition.
Preferred according to the present invention, described nucleotide sequence (ii) described under stringent condition, refer to:
Containing in 6 * SSC damping fluid of 0.5%SDS, at 65 ℃, hybridize, then with the 2 * SSC damping fluid containing 0.1%SDS with containing 1 * SSC damping fluid of 0.1%SDS, respectively wash film once.
Further preferred according to the present invention, described nucleotide sequence is (ii) as SEQ ID NO.5 or SEQ ID NO.6.
A recombinant expression vector has inserted the encoding gene of above-mentioned glycosaminoglycan lyase in expression vector.
Above-mentioned expression vector is selected from coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector.
Recombinant bacterium or a transgenic cell line have inserted the encoding gene of above-mentioned glycosaminoglycan lyase in host cell or clone.
Above-mentioned host cell or clone are selected from e. coli host cell, yeast host cell, Bacillus subtilus host cell, milk-acid bacteria host cell, actinomycetes host cell, filamentous fungal host cell, insect cell or mammalian cell.
For recombinant bacterium or the transgenic cell line of recombinant expressed glycosaminoglycan lyase HCDase, can be that e. coli host cell is (as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cell is (as Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces Iactis etc.), Bacillus subtilus host cell is (as Bacillus subtilis R25, Bacillus subtilis9920 etc.), milk-acid bacteria host cell (as Lactic acid bacteria C0CC101 etc.), actinomycetes host cell (as Streptomyces spp. etc.), filamentous fungal host cell (as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect cell (as Bombyxmori, Antharaea eucalypti etc.) or mammalian cell (as Chinese hamster ovary cell CHO, immature hamster kidney cell BHK, Chinese hamster pneumonocyte CHL etc.).
Above-mentioned glycosaminoglycan lyase is as strengthening the absorption of medicine or sending the application of composition in preparing medicine.
Beneficial effect
Glycosaminoglycan lyase prepared by the present invention is 110,000U/mg, to the specific activity of chondroitin sulfate, is 45,000U/mg hyaluronic specific activity; Enzyme activity be known commercialization glycosaminoglycan lyase (as, CSaseABC, CSaseAC I and II etc.) tens to hundreds of times, can be applicable to the fields such as medicine and makeup, have broad application prospects.
Accompanying drawing explanation
The protein three-dimensional structure model of Fig. 1, glycosaminoglycan lyase HCDase;
The polyacrylamide gel electrophoresis figure (SDS-PAGE) of Fig. 2, restructuring glycosaminoglycan lyase HCDase Expression and purification situation;
Wherein: M, protein molecular weight standard, band from top to bottom size is 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD; Thalline before swimming lane 1, control strain broken wall, applied sample amount 10 μ L, thalline before swimming lane 2, recombinant bacterium broken wall, applied sample amount 10 μ L, supernatant after swimming lane 3, recombinant bacterium broken wall, applied sample amount 10 μ L, swimming lane 4, through the HCDase of ni-sepharose purification, applied sample amount 10 μ L.
Fig. 3, the activity influence curve of p Η value to glycosaminoglycan lyase HCDase;
Fig. 4, the activity influence curve of temperature to glycosaminoglycan lyase HCDase;
Fig. 5, the stability influence curve of temperature to glycosaminoglycan lyase HCDase;
Fig. 6, the stability influence curve of pH value to glycosaminoglycan lyase HCDase;
Fig. 7, the affect column diagram of metal ion on glycosaminoglycan lyase HCDase activity;
The HPLC analysis chart of Fig. 8, glycosaminoglycan lyase HCDase degraded hyaluronic acid and chondroitin sulfate products therefrom;
The HPLC analysis chart of Fig. 9, glycosaminoglycan lyase HCDase different time degraded hyaluronic acid products therefrom.
Embodiment
The elaboration of following examples, is for some common technologies that openly how the present invention implements comprehensively, rather than in order to limit range of application of the present invention.Contriver has tried one's best and has guaranteed in embodiment the accuracy (amount for example, temperature, etc.) of a parameter, but some experimental errors and deviation also should be paid attention to.Except as otherwise noted, in the present invention, molecular weight refers to weight-average molecular weight, and temperature is degree Celsius.
