CN106520728B - A method of improving leech hyaluronidase enzyme activity - Google Patents

A method of improving leech hyaluronidase enzyme activity Download PDF

Info

Publication number
CN106520728B
CN106520728B CN201610982765.9A CN201610982765A CN106520728B CN 106520728 B CN106520728 B CN 106520728B CN 201610982765 A CN201610982765 A CN 201610982765A CN 106520728 B CN106520728 B CN 106520728B
Authority
CN
China
Prior art keywords
lys
leu
ser
asp
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610982765.9A
Other languages
Chinese (zh)
Other versions
CN106520728A (en
Inventor
康振
陈坚
堵国成
原攀红
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to CN201610982765.9A priority Critical patent/CN106520728B/en
Publication of CN106520728A publication Critical patent/CN106520728A/en
Application granted granted Critical
Publication of CN106520728B publication Critical patent/CN106520728B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01036—Hyaluronoglucuronidase (3.2.1.36)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention discloses a kind of methods for improving leech hyaluronidase enzyme activity, belong to technical field of enzyme engineering.The present invention uses leech hyaluronidase, by multizone multidigit point combinatorial mutagenesis, constructs the hyaluronic acid enzyme mutant of enzyme activity raising.And the efficient secretory expression of hyaluronidase enzyme activity is realized in bacillus subtilis expression system.After fermentation 48 hours, hyaluronidase enzyme activity reaches 6030U/mL, improves 500U/mL compared to common bacterial strain, plays the role of important progress to the industrialized production of hyaluronidase.

