CN102559638A - Alkaline pectinase poly lactic acid (PLA) and gene and application thereof - Google Patents
Alkaline pectinase poly lactic acid (PLA) and gene and application thereof Download PDFInfo
- Publication number
- CN102559638A CN102559638A CN2010106092225A CN201010609222A CN102559638A CN 102559638 A CN102559638 A CN 102559638A CN 2010106092225 A CN2010106092225 A CN 2010106092225A CN 201010609222 A CN201010609222 A CN 201010609222A CN 102559638 A CN102559638 A CN 102559638A
- Authority
- CN
- China
- Prior art keywords
- pla
- polygalacturonase
- gene
- alkaline
- alkaline pectinase
- 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.)
- Granted
Links
Images
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the field of gene engineering, in particular to alkaline pectinase poly lactic acid (PLA) and a gene and application thereof. According to the alkaline pectinase PLA, the amino acid sequence of the alkaline pectinase PLA is show as the sequence identity number 1 or 2 (SEQ ID NO. 1 or 2). The optimum potential of hydrogen (pH) for alkaline pectinase is 9.0, and more than 80% of enzymatic activity exists between pH 7.5 to pH 10.0. The alkaline pectinase PLA has good pH stability and is stabilized between the pH5.0 to pH10.5. Specific activity of the alkaline pectinase PLA is 797U/mg, and the alkaline pectinase PLA is good in alkaline protease resistance and easy in industrial fermenting production. Serving as a novel enzyme preparation, the alkaline pectinase PLA has application values in the industries of textile, papermaking, detergent, plant fiber processing, tea and coffee fermentation, oil extraction, treatment of industrial waste water containing pectin, biological technology and the like.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of alkaline pectase PLA and gene and application.
Background technology
Pectin substance is the one group of polysaccharide that extensively is present in higher plant primary wall and the intercellular substance, in the cell tissue of plant, plays a part " bonding ".The elementary cell that constitutes pectin substance is the galacturonic acid of different Zhi Huadus; The D-galacturonic acid is with α-1; 4 glycosidic links are connected as main chain; And be connected on its main chain as side chain (Thakur BR, et al.Crit Rev Food Sci Nutr.37:47-73,1997) with rhamnosyl, pectinose, semi-lactosi, wood sugar and other carbohydrates.
Polygalacturonase (pectinases) is the general name of the plurality of enzymes of decompose pectin matter.Because the chemical structure more complicated of pectin substance. polygalacturonase that can its decomposition of catalysis also is of a great variety.Polygalacturonase can be divided into according to the character of reaction property that decomposes glycosidic link or degraded substrate: pectin hydrolase (pectin hydrolases), pectin lyase (pectin lyases; PL), Rohapect MPE (pectin esterases; PE) and protopectinase (Alkorta I; Et al.Process Biochem33:21-28,1998).Polygalacturonase is divided into acid pectase and alkaline pectase by its effect ph optimum.Studying and use more at present is acid pectase, and its enzyme effect ph optimum is in the slant acidity scope, is mainly used in that fruit is squeezed the juice and juice clarification.Alkaline pectase (Alkaline pectinase) then is meant the polygalacturonase that in alkaline range, has greater activity, generally refers to polygalacturonic acid lyase (Polygalacturonate lyase is called for short PGL) more.Its ph optimum can break off the pectin main polymer chain at 8.0-10.0 under high pH condition, produce oligogalacturonans.Alkaline pectase is one of very important industrial enzyme preparation, has immeasurable potential using value.At present; Alkaline pectase extracts at plant amedica extraction, tea and coffee fermentation, oil; The application that industries such as pectin Industrial Wastewater Treatment and biotechnology were processed, contained to weaving, papermaking, washing composition, vegetable fibre more and more comes into one's own; So become the focus (Jayani RS, et al.Process Biochemistry 40:2931-2944,2005) of people's research.
Alkaline pectase is many to produce (Hayashi K by Bacillaceae in the bacterium and Rhodopseudomonas; Et al.Phytochemistry 45:1359-1363; 1997), actinomycetes (Beg QK, et al.J Ind MicrobiolBiotechnol 24:396-402; 2000) and fungi produce alkaline pectase report (Baracat et al.J IndMicrobiol 11:139-142,1993) also arranged.The alkaline pectase biochemical property difference of different sources is comparatively obvious.Derive from Bacillus licheniformis (Singh SA; Et al.Process Biochem.35:411-417; 1999) and Fusarium oxysporum f.sp.Lycopersci (Pietro AD; The ph optimum of alkaline pectase et al.FEMS Microbiol Lett.145:295-298,1996) is 11.0.The ph optimum that derives from Bacillus alcalophillusNTT33 polygalacturonase is 9.5 (Zhai C, et al.Enzyme and Microbial Technology.33:173-178,2003).The ph optimum that derives from the polygalacturonase of Pseudomonas marginalis CFBP 1287 is 8.5-9.0 (Membre and Burlot, Appl Environ Microbiol.60:2017-2022,1994).The optimum temperuture of the alkaline pectase of report is between 30-70 ℃.The gene of some alkaline pectases is cloned, and has carried out expressing (Zhai C, et al.Enzyme andMicrobial Technology.33:173-178,2003 in the expression system such as intestinal bacteria, pichia spp; Wang Yun etc., microbiology circular, 35 (3): 341-345,2008).But the expression amount of alkaline pectase is still very low, because the cost problem is not used widely so far.