The acquisition of embodiment 1, glycosaminoglycan lyase
Picking vibrios (Vibrio sp.) FC509 inoculation, in 100mL liquid nutrient medium, is that under 28 ℃, the revolution condition that is 200rpm, shaking table is cultivated 12 hours, adds chondroitin sulfate to make mass concentration reach 0.01%, continues to cultivate 48h in temperature; Medium centrifugal is collected to supernatant substratum, the ammonium sulfate precipitation that supernatant is 80% with final concentration, collecting precipitation, precipitation is dialysed and is removed ammonium sulfate with PBS damping fluid, makes glycosaminoglycan lyase.
Above-mentioned vibrios (Vibrio sp.) FC509, within on 03 12nd, 2014, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC NO.8913, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Every liter of component of aforesaid liquid substratum is as follows:
Chondroitin sulfate 5g, Tryptones 10g, yeast extract 5g, NaCl30g, KH 2pO 43g, K 2hPO 47g, (NH 4) 2sO 42g, MgSO 47H 205mg, FeSO 47H 202mg, pH value is 7.2.
Enzyme activity unit definition: per minute catalysis glycosaminoglycan produces the needed enzyme amount of 1 μ moL unsaturated double-bond.According to ultraviolet method, survey glycosaminoglycan and split the method (Yamagata, Saito et al.1968) that enzyme enzyme is lived, recording enzyme work in the liquid nutrient medium of Vibrio sp.FC509 bacterial strain is 3,000U/mL.
The extraction of embodiment 2, vibrios (Vibrio sp.) FC509 strain gene group DNA
By vibrios (Vibrio sp.) FC509 inoculation, to liquid nutrient medium (with embodiment 1), under 30 ℃, the condition of 200rpm, shaking culture is to OD 600=0.8; Get and cultivate bacterium liquid 40mL, centrifugal 25min under 12,000rmp condition, collects bacterial sediment, and with N,O-Diacetylmuramidase damping fluid (the 10mM Tris-HCl pH8.0) washing of 20mL, centrifugal 25min under 12,000rmp condition, collects bacterial sediment;
In above-mentioned bacterial sediment, every pipe adds N,O-Diacetylmuramidase damping fluid 12.0mL, obtains the bacterium liquid of about 14.0mL, and adding respectively concentration is each 560 μ L of N,O-Diacetylmuramidase of 20mg/mL, its final concentration approximately 800 μ g/mL; After ice bath 1.0h, 37 ℃ of temperature are bathed 2h, to solution thickness; Add 10wt%SDS0.82mL, the Proteinase K solution 60 μ L of 100mg/mL, 52 ℃ of water-bath 1.0h; Phenol/chloroform/primary isoamyl alcohol (volume ratio 25:24:1) 15mL that adds Tris-balance to cross, puts upside down and mixes gently, to fully emulsified; Centrifugal 10min under 10,000g, 4 ℃ of conditions, shifts supernatant, adds NaAc-HAc(pH5.2, the 3.0M of 2.0mL) damping fluid, and the dehydrated alcohol of 17.0mL, mix; With the rifle choicest of 1.0mL, go out thread DNA, be transferred in the EP centrifuge tube of 1.5mL, with 70% ethanol (storing in-20 ℃), wash 2 times, micro-ly abandon supernatant after centrifugal; Centrifugal 3min under 10,000g, 4 ℃ of conditions, thoroughly discards supernatant; Sample is in aseptic working platform, and spirit lamp leeward dries up dry; With the resuspended dissolving DNA sample of aseptic deionized water, 4 ℃ are spent the night, and obtain macromolecule genomic dna.
Embodiment 3, the scanning of vibrios (Vibrio sp.) FC509 strain gene group and sequential analysis thereof.
The macromolecule genomic dna that embodiment 2 is made check order (Mei Ji biotech firm).With NCBI(National Center for Biotechnology Information, http://www.ncb1.nlm.nih.gov/) on software sequencing result is analyzed.NCBI analysis software used is Open Reading Frame Finder(ORF Finder, http://www.ncb1.nlm.nih.gov/gorf/gorf.html) and Basic Local Alignment Search Tool(BLAST, http://blast.ncb1.nlm.nih.gov/Blast.cgi).