Description

A method of improving leech hyaluronidase enzyme activity
Technical field
The present invention relates to a kind of methods for improving leech hyaluronidase enzyme activity, belong to technical field of enzyme engineering.
Background technique
Hyaluronidase is the general name of the enzyme of a kind of principal degradation HA, is found and is claimed by Duran Reynals in nineteen twenty-eight For " invasin ", hyaluronidase (hyaluronidase, HAase) was officially named by Chain and Duthie in 1940. HAase is widely present in eucaryote and prokaryotes, is a kind of important physiological activator and in animal body internal reference With many important biological processes, such as cell division, intercellular connection, the activity of reproduction cell, the transfection of DNA, embryo Fetal hair educates, the reparation of wounded tissue and normal cell and tumor cell proliferation etc..According to the source of HAase, catalyst mechanism and Substrate and product specificities, HAase can be divided into three classes: and endo-beta-N-acetyl glucosidase (EC 3.2.1.35, it is main Be present in the venom such as mammal and honeybee, snake, spider), inscribe-beta-glucuronidase enzyme (EC 3.2.1.36, it is main It is present in leech) and HA lyases (EC 4.2.2.1 is primarily present in bacterium, bacteriophage and fungi).
The correlative study of first kind HAases and third class HA lyases have more document report.The first kind is derived from The hyaluronic acid 4- glycosyl hydrolase family of mammal, this kind of HAase (EC 3.2.1.35) are living with hydrolyzing and turning glucosides Property glycosidase, by hydrolyze HA β-Isosorbide-5-Nitrae-glycosidic bond, product is based on tetrose HA molecule.It may also act on chondroitin sulfate Element, chondroitin-4-suleate and 6- chondroitin sulfate generate dermatan sulfate product.It is relatively common in the first kind to have from the food in one's mouth The testis of newborn animal, snake venom, bee venom and lysosome HAases.Third class is microorganism HAases, this kind of HAase (EC 4.2.2.1 β-glycosidic bond that HA) is cracked by beta-elimination reaction, generates with unsaturated disaccharide molecule 2-acetamido-2- Deoxy-3-O- (β-D-gluco-4-enepyranosyluronic acid)-D-glucose is primary product.This kind of HA is split Enzyme source is solved in including the bacterial strains such as Clostridium, Micrococcus, Streptococcus and Streptomyces, and Their substrate specificity is also different.The representative HAase of second class (EC 3.2.1.36) is mainly derived from leech, belongs to Bright matter acid 3- glycosyl hydrolase family, by hydrolyzing β -1 of HA, 3- glycosidic bond, production is with four glycan molecule HA and reducing end has Product based on glucuronic acid.The HAase in leech source is strong to the specificity of substrate, to chondroitin sulfate, 4- chondroitin sulfate Element and 6- chondroitin sulfate do not have activity, and do not have transglycosylation.
HAase can reduce the vicidity of tissue HA, to improve liquid barrier capabilities in tissue.Many pathological changes Journey is usually associated with the variation of HAase and HA, implies that they may play an important role.HAase also can change machine simultaneously The distribution situation of internal some drugs and physiological activator.Therefore, HAase can promote subcutaneously as a kind of pharmacological active substance Infusion, the diffusate of local repertory or blood accelerate diffusion and are conducive to absorb, and are widely used as clinical medicine bleeding agent, promote drug Absorption, promote local edema or hemotoncus after operation and wound to dissipate.Research shows that HAase is aobvious to treatment myocardial infarction effect It writes, the myocardium HA that can degrade substantially reduces interstitial volume, and the Artery resistance as caused by ischemic is prevented to increase, and increases blood flow. HAase is also played an important role in anti-tumor aspect, and HAase can promote the transfer of cancer cell, therefore tie on tumour cell The HAase of conjunction can be used as the target spot of tumour medicine effect.If it can reinforce adriamycin to the anti-cancer ability of breast cancer, reduce The recurrence rate etc. of bladder cancer.In addition, HAase is clinically also used as drug diffusion auxiliary agent, for reducing intraocular after ophthalmologic operation Pressure, Cooperation Anaesthesia agent lidocaine hydrochloride use can be accelerated to enter narcosis and extend anesthesia duration.
