CN102559638B - 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
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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 intercellular substance, plays a part in the cell tissue of plant " bonding ".The elementary cell that consists of pectin substance is the galacturonic acid of different gamma values, 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 with rhamnosyl, pectinose, semi-lactosi, wood sugar and other carbohydrates, et al.Crit Rev Food Sci Nutr.37:47-73,1997).
Polygalacturonase (pectinases) is the general name of the plurality of enzymes of decompose pectin matter.Chemical structure more complicated due to pectin substance. polygalacturonase that can its decomposition of catalysis is also of a great variety.Polygalacturonase can be divided into according to the reaction property that decomposes glycosidic link or degraded Substrate's properties: 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 optimal pH.Studying at present and use more is acid pectase, and its enzyme effect optimal pH is in the slant acidity scope, is mainly used in that fruit is squeezed the juice and juice clarification.Alkaline pectase (Alkaline pectinase) refers to have greater activity in alkaline range polygalacturonase generally refers to polygalacturonic acid lyase (Polygalacturonate lyase is called for short PGL) more.Its optimal pH can disconnect 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 the 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 focus (the Jayani RS of people's research, et al.Process Biochemistry 40:2931-2944,2005).
Alkaline pectase is many produces (Hayashi K by the Bacillaceae in bacterium and Rhodopseudomonas, et al.Phytochemistry 45:1359-1363,1997), actinomycetes (Beg QK, et al.J Ind MicrobiolBiotechnol 24:396-402,2000) and the fungi alkaline pectase report (Baracat et al.J IndMicrobiol 11:139-142,1993) is 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 optimal pH of alkaline pectase et al.FEMS Microbiol Lett.145:295-298,1996) is 11.0.The optimal pH that derives from Bacillus alcalophillusNTT33 polygalacturonase is 9.5 (Zhai C, et al.Enzyme and Microbial Technology.33:173-178,2003).The optimal pH 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, due to Cost Problems, 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 is to provide the alkaline pectase that derives from above-mentioned bacterial strains.
A further object of the present invention is to provide the gene of above-mentioned polygalacturonase.
A further object of the present invention is to provide the recombinant vectors that comprises above-mentioned polygalacturonase.
A further object of the present invention is to provide the recombinant bacterial strain that comprises above-mentioned polygalacturonase gene.
A further object of the present invention is to provide a kind of method for preparing alkaline pectase.
A further object of the present invention is to provide above-mentioned alkaline pectin application of enzymes.
The present invention's technical problem at first to be solved is to overcome the deficiencies in the 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 produces is suitable for extracting in weaving, papermaking, washing composition, vegetable fibre processing, tea and coffee fermentation, oil, use in containing the industries such as pectin Industrial Wastewater Treatment and biotechnology.
The present invention has obtained a kind of alkaline pectase PLA from above-mentioned bacterial strains, its aminoacid sequence is as 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 ripe alkaline pectase PLA is 63.8kDa, and its aminoacid sequence is as 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, is possessing high reactivity under low temperature (0 ℃), normal temperature and higher temperatures, all has the characteristics such as high reactivity, protease inhibitor degraded in alkaline and neutral scope.The polygalacturonase that the klebsiella Klebsiella sp.Y1 that the present invention screens produces, its optimal pH 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; Specific activity 797U/mg; 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 by PCR this alkaline pectase gene plA, gene plA total length 1710bp, the complete genome sequence of this enzyme is as 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.Find that with the nucleotide sequence consistence that derives from the pectin lyase of Enterobacter sp.638 be 78.9%, consensus amino acid sequence is 85.8%.For this reason the transformation of gene and in various heterologous gene expression systems high efficient expression good genetic material is provided.Illustrate that PLA is a kind of new alkaline pectase.This is also to clone for the first time to obtain the alkaline pectase gene from klebsiella.
The present invention also provides the restructuring 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, comprises the following steps:
1) with above-mentioned recombinant vectors transformed host cell, get recombinant bacterial strain; Preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus.
2) cultivate recombinant bacterial strain, induce the expression of restructuring polygalacturonase; And
3) reclaim the also expressed polygalacturonase of purifying.