In NCBI analytical results demonstration Vibrio sp.FC509 strain gene group, carry a glycosaminoglycan lyase gene hcdase, hcdase genes encoding head of district 2436bp, its nucleotide sequence is as shown in SEQ ID NO.1.Whole genome sequence (NCBI number of registration: YP_206952.1), 384 amino acid of hyaluronate lyase gene have 49% homology with Vibrio fischeri ES114.
The glycosaminoglycan lyase HCDase of hcdase genes encoding is comprised of 778 amino acid, and its aminoacid sequence is as shown in SEQ ID NO.2, and the theoretical molecular of protein is about 89kD.With Simple Modular Architecture Research Tool(SMART, http://smart.embl_heidelberg.de/) analyze the structural information of glycosaminoglycan lyase HCDase, result shows that the 1st to the 22nd amino acid of N end is signal peptide sequence, and 23-778 amino acids sequence belongs to glycosaminoglycan lyase superfamily.With SWISS-MODEL homology modeling service device (http://swissmodel.expasy.org), the protein three-dimensional structure of glycosaminoglycan lyase HCDase is carried out to homology modeling, the HCDase protein three-dimensional structure model finally obtaining as shown in Figure 1.
Under stringent condition, there is the protein DNA molecule of glycosaminoglycan lyase activity with sequence hybridization shown in SEQ ID NO.1 and coding, as the DNA molecular of nucleotide sequence as shown in SEQ ID NO.5, SEQ ID NO.6.
DNA molecular by nucleotide sequence as shown in SEQ ID NO.5, SEQ ID NO.6 is in containing 6 * SSC damping fluid (purchased from Kang Wei ShiJi Co., Ltd) of 0.5%SDS, hybridization at 65 ℃, then respectively washes film once with the 2 * SSC damping fluid containing 0.1%SDS with containing 1 * SSC damping fluid of 0.1%SDS.
The protein molecule of the DNA molecule encode shown in above-mentioned SEQ ID NO.5, SEQ ID NO.6, aminoacid sequence is as shown in SEQ ID NO.3 or SEQ ID NO.4, wherein the protein molecule of SEQ ID NO.3 is compared with the protein molecule shown in SEQ ID NO.2, there is 2% difference (amino acid whose substituting), the protein molecule of SEQ ID NO.4 is compared with the protein molecule shown in SEQ ID NO.2, has 3% difference (amino acid whosely substitute, disappearance, insert).
Recombinant expressed in intestinal bacteria of embodiment 4, HCDsae gene
The macromolecule genomic dna that the embodiment 2 of take makes is template, carries out pcr amplification.Primer is as follows:
Forward primer HCDase-F:G cCATGGaTATGCAAGCGATGACCACCAGTTCACTG;
Reverse primer HCDase-R:G cTCGAGtCGCACTGAAAATTGATAACTTTGTCC;
What forward primer underscore marked is restriction enzyme Nco I site, and what reverse primer underscore marked is restriction enzyme Xho I site.Primerstar HS archaeal dna polymerase is purchased from precious biotech firm, and the description of product that PCR reaction system provides according to company operates.
PCR reaction conditions: 94 ℃ of denaturation 5min; 94 ℃ of sex change 30s, 68 ℃ of 1.5min, 35 circulations; 72 ℃ are extended 10min.
By Nco I and Xho I double digestion for PCR product, agarose gel electrophoresis reclaims the PCR product that enzyme is cut.To be purchased from Nco I and the Xho I double digestion for product pET-22b carrier of U.S. Novagen company, agarose gel electrophoresis reclaims enzyme and cuts carrier large fragment.Nco I and Xho I are all purchased from precious biotech firm, the system of enzyme-to-substrate reaction, the description of product operation that temperature and time all provides according to company.
PCR product through double digestion is connected through double digestion pET-22b carrier with same, connect after product transforms e.colistraindh5α and coat on the Luria-Bertani substratum solid plate that contains 50 μ g/mL penbritins, cultivate 14h, picking mono-clonal for 37 ℃; In the liquid Luria-Bertani substratum that mono-clonal access is contained to 50 μ g/mL penbritins, cultivate, extract plasmid; Plasmid is carried out to bacterium liquid PCR checking with forward primer HCDase-F and reverse primer HCDase-R, and result obtains the amplified production that size is correct, and the recombinant plasmid that preliminary proof builds is correct; Then this recombinant plasmid is sent to the order-checking of Sheng Gong biotech firm, result shows, between the Nco of pET-22b I and Xho I restriction enzyme site, insert the hcdsae gene shown in SEQ ID NO.1, and direction of insertion is correct, so the recombinant plasmid that further proof builds is correct, by this recombinant plasmid called after pET22b-HCDase.
