CN104232607A - Preparation and application of mutant enzymes of bacillus thuringiensis chitinase - Google Patents

Preparation and application of mutant enzymes of bacillus thuringiensis chitinase Download PDF

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CN104232607A
CN104232607A CN201410441598.8A CN201410441598A CN104232607A CN 104232607 A CN104232607 A CN 104232607A CN 201410441598 A CN201410441598 A CN 201410441598A CN 104232607 A CN104232607 A CN 104232607A
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chitinase
mutant enzyme
chi9602
chiw50a
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倪红
孙孝文
李林
曾思泉
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Hubei University
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Abstract

The invention provides preparation and application of mutant enzymes of bacillus thuringiensis chitinase. The preparation method comprises the following steps: performing homologous modeling and molecular docking on chitinase Chi9602 genes, and selecting mutation sites; performing site-specific mutagenesis by adopting overlap extension PCR, thereby obtaining mutant enzyme genes chiw50a; recovering the mutant enzyme genes, connecting the mutant enzyme genes to an expression vector pTrcHis B through Bgl II and Pst I double digestion, and constructing plasmids pMB581; transforming the plasmids into Escherichia coli host cells, and performing IPTG inducible expression and affinity chromatography purification, thereby preparing the mutant enzymes Chi W50A. The specific enzyme activity of the mutant enzymes is improved by 62.3 percent compared with that of the original enzymes and reaches 83.46U/mg, the optimum pH value is 7.0, and the mutant enzymes also have high specific enzyme activity (54.69U/mg) at the pH value of 9.0, can be used for biological prevention and control of plant diseases and insect pests and has obvious antifungal and insecticidal synergistic effects.

Description

A kind of mutant enzyme preparation of bacillus thuringiensis chitinase and application thereof
Technical field
The present invention relates to genetically engineered field, be specifically related to a kind of preparation and biological and ecological methods to prevent plant disease, pests, and erosion application thereof of bacillus thuringiensis chitinase mutant enzyme.This mutant enzyme is that the chitinase Chi9602 that one derives from bacillus thuringiensis (Bacillus thuringiensis) is prepared after site-directed point mutation, construction of recombinant vector, carrier prokaryotic expression and purifying.
Background technology
Chitin (chitin) is extensively present in the cell wall of the shell of Crustacean, the crust of insect, nematode and ovum and fungi.Account for second in its standing stock natural polymer on earth, estimate that the resultant quantity of annual nature biotechnology can reach 1 × 10 11ton, is only second to Mierocrystalline cellulose (microorganism chitinase progress, Agriculture of Anhui science [J] (2010) 38 (22): 11685-11688).
Chitinase is can the glycosyl hydrolase of degrade chitin, is found in bacterium, plant and animal (From bacteria to human:A journey into the world of chitinases.Biotechnology Advances (2013) 31:1786 – 1795) widely.Most chitinase belongs to glycosyl hydrolase 18 and 19 family.Wherein the chitinase of glycosyl hydrolase 18 family has typical TIM-tubbiness ((β α) 8bucket) structure and " DXDXE " block.Glutaminic acid residue (E) in block can impel β (1-4) glycosidic link in chitin to rupture, and is hydrolyzed into single N-Acetyl-D-glucosamine (Structure of the D142N mutant of the family18 chitinase Chi B from Serratia marcescens and its complex with allosamidin.Biochimica et Biophysica Acta (2004) 1696:103 – 111).
Chitinase can the growth of effective Antifungi by the chitin of hydrolysis fungal cell wall.Therefore it can be applied to control fungal disease.Chitin is the peritrophic principal structural component of insect midgut.It is the barrier that insect resists medicine, but after being destroyed by chitinase, sterilant can more effectively play a role (Chitinases in biological control.Birkhauser Verlag Basel/Switzerland (1999) pp.171-184).Chitin is also the moiety of nematode and ovum thereof simultaneously.Therefore, chitinase has important purposes biological control disease fungi and plant insect.
As a kind of important biological control resource, bacillus thuringiensis (Bacillus thuringiensis is called for short BT) preparation, has been used successfully to the biological control of various pests, such as: kill bollworm and nematicide etc.And reducing the pollution of chemical insecticide and serving vital role in plant protection.Because chitinase is in the distribution difference to some extent of BT.Therefore the use range that BT chitinase can expand BT preparation is studied, and its insecticidal activity (progress of bacillus thuringiensis chitinase, microbiology circular [J] (2007) 34 (1): 143-147) can be improved further.In practical application, because insect midgut road exists alkaline environment, so the BT chitinase having alkaline chitinases activity will have more efficient insecticidal activity.