Leech HAase has the antibacterial activity of strength, the antibody that energy dissolution of bacteria inside and outside envelope is formed, to enhance medicine Therapeutic effect of the object to host.Especially leech HAase is since its Substratspezifitaet is strong, compared with the HAase in other sources, it Cannot degrade chondroitin or chondroitin sulfate.Although mammal HAase is widely used in drug expansion as " invasin " Auxiliary agent is dissipated, however this kind of HAase activity is vulnerable to effects of heparin.Therefore, theoretically speaking, leech HAase activity is not by heparin shadow It rings, clinically has more medical value meaning as " the drug diffusion factor ", treatment thrombus, glaucoma and other medicinal aspects.
Summary of the invention
The first purpose of the invention is to provide the hyaluronic acid enzyme mutants that a kind of enzyme activity improves, and amino acid sequence is such as Shown in SEQ ID NO.1.
A second object of the present invention is to provide the genes for encoding the hyaluronic acid enzyme mutant.
In one embodiment of the invention, the gene order is as shown in SEQ ID NO.3.
Third object of the present invention is to provide the carriers or cell line of expressing the hyaluronic acid enzyme mutant.
Fourth object of the present invention is to provide a kind of genetic engineering bacterium for producing hyaluronidase, is with bacillus subtilis The hyaluronic acid enzyme mutant is expressed using pPNMK as carrier for host.
In one embodiment of the invention, the bacillus subtilis is Bacillus subtilis168.
Fifth object of the present invention is to provide a kind of methods for improving hyaluronidase enzyme activity, are by SEQ ID NO.2 The L of shown hyaluronic acid enzyme amino acid sequence50-G57Region replaces with SLESISPS, and by K120-R125SKSKMQ is replaced with, and By K163-D167Region replaces with EGYGS, and by P280-W287Region replaces with RHKDKPTG, and by V307-L313Region replaces with WSGFLNI, and by T344Replace with K344。
Sixth object of the present invention is to provide a kind of production methods for improving hyaluronidase enzyme activity, are by the gene Engineering bacteria is seeded in fermentation medium, in 30~37 DEG C of 16~72h of culture.
The present invention also provides the hyaluronidase food, biology, chemical industry, field of medicaments application.
In one embodiment of the invention, the application includes preparing pharmaceutical product containing hyaluronic acid, health care Product, cosmetics.
It is reached the utility model has the advantages that the genetic engineering bacterium MHyal constructed using method of the invention cultivates 48 hours post-fermentation enzyme activity To 6030U/mL, enzyme activity improves 500U/mL compared with control strain.
Detailed description of the invention
Fig. 1 is the enzyme activity trend chart of mutant strain and wild strain.
Specific embodiment
The DNS determination method of hyaluronidase:
The single-minded glucosiduronate key for acting on hyaluronic acid of hyaluronidase, it is glucuronic acid that generating, which has reducing end, Micromolecular polysaccharide.The reduced sugar equivalent generated using 3,5-dinitrosalicylic acid (DNS) colorimetric method for determining hydrolysis hyaluronic acid, Make standard curve with pure glucose is analyzed, enzyme activity is calculated, with the citrate-phosphate disodium hydrogen buffer of pH 5.5,50mM 2mg mL–1Hyaluronic acid substrate, 1mL reaction system contains the enzyme solution of the hyaluronic acid solution of 800 μ L, 100 μ L, and buffer is mended Enough to 1mL.In 38 DEG C of reaction 20min, boiling water terminates reaction immediately, (is equivalent to using the reduced sugar equivalent that the measurement of DNS method generates The reducing power of equivalent glucose), enzyme is calculated than living.To ensure that data are accurate and reliable, the light absorption value of DNS is controlled in 0.25-0.85 Between.