PET22b-HCDase is transformed to coli strain BL21 (DE3) (purchased from U.S. Novagen company), the operation steps then providing according to the said firm glycosaminoglycan lyase HCDase abduction delivering of recombinating.And with Ni Sepharose6Fast Flow(GE) gel carries out purifying to HCDase, and purification condition is according to the product manual operation of GE company.The purifying situation that detects restructuring glycosaminoglycan lyase HCDase with polyacrylamide gel electrophoresis, as shown in Figure 2, the restructuring glycosaminoglycan lyase HCDase after purifying is single band to result on running gel, and the molecular weight of position and prediction matches.
The zymologic property analysis of embodiment 5, restructuring glycosaminoglycan lyase HCDase
The impact on enzymic activity of pH and temperature
By mass concentration, be 150mM HAc-NaAc, the NaH of 1% hyaluronic acid or chondroitin sulfate substrate, HCDase enzyme liquid, different pH values 2pO 4-Na 2hPO 4, Tris-HCl damping fluid and water (pH scope is 5.0～10.0), in 2:1:3:4(volume ratio) ratio mix after, at 30 ℃ of reaction 60min, by aforesaid ultraviolet method survey enzyme activity.Result shows that HCDsae reaches maximum vigor when pH8.0, and the optimal reaction pH that shows HCDase is that 8.0(is as Fig. 3).
Under optimal pH, by mass concentration, be 1% hyaluronic acid or chondroitin sulfate substrate, HCDase enzyme liquid and 150mM NaH 2pO 4-Na 2hPO 4damping fluid (pH8.0) is in 2:1:3:4(volume ratio) ratio mix, respectively at differing temps (0 ℃～90 ℃) reaction 10min, by aforesaid ultraviolet method survey enzyme activity.Result shows that HCDase reaches maximum vigor in the time of 30 ℃, and the optimal reactive temperature that shows HCDase is 30 ℃ (as Fig. 4).
The impact on enzyme stability of pH and temperature
By the HCDase enzyme liquid after the lower thermal treatment 1h of differing temps (0 ℃～70 ℃) and mass concentration, be that 1% hyaluronic acid or chondroitin sulfate substrate solution are in 2:3(volume ratio) ratio mix, then under optimum temperuture and optimal pH, measuring residual enzyme lives, to be defined as 100% relative activity (relativie activity) without the work of heat treated enzyme liquid enzyme, result shows that HCDase has good thermostability (as Fig. 5) at the temperature lower than 60 ℃.
Will be at 30 ℃, different pH(pH5～10) the HCDase enzyme liquid after preincubate 1h and mass concentration are that 1% hyaluronic acid or chondroitin sulfate substrate solution are in 2:3(volume ratio) ratio mix, then under optimum temperuture and optimal pH, measuring residual enzyme lives, with the enzyme liquid enzyme work of processing without pH, be defined as 100% relative activity (relativie activity), result is presented in the scope of pH5-9, HCDase enzyme is lived and still to be kept more than 60%, shows that HCDase is to pH value tolerance range extensively (as Fig. 6).
The impact of metal ion on HCDsae activity
By mass concentration, be that 1% hyaluronic acid or chondroitin sulfate substrate, HCDase enzyme liquid, 150mM Tris-HCl damping fluid and water are in 2:1:3:4(volume ratio) ratio mix after, then in reaction system, add different metal ions, the ion final concentration adding is 1mM or 10mM, then at 30 ℃ of reaction 60min, by aforesaid ultraviolet method, survey enzyme activity.Control group is the activity (being set as 100%) of HCDase while not adding any metal ion, shown in result Fig. 7.Experimental result shows, K +, Na +, Fe 2+, Mn 2+can increase HCDase activity, Li +, Mg 2+, Co 2+, Ni 2+, Cr 3+, Fe 3+active substantially without impact, Ag on HCDase +, Cu 2+, Ca 2+, Fe 2+, Hg 2+, Pb 2+, Zn 2+deng other ions enzyme work, present restraining effect (Fig. 7).