Summary of the invention
The object of the present invention is to provide a kind of bacterial strain Klebsiella sp.Y1 that produces alkaline pectase.
Still a further object of the present invention provides the alkaline pectase that derives from above-mentioned bacterial strains.
A purpose more of the present invention provides the gene of above-mentioned polygalacturonase.
A purpose more of the present invention provides the recombinant vectors that comprises above-mentioned polygalacturonase.
A purpose more of the present invention provides the recombinant bacterial strain that comprises above-mentioned polygalacturonase gene.
A purpose more of the present invention provides a kind of method for preparing alkaline pectase.
A purpose more of the present invention provides the application of above-mentioned alkaline pectase.
The present invention's technical problem at first to be solved is the deficiency that overcomes prior art, provides a kind of character good new enzyme.The inventor screens a kind of natural bacterial strain; It comes from this klebsiella Klebsiella sp., and the polygalacturonase that it produced is suitable for extracting, containing in the industries such as pectin Industrial Wastewater Treatment and biotechnology and use at weaving, papermaking, washing composition, vegetable fibre processing, tea and coffee fermentation, oil.
The present invention has obtained a kind of alkaline pectase PLA from above-mentioned bacterial strains, its aminoacid sequence is shown in SEQ ID NO.1:
1 MKIAKITLAL?GVLSLFSLNA?GAQENERLSS?VKQFADVVLD?KASDRYGHYS
51 PLLANGVDPR?TGKQMEWVFP?DGKVTVLSNF?SAQQNLMRML?VGLSNLTGET
101?KYKQRVAENI?RYYFDHYQDA?SGLLLWGGHR?FVDLKTLQPQ?GPSEKERVHE
151?LKNAYPYYDM?MFAVDDQATA?RFIKAFWNAH?VYDWKTLETS?RHGEYGKPMG
201?ALWQSDFVQQ?PPFFATKGLS?FLNAGNDLIY?SASLLYQYDG?DAGALTWAKR
251?LAEQYVLPRD?KKTGLGVYQF?TQPLKRADTS?DDSDTHSKYG?DRAQRQFGPE
301?LGPDALEGNM?LLKGRTSTLY?SENALMQLAL?AKSLGKDGDD?LKKWTLDGLK
351?AFATYAYDEQ?NNTFRPMLAN?GTDLSDYALK?RDGYYGKKGT?VLKAYPAGNE
401?FLLSYARAWT?LEPDHAIWKV?ARGIAKGQGL?GDIGEPGGTN?RRLNSQTENH
451?QPYAIFALID?LWQSTGQRDY?LTLADRIGAN?IINKQRLNGF?FVDDPEAEYA
501?SIDSIAPYAL?LALEAAFRNT?PDAVAPFLNG?AGFTEGAYRM?ADGTVRYSTR
551?DDELFRLRPG?EQLKPNGKK*
Wherein, 569 amino acid of this enzyme genes encoding and a terminator codon, N holds 22 signal peptide sequences " MKIAKITLALGVLSLFSLNAGA " that amino acid is its prediction.