The present invention also provides above-mentioned alkaline pectin application of enzymes.
The optimal pH of alkaline pectase of the present invention is 9.0, is having the enzyme more than 80% to live between pH7.5-10.0.Have good pH stability, stable between pH5.0-10.5; Specific activity 797U/mg; Fabulous Sumizyme MP resistance and be easy to industrial fermentation production.As a kind of novel zymin, extract in weaving, papermaking, washing composition, vegetable fibre processing, tea and coffee fermentation, oil, contain the industries such as pectin Industrial Wastewater Treatment and biotechnology using value is 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 restructuring polygalacturonase of purifying.
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.
Fig. 5 pectin Thermostability of the present invention.
The metal 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 is all domestic reagent (all can buy from common biochemical reagents company and obtain).
3, substratum:
(1) induce 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) Escherichia coli culture medium LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).
Illustrate: make the experimental methods of molecular biology illustrate in following examples, all carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book, perhaps carry out according to test kit and product description.
The clone of embodiment 1 klebsiella polygalacturonase encoding gene 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 50mg bacterium mud and add 500 μ L sterile water wash, the thalline of centrifuging and taking precipitation.The thalline of precipitation is resuspended in 500 μ L N,O-Diacetylmuramidase mixed solutions, 37 ℃ of incubation 1h, then add enzyme liquid 100 μ L and continue insulation 30min in 45 ℃, after transparent to bacterium liquid, 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 liquor is used respectively equal-volume phenol, phenol: chloroform, chloroform is extracting successively.Get the Virahol normal temperature precipitation 10min that upper solution adds 0.6-1 times of volume.The centrifugal 15min of 16000rpm.Precipitation is cleaned with 70% ethanol, and is slightly centrifugal, and precipitation is drained rear use 30 μ L sterilized water dissolvings, and-20 ℃ save backup.
Find two conserved amino acid sequence conserved sequence GLF (L) Y (L) WGGH and RQFGPE according to the sequence alignment result of the pectin lyase of delivering, and design 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), carry out pcr amplification take total DNA of klebsiella Y1 as template.The PCR reaction parameter is: 95 ℃ of 5min; 94 ℃ of 30sec, 53-48 ℃ of 30sec (wherein after each circulation, the renaturation temperature descends 0.5 ℃), 72 ℃ of 1min, then 10 circulations enter second cycling program: 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 Primers 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 every 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 (upstream 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 by reverse TAIL-PCR, amplification obtains checking order after product reclaims.
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.Result shows, this gene is the gene fragment of a long 1710bp, encode 569 amino acid and a terminator codon.N end 22 amino acid are its signal peptide sequence.Zymoprotein is 85.8% with the consensus amino acid sequence 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 gene 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 the dilution enzyme liquid that 100 μ L are suitable, 900 μ L substrates, and reaction 10min adds 1.5mL DNS termination reaction, boiling water boiling 5min.Cooling rear 540nm measures the OD value.1 enzyme unit (U) that lives is defined as the enzyme amount that under given condition per minute discharges 1 μ mol reducing sugar.
The preparation of embodiment 3 restructuring 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 after double digestion, and preparation BL21 (DE3) competent cell transforms Host Strains 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 respectively in 3mL LB (being added with the penbritin of 100 μ g/mL) nutrient solution 37 ℃ of quick oscillation overnight incubation.Get respectively in the 10mL LB nutrient solution (1% inoculum size) that 100 μ L incubated overnight liquid add the penbritin that contains 100 μ g/mL, 30 ℃ of shaking culture are 2-3h (OD approximately
600Reach 0.6-0.8) after add the inductor IPTG of final concentration 0.8mmol/L, 8h are cultivated in 30 ℃ of 180rpm concussions.Nutrient solution 12, the centrifugal 5min of 000rpm collects respectively cleer and peaceful precipitation on substratum, and cell precipitation is resuspended with the damping fluid of pH9.0, and after ultrasonication 12, the centrifugal 10min of 000rpm, collection supernatant are total protein of cell (CTP).Detect respectively cleer and peaceful CTP enzyme work on substratum by above-mentioned enzyme activity determination method.Recombinase mainly is secreted into supernatant as a result, and the enzymic activity in supernatant is 0.12U/ml.Through the SDS-PAGE electrophoresis detection, can see an obvious specific band (Fig. 1, LANE2, LANE3).