The much research about bacterium chitinase shows, rite-directed mutagenesis changes the change that amino-acid residue can cause the character of enzyme.Use the method for genetically engineered and protein engineering to carry out orthogenesis to bacterium chitinase, the mutant enzyme that chitinase specific activity improves can be obtained.This mutant enzyme preparation is suitable for desinsection and the multiple-effect biological and ecological methods to prevent plant disease, pests, and erosion such as antimycotic application more than protoenzyme.
Summary of the invention
The object of the invention is to use the method for genetically engineered and protein engineering to carry out orthogenesis to bacterium chitinase, propose a kind of bacillus thuringiensis chitinase mutant enzyme, its aminoacid sequence SEQ ID NO.1 is as follows:
Mamrsqkftllllslllflplfltnfitpnlaladspkqsqkivgyfpsagvygrnyqvadidasklthlnyafadic?wngkhgnpsthpdnpnkqtwnckesgvplqnkevpngtlvlgepwadvtksypgsgttwedcdkyarcgnfgel?krlkakyphlktiisvggwtwsnrfsdmaadektrkvfaestvaflraygfdgvdldweypgvetipggsyrpedkqn?ftlllqdvrnalnkagaedgkqylltiasgasqryadhtelkkisqildwisimtydfhggweatsnhnaalykdpndpa?antnfyvdgainvytnegvpvdklvlgvpfygrgwkscgkenngqyqpckpgsdgklaskgtwddystgdtgvyd?ygdlaanyvnkngfvrywndtakvpylynattgtfisyddnesmkyktdyiktkglsgamfwelsgdcrtspkyscs?gpklldtlvkellggpinqkdtepptnvknivvtnknsnsvqlnwtastdnvgvteyeitageekwstttnsitiknlkp?nteykfsiiakdaagnksqptaltvktdeanttppdgngtatfsvtsnwgsgynfsiiiknngtnpiknwklefdysgnlt?qvwdskissktnnhyvitnagwngeippggsitiggagtgnpaellnavisen
It is characterized in that theoretical molecular is 74.45 kDa, belong to glycosyl hydrolase the 18th family, by chitinase catalytic domain CCD (Lys42-Asp452), class fibronectin III type territory Fn3D (Gln504-Asn558)with chitinase binding domain CBD (Thr582-Asn646)three parts are formed.Conserved sequence is " DGVDLDWE ".Containing a signal peptide sequence SP be made up of 34 amino-acid residues in N-terminal structural domain (Met1-Ala34).
The gene order SEQ ID NO.2 that invention also provides a kind of bacillus thuringiensis chitinase mutant enzyme is as follows:
atggctatgaggtctcaaaaattcacactgctattactatccctactacttttcttacctctttttctcacaaattttattactc?caaatctcgcattagcagattcaccaaagcaaagtcaaaaaattgttgggtactttccttcggcgggcgtttacggacgtaatt?atcaagttgctgacattgatgcatcaaaacttactcaccttaactatgctttcgcggatatttgttggaatggaaaacatggaaa?cccttctactcatcctgataatccaaataaacaaacgtggaactgtaaagaatctggtgtaccattgcaaaataaagaggttcc?taatggtactctcgtactcggggaaccatgggctgatgttaccaaatcgtatcctggctcagggacaacttgggaagattgc?gataaatatgcccgttgcggaaatttcggggaactaaaacgattaaaagctaaatatcctcacttaaaaacaattatttccgttg?gtggctggacttggtctaaccgcttttctgatatggccgctgatgaaaaaacaagaaaagtatttgctgaatctacagtagcttt?tcttcgcgcatatgggtttgatggcgtagatttagactgggaatatccgggcgttgaaacgattcctggtggtagttatcgtcct?gaagacaaacaaaatttcactctccttcttcaagacgtccgaaatgctttgaataaagcaggtgctgaagatggcaaacaata?tttactaacaatcgcttcaggtgcaagccaacgctacgctgatcatacagaactaaagaaaatttctcaaatactcgattggat?tagtattatgacatatgatttccacggcggatgggaagctacttctaatcataatgcagctctatataaggatccaaatgatcca?gcagcaaatacgaatttttacgtagatggtgctataaatgtttatacaaatgaaggtgttccagtcgataaactagtattaggcg?tacccttttacggacgtggctggaaaagttgtggcaaagaaaataacggacaatatcaaccttgcaaaccaggtagtgatgg?gaaacttgcttctaaaggtacttgggatgattactctaccggtgacacaggtgtctatgattacggtgatttagcagccaattac?gttaataaaaatggttttgtacgctactggaatgacacagctaaagtaccttacttatataatgcaactacaggcacatttattag?ctacgatgacaatgaatctatgaaatacaaaacagactatataaagacgaaaggtttaagtggagcaatgttttgggaactaa?gcggagattgccgtacaagtccaaaatatagttgcagtggtccaaaattacttgatacgctagtaaaagaattacttggtgga?cctattaatcaaaaagatactgagccaccaacgaatgttaaaaacattgtagttacgaataaaaattcaaactcagttcaattaa?actggactgcatctactgataacgtaggagttacggaatatgaaattactgctggagaagagaaatggagtacaacaacaa?atagcattacaattaaaaacttaaaacctaatacggaatacaaattttcaataattgccaaagatgctgctggaaataaatcaca?acctaccgctcttactgtcaaaacggatgaagctaatacgacacctcctgatggaaatggtactgctacattttcagtcacttc?gaattggggcagcggttataacttctcgattataatcaaaaataatggaacgaaccctattaaaaattggaaattagaatttgat?tatagcggcaatttaacacaagtttgggattctaaaattagtagtaaaacaaataatcattatgtaattacgaacgcaggatgg?aatggtgaaattcctcctggtggatctattacaattggcggtgcaggaacaggtaatcctgccgaacttttaaatgccgtcatt?agcgaaaactag
The present invention is achieved in that the preparation process of the mutant enzyme of preparation bacillus thuringiensis chitinase (Chi9602) is:
(1) homology modeling and molecular docking are carried out to chitinase Chi9602 gene, choose 50 tryptophanes as mutational site.
(2) adopt Overlap extension PCR to carry out rite-directed mutagenesis, jump reaction is divided into two steps.The first step, the gene order at amplification two ends, mutational site, is had two groups of PCR primer of overlapping fragments each other; Two groups of PCR primer are carried out next round pcr amplification as template by second step, obtain the object fragment needed.Described object fragment does not comprise N end signal peptide sequence.Design four primers as follows:
Primer P1:5'-CAG aGATCTgATTCACCAAAGCAAAGT-3'
Bgl?II
Primer P2:5'-CAG cTGCAGcTAGTTTTCGCTAATGACG-3'
Pst?I
Primer P3:5'-TGGGTACTTTCCTTCGGCGGGCGTTTACGGACGTAA-3'
Primer P4:5'-GCCGAAGGAAAGTACCCAACAATTTTTTGACTTTGC-3'
With the STb gene of Bacillus thuringiensis subsp.tenebronis 9602 bacterial strain for pcr template, use primer P1 and P2 to complete first round PCR and react.After the chi9602 fragment Bgl II obtained increasing and Pst I carries out double digestion, be connected on the expression vector pTrcHis B after same Bgl II and the recovery of Pst I double digestion.By this plasmid called after pMB332.
With the pMB332 containing chi9602 fragment for template, overlap extension pcr first round PCR is adopted to react.Complete the rite-directed mutagenesis of the 50th amino acids, obtain chitinase mutant enzyme gene W50A.Described overlap extension pcr as shown in Figure 1.Obtain after mutant nucleotide sequence reclaims, use after BglII and PstI carry out double digestion, be connected to same Bgl II and Pst I double digestion reclaim after expression vector pTrcHis B on.By this plasmid called after pMB581.
(3), after plasmid sequence being checked order, compare with chi9602 gene.PMB581 plasmid is the prokaryotic expression recombinant vectors containing mutant enzyme gene, shown in its mutant enzyme called after ChiW50A, its aminoacid sequence SEQ ID NO.1.
(4) with the recombinant vectors transformed host cell comprising said mutation enzyme gene, prepare recombinant bacterial strain, wherein in recombinant vectors, prokaryotic expression carrier is pTrcHis B, and host cell is intestinal bacteria Escherichia coli TOP10.
(5) cultivate recombinant bacterial strain, induced mutation enzyme ChiW50A expresses.