Therefore, the theoretical molecular of sophisticated alkaline pectase PLA is 63.8kDa, and its aminoacid sequence is shown in SEQID NO.2:
1 QENERLSSVK?QFADVVLDKA?SDRYGHYSPL?LANGVDPRTG?KQMEWVFPDG
51 KVTVLSNFSA?QQNLMRMLVG?LSNLTGETKY?KQRVAENIRY?YFDHYQDASG
101?LLLWGGHRFV?DLKTLQPQGP?SEKERVHELK?NAYPYYDMMF?AVDDQATARF
151?IKAFWNAHVY?DWKTLETSRH?GEYGKPMGAL?WQSDFVQQPP?FFATKGLSFL
201?NAGNDLIYSA?SLLYQYDGDA?GALTWAKRLA?EQYVLPRDKK?TGLGVYQFTQ
251?PLKRADTSDD?SDTHSKYGDR?AQRQFGPELG?PDALEGNMLL?KGRTSTLYSE
301?NALMQLALAK?SLGKDGDDLK?KWTLDGLKAF?ATYAYDEQNN?TFRPMLANGT
351?DLSDYALKRD?GYYGKKGTVL?KAYPAGNEFL?LSYARAWTLE?PDHAIWKVAR
401?GIAKGQGLGD?IGEPGGTNRR?LNSQTENHQP?YAIFALIDLW?QSTGQRDYLT
451?LADRIGANII?NKQRLNGFFV?DDPEAEYASI?DSIAPYALLA?LEAAFRNTPD
501?AVAPFLNGAG?FTEGAYRMAD?GTVRYSTRDD?ELFRLRPGEQ?LKPNGKK*
This polygalacturonase PLA has good pH stability simultaneously, under low temperature (0 ℃), normal temperature and higher temperatures, possesses high reactivity, in alkalescence and neutral scope, all has characteristics such as high reactivity, protease inhibitor degraded.The polygalacturonase that the klebsiella Klebsiella sp.Y1 that the present invention screens is produced, its ph optimum 9.0 is kept the enzymic activity more than 80% in the scope of pH7.5-10.0; 50 ℃ of optimum temperutures have the enzyme more than 85% to live between 20-60 ℃, live at the 0 ℃ of enzyme that can keep more than 35%.Have good pH stability, stable between pH5.0-10.5; Than 797U/mg alive; Fabulous Sumizyme MP resistance and be easy to industrial fermentation production.
The present invention also provides the gene of the above-mentioned alkaline pectase of encoding.The method separating clone of the present invention through PCR this alkaline pectase gene plA, gene plA total length 1710bp, the complete genome sequence of this enzyme is shown in SEQ ID NO.3:
1 ATGAAGATTG?CAAAAATCAC?TCTGGCATTG?GGCGTTCTGA?GTCTCTTCTC?ACTGAATGCG
61 GGTGCCCAGG?AGAACGAGCG?CCTGAGCAGC?GTAAAGCAGT?TCGCCGACGT?CGTGCTGGAC
121?AAAGCAAGCG?ATCGGTACGG?GCATTACTCT?CCCCTGCTGG?CTAATGGCGT?GGACCCACGC
181?ACCGGCAAAC?AAATGGAATG?GGTATTTCCG?GACGGTAAAG?TGACCGTGCT?GTCGAACTTC
241?TCTGCCCAGC?AAAACTTGAT?GCGGATGCTG?GTAGGGTTAA?GCAACCTGAC?TGGCGAAACA
301?AAATATAAAC?AGCGGGTCGC?GGAGAATATA?CGCTACTACT?TTGACCACTA?TCAGGATGCA
361?AGCGGGCTGC?TGCTATGGGG?AGGGCATCGT?TTTGTTGATT?TAAAAACGCT?GCAGCCTCAA
421?GGCCCAAGTG?AAAAAGAGCG?GGTTCATGAA?CTTAAGAATG?CCTATCCCTA?TTACGACATG
481?ATGTTTGCCG?TCGATGACCA?GGCGACCGCG?CGCTTTATCA?AGGCTTTCTG?GAATGCCCAT
541?GTTTACGACT?GGAAAACGCT?GGAAACCAGC?CGCCACGGGG?AGTACGGCAA?ACCGATGGGC
601?GCGCTCTGGC?AAAGTGATTT?TGTCCAACAG?CCGCCCTTTT?TCGCCACCAA?AGGTCTGAGC
661?TTTCTCAATG?CCGGCAACGA?CCTGATCTAC?TCCGCATCGC?TGCTGTATCA?ATACGATGGT
721?GACGCTGGCG?CACTGACGTG?GGCAAAACGG?CTGGCCGAAC?AGTACGTTCT?GCCGCGAGAT
781?AAGAAGACCG?GGCTGGGCGT?ATACCAGTTT?ACCCAACCGT?TAAAACGCGC?CGATACCAGC
841 GATGACAGCG?ACACGCATTC?GAAATACGGC?GACCGCGCGC?AGCGTCAGTT?TGGCCCGGAA
901 CTGGGCCCGG?ACGCGCTGGA?AGGCAATATG?CTGCTTAAGG?GCCGAACCAG?TACGCTCTAT
961 TCAGAAAATG?CGCTGATGCA?GCTTGCCCTG?GCAAAATCGC?TCGGCAAAGA?CGGTGACGAT
1021?CTCAAAAAAT?GGACTCTTGA?TGGCCTCAAG?GCCTTCGCGA?CCTACGCTTA?CGATGAGCAG
1081?AATAATACCT?TCCGCCCGAT?GCTGGCCAAC?GGCACGGATC?TCTCCGACTA?CGCGTTAAAA
1141?CGCGATGGCT?ATTATGGAAA?AAAAGGGACG?GTGCTCAAAG?CCTATCCGGC?GGGAAATGAA
1201?TTTCTTCTCT?CCTATGCCCG?CGCCTGGACG?CTTGAACCGG?ATCATGCCAT?CTGGAAGGTC
1261?GCCCGCGGCA?TCGCGAAAGG?CCAGGGGTTA?GGCGATATCG?GTGAACCCGG?CGGCACAAAC
1321?CGCAGGCTGA?ATAGCCAAAC?GGAGAATCAT?CAGCCGTATG?CGATTTTCGC?GCTTATCGAC
1381?CTGTGGCAGT?CCACCGGGCA?GCGGGATTAT?TTGACGCTGG?CGGATCGCAT?CGGCGCGAAC
1441?ATCATCAACA?AGCAGCGCCT?CAACGGCTTT?TTTGTCGATG?ATCCTGAGGC?AGAATATGCC