1 primer of table
Embodiment 1: functional area is mutated using the method for mutation
On the basis of the hyaluronidase shown in SEQ ID NO.1, fixed point saturation mutation is carried out.With Hyal gene (SEQ Shown in ID NO.4) it is template, design primer is respectively primer (such as 1 institute of table using F0 and R2, F1 and R4, F3 and R6, F5 and R0 Show), carry out PCR.Specified operational procedure is as follows: using Hyal gene as 1 μm of ol of template (gene shown in SEQ ID NO.4), up and down Each 1 μ L of primer, 25 Primestar μ L, 22 μ L of sterile water are swum, is mixed in PCR pipe, mixing is placed on PCR instrument, by following journey Sort run: 94 DEG C of 3min, [94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s] × 30,72 DEG C of 5min.PCR product is subjected to column recycling, is returned It receives product and carries out Gibson recombination, operation sequence is as follows: PCR product (volume=0.02 × fragment length/DNA concentration μ is added L), 2 μ L, 5 × CE buffer, 1 μ L is added in ExnaseII, and sterile water is added and supplies 10 μ L systems, with plasmid pPNMK at 37 DEG C 30min, 0 DEG C of 5min connection, then gene mutation plasmid pP43-MHyal is transferred to e. coli jm109, the single bacterium that picking is grown It falls to be seeded in the LB liquid medium containing 100 μ L/100mL ammonia benzyls and cultivates 12h, thalline were collected by centrifugation, extracts plasmid.
The plasmid pP43-MHyal of extraction is transferred to Bacillus subtilis 168, picking positive colony MHyal, and Be seeded to the card containing 100 μ L/100mL receive chloramphenicol resistance LB liquid medium in cultivate 12h, then 80 μ L bacterium solutions are added Containing 800 μ L fermentation mediums, (fermentation medium group is divided into 30g/L sucrose, 20g/L yeast extract, 2g/L peptone, 15.6g/L phosphorus Sour disodium hydrogen, 5.94g/L potassium dihydrogen phosphate) 96 deep-well plates, cultivate 48h under the conditions of 37 DEG C, 220rpm.By fermentation liquid 3000rpm is centrifuged 20min, is screened using the method for DNS by enzyme activity, filters out the highest bacterial strain of enzyme activity and ferment, It samples at regular intervals, survey enzyme activity, and draw out enzyme activity trend chart (as shown in Figure 1).
Using carrier identical with genetic engineering bacterium and host expresses Hyal gene (shown in SEQ ID NO.4), with its work For control strain, other embodiment is identical as genetic engineering bacterium.
Compared with control strain, the genetic engineering bacterium MHyal that the present invention constructs cultivates 48 hours post-fermentation enzyme activity and reaches 6030U/mL improves 500U/mL compared to control strain (enzyme activity improves 10%).
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of method for improving leech hyaluronidase enzyme activity
<160> 17
<170> PatentIn version 3.3
<210> 1
<211> 488
<212> PRT
<213>artificial sequence
<400> 1
Met Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser
1 5 10 15
Val Ser Glu Ser Phe His Gly Val Ala Phe Asp Ala Ser Leu Phe Ser
20 25 30
Pro Lys Gly Leu Trp Ser Phe Val Asp Ile Thr Ser Pro Lys Leu Phe
35 40 45
Lys Ser Leu Glu Ser Ile Ser Pro Ser Tyr Phe Arg Val Gly Gly Thr
50 55 60
Phe Ala Asn Trp Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys
65 70 75 80
Asp Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr
85 90 95
Arg Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe
100 105 110
Asp Asp Leu Val Lys Leu Thr Ser Lys Ser Lys Met Gln Leu Leu Phe
115 120 125
Asp Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Met
130 135 140
Thr Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys
145 150 155 160
Val Ser Glu Gly Tyr Gly Ser Asn Ile Asp Trp Glu Leu Gly Asn Glu
165 170 175
Pro Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu
180 185 190
Asp Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn
195 200 205
Lys Gly Ser Leu Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Tyr
210 215 220
Val Lys Gly Leu Ala Asp Gly Ala Gly Asp His Val Thr Ala Phe Thr
225 230 235 240