Intestinal bacteria induce the polygalacturonase PLA of generation through Ni-NTA column purification (NEB), and specific activity is increased to 797U/mg.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 optimal pH under different pH.Damping fluid used is the glycine-NaOH damping fluid of the 0.1mol/L of the McIlvaine damping fluid of 0.1mol/L of pH 2.0～8.0 and pH 9.0～11.0.The polygalacturonase PLA of purifying is in the buffer system of different pH, and the pH adaptive result (Fig. 2) of measuring under 50 ℃ shows: the suitableeest action pH of PLA is 9.0, in the enzyme work that is having between pH7.5-10.0 80% or more.Enzyme liquid is processed 1h in the damping fluid of different pH values, then measure enzymic activity with the pH stability of studying enzyme under 37 ℃.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 at the glycine of the 0.1mol/L that is determined at pH9.0 of optimum temperuture-NaOH buffer solution system and different temperature (0～90 ℃).Enzyme reaction optimum temperuture measurement result (Fig. 4) shows, 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.Temperature higher than 60 ℃ time residue live less than 2% enzyme.
Measure polygalacturonase and be incubated respectively different time mensuration enzyme activity under 50 ℃ and 60 ℃, draw the Thermostability curve.The enzyme residue approximately 70% of living after processing 10min under 50 ℃, better heat stability (Fig. 5).
The impact that different metal ion chemistry reagent is lived on the PLA enzyme is determined as follows:
The different metal ions and the chemical reagent that add different concns in enzymatic reaction system are studied it to the impact of enzymic activity, and various material final concentrations are 1 and 5mmol/L.Measure enzymic activity under 50 ℃, pH9.0 condition.Result (Fig. 6) shows, the Cu of lower concentration
2+With beta-mercaptoethanol and 1mmol/L Co
2+Polygalacturonase PLA is had obvious 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 is lived not to be affected or certain activation is arranged.
Polygalacturonic acid (PGA) with different concns (1-20mg/mL) is substrate, in the glycine of pH 9.0-NaOH reaction system, measures enzymic activity under 50 ℃, calculates its K under 50 ℃
mValue.After measured, this polygalacturonase apparent K take PGA as substrate 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.Take PLA to the vigor of PGA as 100%, be that substrate is measured the polygalacturonase relative activity and is respectively 92%, 23.9% and 4.5% with 34%, 70% and 85% citrus pectin.
Claims (9)
1. an alkaline pectase PLA, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.1.
2. an alkaline pectase PLA, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2.
3. an alkaline pectase gene plA, is characterized in that, the described polygalacturonase PLA of coding claim 1 or 2.
4. polygalacturonase gene plA as claimed in claim 3, is characterized in that, its base sequence is as shown in SEQ ID NO.3.
5. the recombinant vectors that comprises the described polygalacturonase gene of claim 3 or 4 plA.
6. the recombinant bacterial strain that comprises the described polygalacturonase gene of claim 3 or 4 plA.
7. a method for preparing polygalacturonase, is characterized in that, comprises the following steps:
1) with claim 5 recombinant vectors transformed host cell, get recombinant bacterial strain;
2) cultivate recombinant bacterial strain, induce the restructuring 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, described host cell is intestinal bacteria, yeast, genus bacillus or lactobacillus.
9. the described polygalacturonase PLA of claim 1 or 2 is used for the application of depolymerized pectin.
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| 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 |
| 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 |
| CN105754884A (en) * | 2016-03-23 | 2016-07-13 | 江南大学 | Strain capable of efficiently expressing alkaline pectinase and application of strain |
| CN109735524B (en) * | 2019-03-20 | 2020-11-06 | 青岛大学 | Alkaline pectinase with psychrophilic property and application thereof |
| 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 |
| 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 |
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