1501?AGTATCGATA?GTATCGCCCC?CTATGCGCTT?CTCGCCCTGG?AAGCCGCCTT?CCGCAACACG
1561?CCGGACGCGG?TCGCTCCGTT?CCTGAACGGC?GCTGGATTCA?CGGAAGGCGC?TTATCGGATG
1621?GCTGACGGAA?CCGTGCGCTA?CTCCACCCGA?GATGACGAGC?TGTTCAGGCT?CAGACCCGGT
1681?GAGCAGCTGA?AACCGAACGG?CAAAAAATAA
Wherein, the base sequence of signal peptide is: ATGAAGATTG CAAAAATCAC TCTGGCATTG GGCGTTCTGA
GTCTCTTCTC?ACTGAATGCG?GGTGCC。
The maturation protein theoretical molecular is 63.8kDa.Alkaline pectase gene plA dna sequence dna and the aminoacid sequence derived are carried out the BLAST comparison in GenBank.The nucleotide sequence consistence of finding and derive from the pectin lyase of Enterobacter sp.638 is 78.9%, and consensus amino acid sequence property is 85.8%.The transformation of gene and in various heterologous gene expression systems, efficiently express the genetic material that provides good for this reason.Explain that PLA is a kind of new alkaline pectase.This also is from klebsiella, to clone for the first time to obtain the alkaline pectase gene.
The present invention also provides the reorganization plA that comprises above-mentioned polygalacturonase gene carrier.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned polygalacturonase gene plA.
The present invention also provides a kind of method for preparing alkaline pectase, may further comprise the steps:
1), gets recombinant bacterial strain with above-mentioned recombinant vectors transformed host cell; Preferred said bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus spp.
2) cultivate recombinant bacterial strain, induce the expression of reorganization polygalacturonase; And
3) reclaim the also expressed polygalacturonase of purifying.
The present invention also provides the application of above-mentioned alkaline pectase.
The ph optimum of alkaline pectase of the present invention is 9.0, is having the enzyme more than 80% to live between the pH7.5-10.0.Have good pH stability, stable between pH5.0-10.5; Than 797U/mg alive; Fabulous Sumizyme MP resistance and be easy to industrial fermentation production.As a kind of novel enzyme preparation, extract in weaving, papermaking, washing composition, vegetable fibre processing, tea and coffee fermentation, oil, contain industries such as pectin Industrial Wastewater Treatment and biotechnology using value is all arranged.
Description of drawings
Fig. 1 plA analyzes at the SDS-PAGE of the polygalacturonase of expression in escherichia coli, and 1, molecular weight standard; 2.3 inducing culture supernatants; 4, penetrate peak 5, NTA-0; 6, NTA-20; 7, NTA-40; 8, NTA-60; 9, NTA-80; The purified recombinant polygalacturonase.
The recombinate optimum pH of polygalacturonase of Fig. 2 the present invention.
The pH stability of Fig. 3 polygalacturonase of the present invention.
Fig. 4 polygalacturonase optimal reactive temperature of the present invention.
The thermostability of Fig. 5 polygalacturonase of the present invention.
The metals ion resistance of Fig. 6 polygalacturonase of the present invention
Embodiment
Test materials and reagent
1, bacterial strain and carrier: host e. coli (Escherichia coli) BL21 (DE3), carrier pET-22b (+) is available from Novagen company.
2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Pectin, galacturonic acid, polygalacturonic acid be available from Sigma company, and other all is domestic reagent (all can buy from common biochemical reagents company and obtain).
3, substratum:
(1) induces the selection substratum: 0.5% pectin, 0.5% peptone, 0.5% yeast extract, 0.1%KH
2PO
4, 0.2%CuSO
4, 0.1%CaCl
2, pH9.0.