(6) the mutant enzyme ChiW50A after expression is reclaimed and adopts affinitive layer purification to prepare mutant enzyme ChiW50A.
After tested, the biological activity of mutant enzyme ChiW50A of the present invention, apparently higher than chitinase Chi9602, is shown in accompanying drawing 3.This mutant enzyme ChiW50A comparatively protoenzyme Chi9602 specific activity improves 62.3%.
By the biological pest control of mutant enzyme ChiW50A of the present invention for plant, there is obvious synergism.
Adopt face-off method to measure mutant enzyme ChiW50A to the Inhibition test of rice sheath blight disease bacteria growing, result illustrates that it has obvious restraining effect to rice sheath blight disease bacteria growing, sees accompanying drawing 4.
Brilliant for YBT-1520 spore containing mutant enzyme ChiW50A mixture is used for killing bollworm there is synergism, see enforcement table 1.
Brilliant for YBT-020 spore containing mutant enzyme ChiW50A mixture is used for nematicide there is synergism.See accompanying drawing 5.
The present invention has a significant effect.Mutant enzyme ChiW50A of the present invention improves a lot than the biological activity of protoenzyme Chi9602, is used on biological pest control, has good synergism.
Accompanying drawing explanation
Fig. 1 Overlap extension PCR schematic diagram.
The rite-directed mutagenesis of Fig. 2 chitinase gene Chi9602, the structure of prokaryotic expression carrier and expression transform.
(a) pTrcHisB; (b) PCR primer agarose electrophoresis; (c) recombinant plasmid pMB581;
D () pMB581 double digestion is verified; (e) intestinal bacteria TOP10; (f) SDS-PAGE (swimming lane M1: nucleic acid Maker 1; Swimming lane M2: nucleic acid Maker 2; Swimming lane M3: albumen Maker 3; Swimming lane 1:PCR product; Swimming lane 2:Bgl II single endonuclease digestion; Swimming lane 3:Pst I single endonuclease digestion; Swimming lane 4:Bgl II/Pst I double digestion; Swimming lane 5:MB581; Swimming lane 6:MB581+IPTG; The mutant enzyme ChiW50A of swimming lane 7:Ni column purification)
Fig. 3 chitinase Chi9602 and mutant enzyme ChiW50A zymologic property.
A () differing temps is on the impact of the chitinase Chi9602 of purifying and mutant enzyme ChiW50A enzyme specific activity.
B () different pH is on the impact of the chitinase Chi9602 of purifying and mutant enzyme ChiW50A enzyme specific activity.
The mutant enzyme ChiW50A of Fig. 4 purifying suppresses rice sheath blight disease bacteria growing.
A () PBS contrasts; (b) 2.5 mutant enzyme ChiW50A of μ g;
(c) 5.0 μ g mutant enzyme ChiW50A; (d) 7.5 μ g mutant enzyme ChiW50A.
The mutant enzyme W50A of the brilliant mixture of Fig. 5 YBT-020 born of the same parents and preparation is composite kills L4 phase Caenorhabditis elegans N2 synergism.
Embodiment
Below in conjunction with specific examples, detailed specific description is done to the present invention, but protection scope of the present invention is not limited to following instance.
In this example, Bacillus thuringiensis subsp.tenebronis 9602 bacterial strain, prokaryotic expression carrier pTrcHis B and intestinal bacteria Escherichia coli TOP10 come from agricultural microorganism key lab of Hua Zhong Agriculture University.Restriction enzyme and ligase enzyme come from Invitrogen company.Chitin derives from Sigma company, and other are domestic analytical grade reagent.
In this example, LB culture medium prescription is as follows: 10.0g/L peptone, 5g/L yeast leaching powder, 10g/L sodium-chlor, pH 7.2-7.4.In above-mentioned formula, the agar powder of 2% is added during preparation solid LB media.
PDA culture medium prescription is as follows: potato 200g/L, glucose 20g/L, agar 20g/L.Nature pH.
PM liquid culture based formulas is as follows: glucose 10g, Tryptones 10g, yeast powder 2g, KH 2pO 41g, K 2hPO 41g, MgSO 47H 2o 0.02g, FeSO 47H 2o 0.02g, ZnSO 47H 2o 0.02g, MnSO 47H 2o 0.02g, is settled to 1L with distilled water.Sodium-chlor 3.0g, peptone 2.5g, agar powder 17g, is settled to 975mL with distilled water, autoclaving, when its temperature drops to 55 DEG C, adds 1mL 1MCaCl 2, 1mL 1M MgSO 4, 1mL 5mgmL -1cholesterol (with anhydrous alcohol solution), 25mL 1M potassium phosphate buffer (KH 2pO 4108.3g, K 2hPO 435.6g, is settled to 1L with distilled water.