The enzyme activity determination of embodiment 6, HCDase
By mass concentration, be that 1% hyaluronic acid or chondroitin sulfate substrate, HCDase enzyme liquid, 150mM Tris-HCl damping fluid and water are in 2:1:3:4(volume ratio) ratio mix after, under optimum temperuture and optimal pH, react 2-10min, by aforesaid ultraviolet method, survey enzyme activity (Yamagata, Saito et al.1968), simultaneously with the protein content that is purchased from the quantification of protein kit measurement HCDase enzyme liquid of Kang Wei ShiJi Co., Ltd, the result HCDase that shows to recombinate is 110 to hyaluronic specific activity, 000U/mg, to the specific activity of chondroitin sulfate, be 45,000U/mg.
Albumen shown in SEQ ID NO.3 is 100,000U/mg, to the specific activity of chondroitin sulfate, is 40,000U/mg hyaluronic specific activity;
Albumen shown in SEQ ID NO.4 is 89,000U/mg, to the specific activity of chondroitin sulfate, is 37,000U/mg hyaluronic specific activity;
The high performance liquid phase (HPLC) of embodiment 7, HCDase degraded glycosaminoglycan degraded product is analyzed
By mass concentration, be that 1% hyaluronic acid or chondroitin sulfate substrate, HCDase enzyme liquid, 150mM Tris-HCl damping fluid and water are in 2:1:3:4(volume ratio) ratio mix after, at pH8.0, under 30 ℃ of conditions, react, the product of choosing different enzymolysis times (0,0.25,1,5,15,30,120min) carries out HPLC analysis.HPLC analysis condition is gel column: superdex peptide10/300GL(GE); Moving phase: 0.2M bicarbonate of ammonia; Flow velocity: 0.4mL/min; Testing conditions: UV232nm.
As shown in Figure 8 and Figure 9, along with the increase of degradation time, the product polymerization degree is fast reducing with the prolongation of degradation time, is finally converted into disaccharides as can be seen from Figure 9 for result.This result shows that HCDase belongs to inscribe glycosaminoglycan lyase, can be used to preparation and the glycosaminoglycan structure activity study of glycosaminoglycan oligosaccharides.
Embodiment 8, the application of glycosaminoglycan lyase HCDase in medicine
Glycosaminoglycan lyase HCDase can, for strengthening the absorption of medicine or/and send, include but not limited to permeate through skin approach ancillary drug.Exemplary medicine comprises that reflunomide is as hydrocortisone, prednisolone, beclometasone propionic ester, fluorine compound, triamcinolone, clobetasol propionate etc.; Anodyne and/or anti-inflammatory agent are as paracetamol, vialidon, Flufenamic Acid, diclofenac, diclofenac sodium, Warner-Lambert), Tacote, Phenylbutazone, Ibuprofen BP/EP, flurbiprofen, Whitfield's ointment, menthol, camphor, Naproxen Base etc.; Antihypertensive drug is as pindolol, indenolol, nifedipine etc.; Microbiotic is as penicillin, tsiklomitsin, terramycin, Liu Suanyan NEOMYCIN SULPHATE, erythromycin, paraxin etc.; Narcotic is as lignocaine, Benzocaine etc. and other drug.
By compress, gauze, or other skin is smeared approach and is used glycosaminoglycan lyase.Glycosaminoglycan lyase solution can be coated to suitable apply ointment or plaster upper (as 5-6 layer gauze).Apply ointment or plaster and should cover affected area, and apply ointment or plaster and itself with paraffin paper or bandage, fix.The amount of the preparation of application depends on the area of damage, is generally 5-100IU/cm 2.Apply ointment or plaster and should use 12-24 hour every day, use continuously 10-100 days.