(2) intestinal bacteria substratum LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
Explain: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
The clone of embodiment 1 klebsiella polygalacturonase encoding sox plA
Klebsiella (Klebsiella sp.) derives from the soil sample of Guangxi fruit juice factory; Extract klebsiella (Klebsiella sp.) genomic dna: will be in the LB substratum 30 ℃ of centrifugal 10min of bacterium liquid 10000rpm that cultivated 2d; Take by weighing 50mg bacterium mud and add 500 μ L sterile water wash, the sedimentary thalline of centrifuging and taking.Sedimentary thalline is resuspended in 500 μ L N,O-Diacetylmuramidase mixed solutions, and 37 ℃ of incubation 1h add enzyme liquid 100 μ L again and continue insulation 30min in 45 ℃; To bacterium liquid transparent after; Add 10%SDS to final concentration 2%, stir about 5min significantly descends to the bacterium fluid viscosity, and 15000rpm is centrifugal, and 10min removes fragment.Supernatant is used equal-volume phenol respectively, phenol: chloroform, chloroform is extracting successively.Get the Virahol normal temperature deposition 10min that upper solution adds 0.6-1 times of volume.The centrifugal 15min of 16000rpm.Deposition is cleaned with 70% ethanol, and is centrifugal slightly, and deposition is drained the back with the dissolving of 30 μ L sterilized waters, and-20 ℃ of preservations are subsequent use.
Sequence alignment result according to the pectin lyase of delivering finds two conserved amino acid sequence conserved sequence GLF (L) Y (L) WGGH and RQFGPE, and designs and synthesized degenerated primer PL9-F, 5 '-CTGTTYTAYTGGGGYGGRCA-3 '; PL9-R, 5 '-CTCNGGNCCRAAYTGTCG-3 ' (Y represents C/T, and R represents G/A, and N represents A/T/C/C), be that template is carried out pcr amplification with total DNA of klebsiella Y1.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 53-48 ℃ of 30sec (wherein each circulation back renaturation temperature descends 0.5 ℃), 72 ℃ of 1min, 10 circulations get into second cycling program then: 94 ℃ of 30sec, 48 ℃ of 30sec, 72 ℃ of 1min are after 25 circulations; 72 ℃ of 10min, agarose electrophoresis detects.The sequencing fragment that obtains.
According to the nucleotide sequence that order-checking obtains, each three TAIL-PCR Auele Specific Primer of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1, and sp3 is positioned at the inboard of sp2.Distance between per two primers does not have strict regulation, the general 22～30nt of primer length, and annealing temperature is at 60～65 ℃.And with they difference called after usp1, usp2, usp3 (upper reaches Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees table 1.
Table 1 polygalacturonase plA TAIL-PCR Auele Specific Primer
Obtain the flanking sequence of known sequence through reverse TAIL-PCR, amplification obtains product and reclaims the back order-checking.
The core fragment that degenerated primer is obtained splices with the flanking sequence that obtains through TAIL-PCR and obtains the plA full-length gene.The result shows that this gene is the gene fragment of a long 1710bp, encode 569 amino acid and a terminator codon.It is its signal peptide sequence that N holds 22 amino acid.Zymoprotein is 85.8% with the consensus amino acid sequence property that derives from the pectin lyase of Enterobacter sp.638.With the sequence identity of the pectin lyase that derives from Pectobacterium wasabiae WPP163 be 71%.This encoding sox is a new gene.
The enzyme activity assay of embodiment 2 polygalacturonases
Activity determination method adopts the DNS method.At pH9.0, under 50 ℃ of conditions, the reaction system of 1mL comprises 100 μ L suitable dilution enzyme liquid, 900 μ L substrates, and reaction 10min adds 1.5mL DNS termination reaction, and boiling water boils 5min.Cooling back 540nm measures the OD value.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition PM discharges 1 μ mol reducing sugar.
The preparation of embodiment 3 reorganization polygalacturonases.