Leu His Gln Tyr Tyr Phe Asp Gly Asn Thr Ser Asp Val Ser Thr Tyr
245 250 255
Leu Asp Ala Thr Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val
260 265 270
Lys Asp Val Leu Lys Asn Ser Arg His Lys Asp Lys Pro Thr Gly Leu
275 280 285
Gly Glu Thr Ser Ser Gly Tyr Asn Ser Gly Thr Lys Asp Val Ser Asp
290 295 300
Arg Tyr Trp Ser Gly Phe Leu Asn Ile Asp Lys Leu Gly Leu Ser Ala
305 310 315 320
Ala Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Asn Gly Tyr
325 330 335
Tyr Gly Leu Leu Asp Lys Asn Lys Leu Glu Pro Asn Pro Asp Tyr Trp
340 345 350
Leu Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val
355 360 365
Asp Val Ser Asp Pro Thr Asn Lys Ala Arg Val Tyr Ala Gln Cys Thr
370 375 380
Lys Thr Asn Ser Lys His Thr Gln Ser Arg Tyr Tyr Lys Gly Ser Leu
385 390 395 400
Thr Ile Phe Ala Leu Asn Val Gly Asp Glu Asp Val Thr Leu Lys Ile
405 410 415
Asp Gln Tyr Ser Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu
420 425 430
Gly Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu
435 440 445
Lys Leu Val Ser Asp Gln Leu Pro Glu Leu Asn Ala Asp Glu Ser Lys
450 455 460
Thr Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser
465 470 475 480
Asp Ala Asn Val Glu Ala Cys Lys
485
<210> 2
<211> 489
<212> PRT
<213>artificial sequence
<400> 2
Met Lys Glu Ile Ala Val Thr Ile Asp Asp Lys Asn Val Ile Ala Ser
1 5 10 15
Val Ser Glu Ser Phe His Gly Val Ala Phe Asp Ala Ser Leu Phe Ser
20 25 30
Pro Lys Gly Leu Trp Ser Phe Val Asp Ile Thr Ser Pro Lys Leu Phe
35 40 45
Lys Leu Leu Glu Gly Leu Ser Pro Gly Tyr Phe Arg Val Gly Gly Thr
50 55 60
Phe Ala Asn Trp Leu Phe Phe Asp Leu Asp Glu Asn Asn Lys Trp Lys
65 70 75 80
Asp Tyr Trp Ala Phe Lys Asp Lys Thr Pro Glu Thr Ala Thr Ile Thr
85 90 95
Arg Arg Trp Leu Phe Arg Lys Gln Asn Asn Leu Lys Lys Glu Thr Phe
100 105 110
Asp Asp Leu Val Lys Leu Thr Lys Gly Ser Lys Met Arg Leu Leu Phe
115 120 125
Asp Leu Asn Ala Glu Val Arg Thr Gly Tyr Glu Ile Gly Lys Lys Met
130 135 140
Thr Ser Thr Trp Asp Ser Ser Glu Ala Glu Lys Leu Phe Lys Tyr Cys
145 150 155 160
Val Ser Lys Gly Tyr Gly Asp Asn Ile Asp Trp Glu Leu Gly Asn Glu
165 170 175
Pro Asp His Thr Ser Ala His Asn Leu Thr Glu Lys Gln Val Gly Glu
180 185 190
Asp Phe Lys Ala Leu His Lys Val Leu Glu Lys Tyr Pro Thr Leu Asn
195 200 205
Lys Gly Ser Leu Val Gly Pro Asp Val Gly Trp Met Gly Val Ser Tyr
210 215 220
Val Lys Gly Leu Ala Asp Gly Ala Gly Asp His Val Thr Ala Phe Thr
225 230 235 240
Leu His Gln Tyr Tyr Phe Asp Gly Asn Thr Ser Asp Val Ser Thr Tyr
245 250 255
Leu Asp Ala Thr Tyr Phe Lys Lys Leu Gln Gln Leu Phe Asp Lys Val
260 265 270
Lys Asp Val Leu Lys Asn Ser Pro His Lys Asp Lys Pro Leu Trp Leu
275 280 285
Gly Glu Thr Ser Ser Gly Tyr Asn Ser Gly Thr Lys Asp Val Ser Asp
290 295 300
Arg Tyr Val Ser Gly Phe Leu Thr Leu Asp Lys Leu Gly Leu Ser Ala
305 310 315 320
Ala Asn Asn Val Lys Val Val Ile Arg Gln Thr Ile Tyr Asn Gly Tyr
325 330 335
Tyr Gly Leu Leu Asp Lys Asn Thr Leu Glu Pro Asn Pro Asp Tyr Trp
340 345 350
Leu Met His Val His Asn Ser Leu Val Gly Asn Thr Val Phe Lys Val
355 360 365
Asp Val Ser Asp Pro Thr Asn Lys Ala Arg Val Tyr Ala Gln Cys Thr
370 375 380
Lys Thr Asn Ser Lys His Thr Gln Ser Arg Tyr Tyr Lys Gly Ser Leu
385 390 395 400
Thr Ile Phe Ala Leu Asn Val Gly Asp Glu Asp Val Thr Leu Lys Ile
405 410 415
Asp Gln Tyr Ser Gly Lys Lys Ile Tyr Ser Tyr Ile Leu Thr Pro Glu
420 425 430
Gly Gly Gln Leu Thr Ser Gln Lys Val Leu Leu Asn Gly Lys Glu Leu
435 440 445
Lys Leu Val Ser Asp Gln Leu Pro Glu Leu Asn Ala Asp Glu Ser Lys
450 455 460
Thr Ser Phe Thr Leu Ser Pro Lys Thr Phe Gly Phe Phe Val Val Ser
465 470 475 480
Asp Ala Asn Val Glu Ala Cys Lys Lys
485
<210> 3
<211> 1470
<212> DNA
<213>artificial sequence
<400> 3
atgaaagaga tcgcggtgac aatagacgat aagaacgtta ttgcctctgt ctccgaaagt 60
ttccatggcg ttgcctttga tgcgagttta ttttcaccga aagggttgtg gtcatttgtt 120
gacatcacat caccgaaatt gtttaagagc ttggagagca tctctccaag ctatttccgc 180
gtcggaggga cgtttgctaa ctggttattc tttgacctcg atgaaaacaa caaatggaaa 240
gactattggg cgtttaaaga taagacacct gagacagcaa cgatcacacg ccggtggctt 300
tttcgaaaac agaacaacct gaagaaagaa acttttgatg atctggtcaa actcaccagc 360
aagagcaaaa tgcagctgct gtttgattta aacgctgaag tcagaacagg ttatgaaatt 420
ggaaagaaaa tgacatccac atgggatagc agtgaagctg aaaaactctt caagtactgt 480
gtctccgagg gctatggaag caacattgac tgggaacttg gtaatgaacc ggatcatacg 540