For part, use solution, suspension, gelifying agent, paste, ointment, other formulas (be with or without other promoting agent, can use) of emulsifiable paste or glycosaminoglycan lyase.In the formulas such as solution, comprise pharmacy and cosmetic field and use acceptable thinner for part, auxiliary and vehicle, these other formula mainly comprises that buffer reagent is as phosphoric acid salt, Citrate trianion, acetate and other organic acid salt, antioxidant be if xitix, peptide, protein are as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is as polyvinylpyrrolidone, natural or synthetic oil; Amino acid is as glycine, L-glutamic acid, aspartic acid, or arginine; Monose, disaccharides and other carbohydrate, comprise Mierocrystalline cellulose or derivatives thereof, glucose, lactose, seminose or dextrin; Sequestrant is as EDTA; Sugar alcohol is as mannitol or Sorbitol Powder; Inorganic salt, as sodium-chlor, and nonionogenic tenside is as tween, polyoxyethylene glycol.
A kind of preferred, glycosaminoglycan lyase preparation is that the lyophilized powder of glycosaminoglycan lyase HCDase suspends or is dissolved in the solvent that contains 0.1%-10% sucrose, 1%-20% glycerine, and the pH of solution remains within the scope of 6.5-8.5.
Glycosaminoglycan lyase composition can pass through oral administration or administered parenterally, and glycosaminoglycan lyase composition can be combined with suitable delivery vector rear administration.
Can obtain the glycosaminoglycan lyase pharmaceutical preparation for orally using by the combination of active compound and solid excipient.Selecting above-mentioned mixture to add suitable attached auxiliary agent to mix uses.If necessary, suitable vehicle is carbohydrate or protein, such as sugar, Mierocrystalline cellulose, bovine serum albumin etc.
Glycosaminoglycan lyase enzyme and medicine (as hydrocortisone, microbiotic, VITAMIN) combination.With oral gel, paste, or the form of suspension is by intravenously, subcutaneous or intramuscularly administration; Or with ointment, ointment, face shield topical; And as makeup auxiliary, for strengthening the absorption of skin.
The reference relating in specification sheets
1、Bradbury,E.J.and L.M.Carter(2011)."Manipulating the glial scar:Chondroitinase ABC as a therapy for spinal cord injury." Brain Research Bulletin84(4-5):306-316.
2、Bradbury,E.J.,et al.(2002)."Chondroitinase ABC promotes functional recovery after spinal cord injury." Nature416(6881):636-640.
3、Jiang,D.,et al.(2007).Hyaluronan in tissue injury and repair. Annual Review of Cell and Developmental Biology.23:435-461.
4、Karbownik,M.S.and J.Z.Nowak(2013)."Hyaluronan:Towards novel anti-cancer therapeutics." Pharmacological Reports65(5):1056-1074.
5、Knudson,C.B.and W.Knudson(1993)."Hyaluronan-binding proteins in development,tissue homeostasis,and disease." FASEB journal:official publication of the Federation of American Societies for Experimental Biology7(13):1233-1241.
6、Kwok,J.C.F.,et al.(2008)."Proteoglycans in the central nervous system:Plasticity,regeneration and their stimulation with chondroitinase ABC." Restorative Neurology and Neuroscience26(2-3):131-145.
7、Lee,A.,et al.(2010)."Hyaluronidase." Dermatologic Surgery36(7):1071-1077.
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14、Yamada,S.,et al.(2011)."Evolution of glycosaminoglycans:Comparative biochemical study." Communicative&Integrative Biology4(2):150-158.
15、Yamagata,T.,et al.(1968)."Purification and properties of bacterial chondroitinases and chondrosulfatases." The Journal of biological chemistry243(7):1523-1535.
16、Zaneveld,L.J.,et al.(1973)."Properties of acrosomal hyaluronidase from bull spermatozoa.Evidence for its similarity to testicular hyaluronidase." The Journal of biological chemistry248(2):564-570。

Claims (10)

1. a glycosaminoglycan lyase, is characterized in that, aminoacid sequence is as (a) or (b):
(a) aminoacid sequence is as shown in SEQ ID NO.2;
(b) aminoacid sequence in (a) through replacement, lack or add one or several amino acid and have glycosaminoglycan lyase activity by (a) derivative protein.
2. glycosaminoglycan lyase as claimed in claim 1, is characterized in that, described aminoacid sequence (b) is as shown in SEQ ID NO.3 or SEQ ID NO.4.