Damping fluid I: 0.5M NaH 2pO 419ml, 0.5M Na 2pO 481ml, NaCl 29.3g, adds water and is settled to 1L.
Damping fluid II: 0.5M NaH 2pO 419ml, 0.5M Na 2hPO 481ml, NaCl 29.3g and imidazoles 34g, is settled to 1L.
PBS damping fluid: 8g sodium-chlor, 0.2g Repone K, 1.44g Sodium phosphate dibasic, 0.24g potassium primary phosphate, distilled water is settled to 1L.
The preparation method of the brilliant mixture of bacillus thuringiensis YBT-1520 born of the same parents: YBT-1520 is inoculated in PM liquid nutrient medium 28 DEG C of 200rpm shaking tables and cultivates 3 days, 8000rpm collected by centrifugation, the brilliant mixture of YBT-1520 born of the same parents is made in lyophilize.
The preparation method of the brilliant mixture of bacillus thuringiensis YBT-020 born of the same parents: YBT-020 is inoculated in PM liquid nutrient medium 28 DEG C of 200rpm shaking tables and cultivates 3 days, 8000rpm collected by centrifugation, the brilliant mixture of YBT-020 born of the same parents is made in lyophilize.
Above-mentioned bacillus thuringiensis YBT-1520 is that agricultural microorganism key lab of Hua Zhong Agriculture University is separated the bacterial strain lepidopterous insects such as bollworm, small cabbage moth to high poison.
Above-mentioned bacillus thuringiensis YBT-020 is that agricultural microorganism key lab of Hua Zhong Agriculture University is separated bacterial strain nematode to high poison.
The molecule experiments method do not elaborated in this example, with reference to " molecule experiments guide " third edition, or reference reagent box specification sheets carries out.
It is as follows that embodiment 1 prepares directed mutagenesis method:
Carry out homology modeling and molecular docking with the Bacillus thuringiensis subsp.tenebronis 9602 bacterial strain chitinase Chi9602 gene obtained, and mutational site is designed to 50 tryptophanes and replaces with L-Ala.Relate to primer as follows:
Primer P1:5'-CAG aGATCTgATTCACCAAAGCAAAGT-3'
Bgl?II
Primer P2:5'-CAG cTGCAGcTAGTTTTCGCTAATGACG-3'
Pst?I
Primer P3:5'-TGGGTACTTTCCTTCGGCGGGCGTTTACGGACGTAA-3'
Primer P4:5'-GCCGAAGGAAAGTACCCAACAATTTTTTGACTTTGC-3'
With the STb gene of Bacillus thuringiensis subsp.tenebronis 9602 bacterial strain for template, use primer P1 and P2, complete PCR reaction.It is as follows that this PCR reacts actual conditions:
Circulating system is as follows:
After the chi9602 fragment Bgl II obtained increasing and Pst I carries out double digestion, be connected on the expression vector pTrcHis B after same Bgl II and the recovery of Pst I double digestion.By this plasmid called after pMB332.
With the pMB332 containing chi9602 fragment for template, adopt overlap extension pcr to complete the rite-directed mutagenesis of the 50th amino acids, obtain chitinase mutant enzyme gene.Chitinase mutant enzyme gene order is as shown in SEQ ID NO.2.Described overlap extension pcr as shown in Figure 1.Obtain after mutant nucleotide sequence reclaims, use after Bgl II and Pst I carries out double digestion, be connected on the expression vector pTrcHis B after reclaiming with Bgl II and Pst I double digestion.By this plasmid called after pMB581.And after sequence is checked order, compare with chi9602 gene.PMB581 plasmid is the prokaryotic expression carrier containing mutant enzyme gene, its mutant enzyme called after ChiW50A expressed, shown in aminoacid sequence SEQ ID NO.1.