tccgcacaca atcttactga aaaacaagta ggagaggact ttaaagccct gcataaagtg 600
ctggaaaaat atccgacgtt gaataaagga tcattagtgg gccctgacgt tggctggatg 660
ggagtgtctt atgtgaaagg actggcggac ggagccgggg atcatgtaac ggccttcacg 720
cttcaccagt actactttga cggcaatacc agcgatgtca gcacatacct ggacgctaca 780
tatttcaaaa agctacagca gctttttgat aaagtgaagg atgtccttaa aaatagcagg 840
cataaagata agccgaccgg cttaggagaa acctcttcag gctataattc tggcaccaaa 900
gatgtttcgg atcgttattg gagcggtttt cttaacatag acaaactggg tctgtctgcc 960
gcgaacaatg ttaaggtagt aattagacaa acgatctata atggatatta cggccttctg 1020
gacaaaaaca aacttgagcc gaatccagat tattggctga tgcatgtgca taactcgtta 1080
gtagggaata cggtctttaa agtggacgtt agtgatccta caaataaagc tagagtgtac 1140
gcacaatgca ccaaaacaaa ctcaaaacac actcagagtc gttactacaa gggctcatta 1200
acgatctttg ctttaaatgt tggggatgag gatgtgacgt tgaaaattga tcaatattct 1260
ggcaagaaga tttattcata tatacttaca cctgaaggcg gccaattgac ttcacaaaaa 1320
gttttattga atggtaaaga attaaaactt gtcagcgatc agttgccgga actgaatgca 1380
gacgagtcga agacctcgtt tactctgtcc ccgaagacat tcgggttctt tgtagtcagc 1440
gatgcaaatg ttgaagcgtg caaaaagtaa 1470
<210> 4
<211> 1468
<212> DNA
<213>artificial sequence
<400> 4
atgaaagaga tcgcggtgac aatagacgat aagaacgtta ttgcctctgt ctccgaaagt 60
ttccatggcg ttgcctttga tgcgagttta ttttcaccga aagggttgtg gtcatttgtt 120
gacatcacat caccgaaatt gtttaagctc ttggagggtc tctctccagg ttatttccgc 180
gtcggaggga cgtttgctaa ctggttattc tttgacctcg atgaaaacaa caaatggaaa 240
gactattggg cgtttaaaga taagacacct gagacagcaa cgatcacacg ccggtggctt 300
tttcgaaaac agaacaacct gaagaaagaa acttttgatg atctggtcaa actcaccaaa 360
ggatctaaaa tgcggctgct gtttgattta aacgctgaag tcagaacagg ttatgaaatt 420
ggaaagaaaa tgacatccac atgggatagc agtgaagctg aaaaactctt caagtactgt 480
gtctccaaag gctatggaga taacattgac tgggaacttg gtaatgaacc ggatcatacg 540
tccgcacaca atcttactga aaaacaagta ggagaggact ttaaagccct gcataaagtg 600
ctggaaaaat atccgacgtt gaataaagga tcattagtgg gccctgacgt tggctggatg 660
ggagtgtctt atgtgaaagg actggcggac ggagccgggg atcatgtaac ggccttcacg 720
cttcaccagt actactttga cggcaatacc agcgatgtca gcacatacct ggacgctaca 780
tatttcaaaa agctacagca gctttttgat aaagtgaagg atgtccttaa aaatagccca 840
cataaagata agccgctctg gttaggagaa acctcttcag gctataattc tggcaccaaa 900
gatgtttcgg atcgttatgt aagcggtttt cttactcttg acaaactggg tctgtctgcc 960
gcgaacaatg ttaaggtagt aattagacaa acgatctata atggatatta cggccttctg 1020
gacaaaaaca cacttgagcc gaatccagat tattggctga tgcatgtgca taactcgtta 1080
gtagggaata cggtctttaa agtggacgtt agtgatccta caaataaagc tagagtgtac 1140
gcacaatgca ccaaaacaaa ctcaaaacac actcagagtc gttactacaa gggctcatta 1200
acgatctttg ctttaaatgt tggggatgag gatgtgacgt tgaaaattga tcaatattct 1260
ggcaagaaga tttattcata tatacttaca cctgaaggcg gccaattgac ttcacaaaaa 1320
gttttattga atggtaaaga attaaaactt gtcagcgatc agttgccgga actgaatgca 1380
gacgagtcga agacctcgtt tactctgtcc ccgaagacat tcgggttctt tgtagtcagc 1440
gatgcaaatg ttgaagcgtg caaaaagt 1468
<210> 5
<211> 8
<212> PRT
<213>artificial sequence
<400> 5
Ser Leu Glu Ser Ile Ser Pro Ser
1 5
<210> 6
<211> 6
<212> PRT
<213>artificial sequence
<400> 6
Ser Lys Ser Lys Met Gln
1 5
<210> 7
<211> 5
<212> PRT
<213>artificial sequence
<400> 7
Glu Gly Tyr Gly Ser
1 5
<210> 8
<211> 8
<212> PRT
<213>artificial sequence
<400> 8
Arg His Lys Asp Lys Pro Thr Gly
1 5
<210> 9
<211> 7
<212> PRT
<213>artificial sequence
<400> 9
Trp Ser Gly Phe Leu Asn Ile
1 5
<210> 10
<211> 46
<212> DNA
<213>artificial sequence
<400> 10
ccggaattcc accaccacca ccaccacatg aaagagatcg cggtga 46
<210> 11
<211> 40
<212> DNA
<213>artificial sequence
<400> 11
attgtttnnb nnbttggagn nbnnbtctcc annbtatttc 40
<210> 12
<211> 35
<212> DNA
<213>artificial sequence
<400> 12
gtgtctccnn bggctatgga nnbaacattg actgg 35
<210> 13
<211> 44
<212> DNA
<213>artificial sequence
<400> 13
tttcggatcg ttatnnbagc ggttttcttn nbnnbgacaa actg 44
<210> 14
<211> 42
<212> DNA
<213>artificial sequence
<400> 14
cgcggatccg cggccgctta ctttttgcac gcttcaacat tt 42
<210> 15
<211> 40
<212> DNA
<213>artificial sequence
<400> 15
ttaaatcaaa nnbcagccgn nbnnbnntcc tttggtgagt 40
<210> 16
<211> 41
<212> DNA
<213>artificial sequence
<400> 16
ttctcctaan nbnnbcggct tatctttatg nnbgctattt t 41
<210> 17
<211> 34
<212> DNA
<213>artificial sequence
<400> 17
aataatctgg attcggctca agnnbgtttt tgtc 34