3. an encoding gene for glycosaminoglycan lyase, is characterized in that, nucleotide sequence is as (i) or (ii):
(i) nucleotide sequence is as shown in SEQ ID NO.1;
(ii) the protein DNA molecule that there is glycosaminoglycan lyase activity with the DNA sequence dna hybridization (i) limiting and coding under stringent condition.
4. encoding gene as claimed in claim 3, is characterized in that, described nucleotide sequence (ii) described under stringent condition, refer to:
Containing in 6 * SSC damping fluid of 0.5%SDS, at 65 ℃, hybridize, then with the 2 * SSC damping fluid containing 0.1%SDS with containing 1 * SSC damping fluid of 0.1%SDS, respectively wash film once.
5. encoding gene as claimed in claim 4, is characterized in that, described nucleotide sequence is (ii) as SEQ ID NO.5 or SEQ ID NO.6.
6. a recombinant expression vector has inserted the encoding gene of glycosaminoglycan lyase claimed in claim 3 in expression vector.
7. recombinant expression vector as claimed in claim 6, it is characterized in that, described expression vector is selected from coli expression carrier, Yeast expression carrier, Bacillus subtilus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, filamentous fungus expression vector, plant expression vector, insect expression vector or mammalian cell expression vector.
8. recombinant bacterium or a transgenic cell line have inserted the encoding gene of glycosaminoglycan lyase claimed in claim 3 in host cell or clone.
9. recombinant bacterium as claimed in claim 8 or transgenic cell line, it is characterized in that, described host cell or clone are selected from e. coli host cell, yeast host cell, Bacillus subtilus host cell, milk-acid bacteria host cell, actinomycetes host cell, filamentous fungal host cell, insect cell or mammalian cell.
Described in claim 1 glycosaminoglycan lyase in glycosaminoglycan oligosaccharides preparation and himself is as strengthening the absorption of medicine or sending composition and preparing the application in medicine.
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CN105713890A (en) * 2016-04-13 2016-06-29 山东大学 Exo-GAG (glycosaminoglycan) lyase as well as encoding gene and application thereof
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CN116515803A (en) * 2022-02-08 2023-08-01 河北农业大学 Hyaluronidase and gene expression and application thereof
CN118028270A (en) * 2024-02-04 2024-05-14 北京华妍生物科技有限公司 Glycosaminoglycan degrading enzyme and application thereof
CN118406592A (en) * 2024-04-22 2024-07-30 北京华妍生物科技有限公司 Microorganism and application thereof

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CN104830881B (en) * 2015-05-29 2018-01-12 山东大学 A kind of glycosaminoglycan lyases and its encoding gene and application
CN105713890A (en) * 2016-04-13 2016-06-29 山东大学 Exo-GAG (glycosaminoglycan) lyase as well as encoding gene and application thereof
CN105713890B (en) * 2016-04-13 2019-06-11 山东大学 A kind of circumscribed-type glycosaminoglycan lyases and its encoding gene and application
CN107312765B (en) * 2017-07-05 2020-01-21 山东大学 Glycosaminoglycan lyase and coding gene and application thereof
CN107312765A (en) * 2017-07-05 2017-11-03 山东大学 A kind of glycosaminoglycan lyases for being difficult to degraded CS E and its encoding gene and application
WO2019007033A1 (en) * 2017-07-05 2019-01-10 山东大学 Glycosaminoglycan lyase having difficulty degrading cs-e and encoding gene and use thereof
CN107460179A (en) * 2017-09-22 2017-12-12 青岛农业大学 A kind of polysaccharide degrading enzyme and its encoding gene and application
CN110408566A (en) * 2019-07-29 2019-11-05 山东大学 A kind of circumscribed-type chondroitin sulfate degrading enzyme and its encoding gene and application
CN110408566B (en) * 2019-07-29 2022-09-30 山东大学 Externally tangent chondroitin sulfate degrading enzyme and coding gene and application thereof
CN116515803A (en) * 2022-02-08 2023-08-01 河北农业大学 Hyaluronidase and gene expression and application thereof
CN118028270A (en) * 2024-02-04 2024-05-14 北京华妍生物科技有限公司 Glycosaminoglycan degrading enzyme and application thereof
CN118406592A (en) * 2024-04-22 2024-07-30 北京华妍生物科技有限公司 Microorganism and application thereof

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