Prokaryotic expression carrier pMB581 containing mutant enzyme ChiW50A gene is used and is converted in host cell E. coli Escherichia coli TOP10.Obtain recombinant bacterial strain MB581
Picking recombinant bacterium list colony inoculation is (containing ammonia benzyl microbiotic) in 5ml LB substratum, 37 DEG C, 155rpm/min shaking table overnight incubation.Inoculum size by 1% is transferred (containing ammonia benzyl microbiotic) in 200ml LB substratum, 37 DEG C, and it is thin to bacterium liquid that about 1.5h cultivated by 155rpm/min shaking table, adds 200 μ l IPTG, 37 DEG C, 155rpm/min inducing culture 6h.
The affinitive layer purification of chitinase mutant enzyme ChiW50A: 4 DEG C, the centrifugal 5min of 6000rpm/min collects thalline, with 20ml PBS (pH7.4) suspension thalline, is dispensed in the centrifuge tube of 50ml.The centrifugal 20min of 12000rpm/min after pressure breaking cell, supernatant liquor is for subsequent use.Get 2ml Ni post damping fluid I rinse three times, add cytoclasis centrifuged supernatant, 4 DEG C in conjunction with 30min.Add in Filter column in conjunction with liquid, with the damping fluid II wash-out foreign protein containing imidazoles 10mM.With the damping fluid II wash-out target protein containing concentration imidazoles 250mM/L.
Bacillus thuringiensis chitinase Chi9602 gene successfully passes Overlap extension PCR and carries out rite-directed mutagenesis, the structure of prokaryotic expression carrier and Expression and purification.Obtain the 74.5kDa expression product conformed to theoretical molecular size, i.e. mutant enzyme ChiW50A.The results are shown in accompanying drawing 2.
The enzyme property of chitinase Chi9602 and mutant enzyme ChiW50A.
Chitinase enzyme specific activity measures: the chitinase solution adding 500 μ L in 1.5mL centrifuge tube, add the tobacco brown spot pathogen solution of 200 μ L2.5% again, PBS (pH7.4) complements to 1mL, and blank 500 μ LPBS (pH7.4) replace the chitinase solution of purifying.37 DEG C of water-bath 1h, after cooling, the centrifugal 5min of 12000rpm/min, gets 1mL and resets and add into 1mL DNS, boiling water bath 10min, measures the light absorption value at 535nm place after cooling.The enzyme work of a unit is defined as 1h at 37 DEG C and produces the enzyme amount of 1 μm of ol NAG.
Chitinase stability test: respectively under different reaction system conditions, (pH is 3,4,5,6,7,8 and 9; Temperature is 25 DEG C, 30 DEG C, 35 DEG C, 37 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 70 DEG C and 80 DEG C) measure chitinase enzyme specific activity.
Test mutant enzyme ChiW50A equally, test result is shown in accompanying drawing 3.Chitinase Chi9602 mutant enzyme ChiW50A optimal reactive temperature is 37 DEG C as seen from the figure.Chitinase Chi9602 mutant enzyme ChiW50A optimal reaction pH is 7.0.This mutant enzyme ChiW50A comparatively protoenzyme Chi9602 specific activity improves 62.3%.When pH 9.0, still there is high enzyme specific activity (54.69U/mg).To there is the effect of good degrade chitin in insect gut alkaline environment.
The compound method of 2.5% described tobacco brown spot pathogen solution is as follows: 2 grams of chitins (powder) are dissolved with the concentrated hydrochloric acid 45mL of precooling, magnetic stirring apparatus are stirred, now yellowly.After stirring 2h, put into 4 degrees Celsius, refrigerator, leave standstill 24h.Hydrochloric acid mixed solution is joined the alcohol (precooling is good) of 50% of 300mL, stirring 2h, is now opaque milky white solution, 4000 leave the heart 20 minutes, see milky white precipitate, repeatedly wash about pH to 6.5 with distilled water, or add water in centrifuge tube, stir, centrifugal.Finally add 40mL distilled water 115 DEG C, preserve after 15min autoclaving.
The preparation of described DNS solution: take 6.3g 3,5-dinitrosalicylic acid, be dissolved in the NaOH solution of 262ml 2mol/L, again this solution is mixed with the hot water that 500ml contains 182g Seignette salt, add 5g re-distilled phenol and 5g S-WAT, fully stir, be settled to 1L after cooling.