Design primer according to sequencing result:
PLAF (5 '-CTG
CCATGGCAGGAGAACGAGCGCCTGAGCAGCGTAAAGC-3 ', underscore are Nco I site) and PLAR (5 '-CTT
GCGGCCGCTTATTTTTTGCCGTTCGGTTTCAGCTGCTCAC-3 '), underscore is Not I site), carry out pcr amplification.Plasmid pET22b (+) and gene segment carry out being connected to form carrier pET22b (+)-plA behind the double digestion, and preparation BL21 (DE3) competent cell transforms the host bacterium with recombinant plasmid pET22b-plA electric shock.Identify positive recombinant, and induce the detection enzyme to live.Get BL21 (DE3) bacterial strain that contains plasmid recombinant and BL21 (DE3) bacterial strain (comparing) that contains pET22b (+) empty plasmid, be inoculated in 3mL LB (being added with the penbritin of the 100 μ g/mL) nutrient solution 37 ℃ of quick oscillation overnight cultures respectively.Get in the 10mL LB nutrient solution (1% inoculum size) that 100 μ L incubated overnight liquid add the penbritin contain 100 μ g/mL 30 ℃ of about 2-3h (OD of shaking culture respectively
600Reach 0.6-0.8) the back inductor IPTG that adds final concentration 0.8mmol/L, 8h are cultivated in 30 ℃ of 180rpm concussions.Nutrient solution 12, the centrifugal 5min of 000rpm collects supernatant of culture medium and deposition respectively, and cell precipitation is resuspended with the damping fluid of pH9.0, and after the ultrasonication 12, the centrifugal 10min of 000rpm, collection supernatant are total protein of cell (CTP).Detect supernatant of culture medium and the work of CTP enzyme respectively by above-mentioned enzyme activity determination method.Recombinase mainly is secreted into supernatant as a result, and the enzymic activity in the supernatant is 0.12U/ml.Through the SDS-PAGE electrophoresis detection, can see a tangible specific band (Fig. 1, LANE2, LANE3).
Intestinal bacteria induce the polygalacturonase PLA of generation through Ni-NTA column purification (NEB), are increased to 797U/mg than living.Purifying PLA obtains electrophoretically pure single band through SDS-PAGE, and molecular weight is about 63kDa close with predicted molecular weight (Fig. 1, LANE 9).
The zymologic property of embodiment 4 polygalacturonase PLA
Purified polygalacturonase PLA carries out enzymatic reaction to measure its ph optimum under different pH.Used damping fluid is glycocoll-NaOH damping fluid of 0.1mol/L of McIlvaine damping fluid and pH 9.0～11.0 of the 0.1mol/L of pH 2.0～8.0.The polygalacturonase PLA of purifying is in the buffer system of different pH, and 50 ℃ of pH that measure down fit property result (Fig. 2) and show: the righttest action pH of PLA is 9.0, is having the enzyme more than 80% to live between the pH7.5-10.0.Enzyme liquid is handled 1h in the damping fluid of different pH values, measure the pH stability of enzymic activity again under 37 ℃ with the research enzyme.The result shows (Fig. 3), and PLA is very stable between 5.0-10.5 in the pH scope, keeps the enzyme more than 80% to live.
Carry out enzymatic reaction under the glycocoll of the 0.1mol/L that is determined at pH9.0 of optimum temperuture-NaOH buffer solution system and the different temperature (0～90 ℃).The enzyme reaction optimum temperuture is measured result (Fig. 4) and is shown, 50 ℃ of the optimum temperatures of PLA between 30 ℃ and 60 ℃, keep the enzyme more than 85% to live.At 0 ℃ 35% enzyme work is arranged still.Residue was lived less than 2% enzyme when temperature was higher than 60 ℃.
The mensuration polygalacturonase is incubated different time respectively and measures relative enzyme activity under 50 ℃ and 60 ℃, draw the thermostability curve of enzyme.50 ℃ down handle 10min after the enzyme residue of living about 70%, better heat stability (Fig. 5).
The influence that different metal ion chemistry reagent is lived to the PLA enzyme is measured as follows:
In enzymatic reaction system, add the different metallic ion and the chemical reagent of different concns, study its influence to enzymic activity, various material final concentrations are 1 and 5mmol/L.Under 50 ℃, pH9.0 condition, measure enzymic activity.Result (Fig. 6) shows, the Cu of lower concentration
2+With beta-mercaptoethanol and 1mmol/L Co
2+Polygalacturonase PLA is had tangible activation.The Co of 5mmol/L
2+, Ni
2+, Fe
3+, Zn
2+, Mn
2+, Pb
2+, Hg
2+, the activity of EDTA and SDS strongly inhibited PLA.Li
+, Na
+, K
+And Mg
2+Enzyme lived not to be influenced or certain activation is arranged.
Polygalacturonic acid (PGA) with different concns (1-20mg/mL) is a substrate, in glycocoll-NaOH reaction system of pH 9.0, measures enzymic activity down, calculates its K under 50 ℃ for 50 ℃
mValue.Through measuring, this polygalacturonase is the apparent K of substrate with PGA under 50 ℃
mValue is 13.31mg/ml, maximum reaction velocity V
MaxBe 253.4 μ mol/min/mg.
The substrate specificity of embodiment 5 polygalacturonase PLA
Being respectively 34%, 70% and 85% citrus pectin with PGA and degree of esterification respectively is that substrate is measured pectinase activity.Is 100% with PLA to the vigor of PGA, is that substrate is measured the relative vigor of polygalacturonase and is respectively 92%, 23.9% and 4.5% with 34%, 70% and 85% citrus pectin then.
Claims (9)
1. an alkaline pectase PLA is characterized in that, its aminoacid sequence is shown in SEQ ID NO.1.