Claims (9)

1. the hyaluronic acid enzyme mutant that a kind of enzyme activity improves, which is characterized in that amino acid sequence is as shown in SEQ ID NO.1.
2. encoding the gene of hyaluronic acid enzyme mutant described in claim 1.
3. expressing the carrier or cell line of hyaluronic acid enzyme mutant described in claim 1.
4. a kind of genetic engineering bacterium for producing hyaluronidase, which is characterized in that with bacillus subtilis be host, be with pPNMK Carrier expresses hyaluronic acid enzyme mutant described in claim 1.
5. genetic engineering bacterium according to claim 4, which is characterized in that the bacillus subtilis includes Bacillus Subtilis168, Bacillus subtilis WB600.
6. a kind of method for improving hyaluronidase enzyme activity, which is characterized in that by hyaluronidase amino shown in SEQ ID NO.2 The L of acid sequence50-G57Region replaces with SLESISPS, and by K120-R125Replace with SKSKMQ, and by K163-D167Region replacement For EGYGS, and by P280-W287Region replaces with RHKDKPTG, and by V307-L313Region replaces with WSGFLNI, and by T344It replaces It is changed to K344。
7. a kind of production method for improving hyaluronidase enzyme activity, which is characterized in that by genetic engineering bacterium described in claim 5 It is seeded in fermentation medium, in 30~37 DEG C of 16~72h of culture.
8. the method according to the description of claim 7 is characterized in that the fermentation medium group is divided into 30g/L sucrose, 20g/L Yeast extract, 2g/L peptone, 15.6g/L disodium hydrogen phosphate, 5.94g/L potassium dihydrogen phosphate.
9. hyaluronic acid enzyme mutant described in claim 1 is prepared in food, biology, chemical industry, field of medicaments containing hyaluronic acid Pharmaceutical product, health care product, the application in cosmetics.
CN201610982765.9A 2016-11-08 2016-11-08 A method of improving leech hyaluronidase enzyme activity Active CN106520728B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610982765.9A CN106520728B (en) 2016-11-08 2016-11-08 A method of improving leech hyaluronidase enzyme activity