2. an alkaline pectase PLA is characterized in that, its aminoacid sequence is shown in SEQ ID NO.2.
3. an alkaline pectase gene plA is characterized in that, coding claim 2 or 3 described polygalacturonase PLA.
4. polygalacturonase gene plA as claimed in claim 3 is characterized in that its base sequence is shown in SEQ ID NO.3.
5. the recombinant vectors that comprises claim 3 or 4 said polygalacturonase gene plA.
6. the recombinant bacterial strain that comprises claim 3 or 4 said polygalacturonase gene plA.
7. a method for preparing polygalacturonase is characterized in that, may further comprise the steps:
1), gets recombinant bacterial strain with claim 5 recombinant vectors transformed host cell;
2) cultivate recombinant bacterial strain, induce the reorganization polygalacturonase because of expression; And
3) reclaim the also expressed polygalacturonase PLA of purifying.
8. method as claimed in claim 7 is characterized in that, said host cell is intestinal bacteria, yeast, genus bacillus or lactobacillus spp.
9. the application of claim 1 or 2 described polygalacturonase PLA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010609222 CN102559638B (en) | 2010-12-17 | 2010-12-17 | Alkaline pectinase poly lactic acid (PLA) and gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010609222 CN102559638B (en) | 2010-12-17 | 2010-12-17 | Alkaline pectinase poly lactic acid (PLA) and gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102559638A true CN102559638A (en) | 2012-07-11 |
| CN102559638B CN102559638B (en) | 2013-06-05 |
Family
ID=46406246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010609222 Active CN102559638B (en) | 2010-12-17 | 2010-12-17 | Alkaline pectinase poly lactic acid (PLA) and gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102559638B (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368856A (en) * | 2015-12-09 | 2016-03-02 | 吉林大学 | Improved specific activity mutant thermophilic thermophilic alkaline pectate lyase gene, engineering bacterium, enzyme and their application |
| CN105441467A (en) * | 2015-12-09 | 2016-03-30 | 吉林大学 | Thermophilic alkaline pectin lyase gene, engineering bacterium, thermophilic alkaline pectin lyase and application thereof |
| CN105754884A (en) * | 2016-03-23 | 2016-07-13 | 江南大学 | Strain capable of efficiently expressing alkaline pectinase and application of strain |
| CN109735524A (en) * | 2019-03-20 | 2019-05-10 | 青岛大学 | A kind of alkaline pectase and its application with thermophilic cold characteristic |
| CN112111407A (en) * | 2020-09-21 | 2020-12-22 | 天津科技大学 | Synechocystis PCC6803 strain for producing alkaline pectin lyase and construction method thereof |
| CN113913418A (en) * | 2021-11-22 | 2022-01-11 | 山东隆科特酶制剂有限公司 | Antitrypsin alkaline pectinase BPAP-11 and application thereof |
| CN114317364A (en) * | 2021-12-30 | 2022-04-12 | 中国科学院青岛生物能源与过程研究所 | Bacillus altitudinis and application thereof in production of high-stability alkaline pectinase |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1113951A (en) * | 1994-05-24 | 1995-12-27 | 中国科学院遗传研究所 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
| CN101531988A (en) * | 2009-04-24 | 2009-09-16 | 天津科技大学 | Alkaline pectinase genetic engineering bacteria and construction method thereof |
| CN101781641A (en) * | 2009-10-09 | 2010-07-21 | 天津科技大学 | Optimization method of culture conditions of alkaline pectinase gene engineering bacteria |
-
2010
- 2010-12-17 CN CN 201010609222 patent/CN102559638B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1113951A (en) * | 1994-05-24 | 1995-12-27 | 中国科学院遗传研究所 | Bacillus subtilis and producing tech. for solid alkaline degumming enzyme |
| CN101531988A (en) * | 2009-04-24 | 2009-09-16 | 天津科技大学 | Alkaline pectinase genetic engineering bacteria and construction method thereof |
| CN101781641A (en) * | 2009-10-09 | 2010-07-21 | 天津科技大学 | Optimization method of culture conditions of alkaline pectinase gene engineering bacteria |
Non-Patent Citations (1)
| Title |
|---|
| G.KOVTUNOVYCH等: "Correlation between pectate lyase activity and ability of diazotrophic Klebsiella oxytoca VN 13 to penetrate into plant tissues", 《PLANT AND SOIL》, 31 December 1999 (1999-12-31), pages 1 - 6 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105368856A (en) * | 2015-12-09 | 2016-03-02 | 吉林大学 | Improved specific activity mutant thermophilic thermophilic alkaline pectate lyase gene, engineering bacterium, enzyme and their application |