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610982765.9A CN106520728B (en) 2016-11-08 2016-11-08 A method of improving leech hyaluronidase enzyme activity

Publications (2)

Publication Number Publication Date
CN106520728A CN106520728A (en) 2017-03-22
CN106520728B true CN106520728B (en) 2019-06-21

Family

ID=58350268

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610982765.9A Active CN106520728B (en) 2016-11-08 2016-11-08 A method of improving leech hyaluronidase enzyme activity

Country Status (1)

Country Link
CN (1) CN106520728B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1185666A1 (en) * 1999-06-12 2002-03-13 MERCK PATENT GmbH Hyaluronidase from the hirudinaria manillensis, isolation, purification and recombinant method of production
CN104278005A (en) * 2014-10-17 2015-01-14 江南大学 Recombinant bacillus subtilis for expressing hyaluronidase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1185666A1 (en) * 1999-06-12 2002-03-13 MERCK PATENT GmbH Hyaluronidase from the hirudinaria manillensis, isolation, purification and recombinant method of production
CN104278005A (en) * 2014-10-17 2015-01-14 江南大学 Recombinant bacillus subtilis for expressing hyaluronidase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lysase;Songlin Li et al.;《The EMBO Journal》;20001231;第19卷(第6期);1228-1240

Also Published As

Publication number Publication date
CN106520728A (en) 2017-03-22

Similar Documents

Publication Publication Date Title
CN105087456B (en) A method of producing the recombined bacillus subtilis building of specified molecular weight hyaluronic acid
US11807847B2 (en) Strain of Enterobacter for degrading hyaluronic acid and application thereof
CN100424096C (en) Survivin mutant containing HIV transduction structural area and its preparation method and uses
Cao et al. Purification and characterization of alginate lyase from Streptomyces species strain A5 isolated from banana rhizosphere
CN101316608A (en) Bacillus subtilis novel bacterial strain and its usage in preparation of medicament for treating thrombus disease
CN110055218A (en) A kind of artificial reconstructed natural killer cell and its preparation and application
CN100536917C (en) Cow mastitis concatenate inactivated vaccine
CN104278005A (en) Recombinant bacillus subtilis for expressing hyaluronidase
CN101633931A (en) Hyaluronidase expression vector and application thereof
CN105238720B (en) A kind of production fibrinolysin bacillus subtilis and fermentation process and application
CN103695448B (en) A kind of hyaluronic acid enzyme coding gene and fermenting and producing thereof and purification process
CN111961619B (en) Vibrio maritima capable of producing alginate lyase with good thermal stability and application
CN107227284A (en) A kind of restructuring streptococcus zooepidemicus for the micromolecule hyaluronic acid that ferments
CN106520728B (en) A method of improving leech hyaluronidase enzyme activity
CN104388401A (en) High-temperature resistant superoxide dismutase and encoding gene and application thereof
CN109392600A (en) A kind of cultural method of selenium-enriched cordceps militaris
CN106497881A (en) Stability and high efficiency expression recombined human hyaluronidase CHO K1SP cell lines and its construction method
CN108823118A (en) One plant of Le gram bacterium that can inhibit alpha-glucosidase activity and its application
CN108220272A (en) A kind of preparation method of hyaluronidase
Mahesh et al. Optimization and production of hyaluronidase by Streptococcus mitis MTCC 2695
CN110527680B (en) Hyaluronic acid lyase, gene thereof and application thereof
CN106636036A (en) Hyaluronidase mutant and application thereof
CN102181405B (en) Recombinant virus and method for producing L-asparaginase II by using virus
CN101120959A (en) Application of guhong injection for treating lung cancer
Shareef et al. Antifungal Activity Of Purified Dextrnase From Dental Caries Staphylococcus Salivariusagainstoral Candidiasis Caused Bycandida Albicans

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20170322

Assignee: Shuiyang Cosmetics Manufacturing Co.,Ltd.

Assignor: Jiangnan University

Contract record no.: X2024980001574

Denomination of invention: A method to increase the hyaluronidase activity of leeches

Granted publication date: 20190621

License type: Exclusive License

Record date: 20240130