| CN105441467A (en) * | 2015-12-09 | 2016-03-30 | 吉林大学 | Thermophilic alkaline pectin lyase gene, engineering bacterium, thermophilic alkaline pectin lyase and application thereof |
| CN105441467B (en) * | 2015-12-09 | 2018-11-27 | 吉林大学 | A kind of thermo philic alkali pectin lyase enzyme gene, engineering bacteria, thermo philic alkali pectin lyase and its application |
| CN105368856B (en) * | 2015-12-09 | 2018-11-30 | 吉林大学 | A kind of saltant type thermo philic alkali pectin lyase enzyme gene, engineering bacteria, enzyme and its application than raising living |
| CN105754884A (en) * | 2016-03-23 | 2016-07-13 | 江南大学 | Strain capable of efficiently expressing alkaline pectinase and application of strain |
| CN109735524A (en) * | 2019-03-20 | 2019-05-10 | 青岛大学 | A kind of alkaline pectase and its application with thermophilic cold characteristic |
| CN112111407A (en) * | 2020-09-21 | 2020-12-22 | 天津科技大学 | Synechocystis PCC6803 strain for producing alkaline pectin lyase and construction method thereof |
| CN113913418A (en) * | 2021-11-22 | 2022-01-11 | 山东隆科特酶制剂有限公司 | Antitrypsin alkaline pectinase BPAP-11 and application thereof |
| CN113913418B (en) * | 2021-11-22 | 2023-10-13 | 山东隆科特酶制剂有限公司 | Antitrypsin alkaline pectase BPAP-11 and application thereof |
| CN114317364A (en) * | 2021-12-30 | 2022-04-12 | 中国科学院青岛生物能源与过程研究所 | Bacillus altitudinis and application thereof in production of high-stability alkaline pectinase |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102559638B (en) | 2013-06-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102559638B (en) | Alkaline pectinase poly lactic acid (PLA) and gene and application thereof | |
| Ghani et al. | Isolation and characterization of different strains of Bacillus licheniformis for the production of commercially significant enzymes | |
| Li et al. | Cloning, expression, and characterization of a cold-adapted and surfactant-stable alginate lyase from marine bacterium Agarivorans sp. L11 | |
| CN104357427A (en) | High-temperature-resistant alkaline salt-tolerant xylanase XynSL4 as well as gene and application thereof | |
| Zou et al. | Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis | |
| CN102220303B (en) | Xylanase XynAHJ3 with protease resistance and gene thereof | |
| CN107164353B (en) | Low-temperature alkaline pectin lyase and coding gene and application thereof | |
| CN101768581B (en) | Mutant enzyme L20A of L-arabinose isomerase with high D-tagatose production capacity and mutation method thereof | |
| Yuan et al. | A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry | |
| CN102719417B (en) | High-temperature resistance arabinfuranosidease Abf51B8, as well as gene and application thereof | |
| CN102363774B (en) | Beta-mannaseBA-Man5A with wide pH range, gene thereof and application of gene | |
| CN101215554A (en) | Fire resistant L-arabinose isomerase and gene sequence thereof | |
| CN104560833B (en) | A kind of basophilic micrococcus luteus and its alkalescent xylanase and the application of generation | |
| CN102839184A (en) | Recombinant ribose-5-phosphate isomerase and application thereof | |
| CN102245763A (en) | Acidothermus celluloyticus xylanase | |
| CN102399764B (en) | Alkaline pectinase PL-STR as well as gene and application thereof | |
| Irfan et al. | Cloning, purification and characterization of a cellulase-free xylanase from Geobacillus thermodenitrificans AK53 | |
| JPWO2004090127A1 (en) | Agar-degrading enzyme and its use | |
| CN103232949A (en) | Pichia pastoris engineered strain expressing 61-family glycoside hydrolase gene | |
| CN114045241A (en) | Bacillus subtilis HKS018 and application thereof in production of beta-mannase | |
| CN101892207B (en) | Low-temperature alpha-galactosidase GalA17, gene thereof and application thereof | |
| AU2021100409A4 (en) | Recombinant low-temperature catalase, recombinant vector and engineered strain thereof | |
| CN105176943A (en) | Salt-tolerant and organic solvent-tolerant low-temperature alkaline esterase EstSL3 and gene and application thereof | |
| Hassan et al. | Purification and biochemical characterization of a Ca2+-independent, thermostable and acidophilic α-amylase from Bacillus sp. RM16 | |
| Mei et al. | Purification and characterization of β-agarase from Paenibacillus sp. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: No.98, guangguba Road, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee after: Wuhan Sunhy Biology Co., Ltd. Address before: 430074, No. 5, Ling Nan Road, East Lake New Technology Development Zone, Wuhan, Hubei, China Patentee before: Wuhan Sunhy Biology Co., Ltd. |