Face-off method is adopted to measure the Inhibition test of mutant enzyme ChiW50A to rice sheath blight disease bacteria growing as follows:
Cut-off footpath is that the Rhizoctonia solani Kuhn bacterium block of 5mm is inoculated in the dull and stereotyped central authorities of potato dextrose agar (PDA), cultivate after 2 days for 28 DEG C, in distance, sterilized filter paper is placed at 2cm place, mycelia edge, mutant enzyme ChiW50A and PBS of preparation contrast dropped in respectively on the filter paper of surrounding, wherein (a) PBS contrasts; (b) 2.5 mutant enzyme ChiW50A of μ g; (c) 5.0 μ g mutant enzyme ChiW50A; (d) 7.5 μ g mutant enzyme ChiW50A.28 DEG C were continued cultivation after 1 day, observed mutant enzyme ChiW50A to the suppression situation of Rhizoctonia solani Kuhn mycelial growth.See accompanying drawing 4.Chitinase Chi9602 mutant enzyme ChiW50A has obvious restraining effect to Rhizoctonia solani Kuhn at PDA grow on plates as seen from the figure.
The brilliant mixture of mutant enzyme ChiW50A and YBT-1520 born of the same parents is composite to kill raw survey of bollworm second instar larvae and tests
As follows:
Take the brilliant mixture of 2g YBT-1520 born of the same parents, add and prepare mutant enzyme ChiW50A, suspend with the PBS of 10mM, make mutant enzyme ChiW50A final concentration be 35 μ g/mL, population of samples amasss as 20mL.Contrast adds BSA, and its concentration is 35 μ g/mL; Blank only containing YBT-1520, sample volume is 20mL.Sample presentation measures.See attached list 1.Result shows: when mutant enzyme ChiW50A and YBT-1520 spore crystalline substance mixture is composite with 2000:1, YBT-1520 kills tiring of bollworm second instar larvae and improves 1145 units, reaches 4211 units.
Table 1
a: IU.mL -1; b: chitinase concentration (0.35 μ g.mL -1).
The brilliant mixture of mutant enzyme ChiW50A and YBT-020 born of the same parents is composite kills that L4 phase Caenorhabditis elegans N2 is raw to be surveyed
Test as follows:
First by Caenorhabditis elegans synchronization, then cultivate on NGM solid medium, obtain the larva of L4 phase, wash low suspension with M9 buffer, make nematode concentration be 40/5 μ L.With PBS, brilliant for YBT-020 spore mixture is mixed with identical 4 groups, often organizes 5 concentration gradients: 2mg/mL, 5mg/mL, 20mg/mL, 30mg/mL and 40mg/mL.To in the brilliant mixture of first group of spore, add the chitinase Chi9602 of purifying, make the concentration of chitinase be 40 μ g/mL; In second group of brilliant mixture of spore, add the mutant enzyme ChiW50A of purifying, make the concentration of mutant enzyme be 40 μ g/mL; In the 3rd group of brilliant mixture of spore, add BSA, make its concentration be 40 μ g/mL, in contrast; 4th group only containing the brilliant mixture of YBT-020 spore.5th group only adds mutant enzyme ChiW50A.Use 96 orifice plates, in every hole, add 100 μ L samples, 5 μ L 10mM FUdR (suppressing nematode to lay eggs), the L4 phase larva that 5 μ L suspend with M9 damping fluid, the OD that 40 μ L suspend with M9 buffer 600nmvalue is the E.coli OP50 of 0.6,50 μ L M9 buffer.Each sample does three repetitions, in 25 DEG C, after 3d, observes statistics nematode death condition under stereoscopic microscope.The solution of 200 μ L is contained in each hole, and wherein 100 μ L are desinsection sample, is equivalent to diluted sample one times, so the final concentration of chitinase is 20 μ g/mL, the concentration of the brilliant mixture of spore becomes: 1mg/mL, 2.5mg/mL, 10mg/mL, 15mg/mL, 20mg/mL.See accompanying drawing 5.Result shows: when the brilliant mixture 500:1 of chitinase Chi9602 mutant enzyme ChiW50AYBT-020 spore is composite, the mortality ratio that YBT-020 kills the Caenorhabditis elegans N2 of L4 phase significantly improves.

Claims (4)

1. the mutant enzyme W50A of a bacillus thuringiensis chitinase Chi9602, it is characterized in that: the aminoacid sequence of described mutant enzyme is as shown in SEQ ID NO.1, described mutant enzyme theoretical molecular is 74.45kDa, belongs to glycosyl hydrolase the 18th family, by chitinase catalytic domain CCD (Lys42-Asp452), class fibronectin III type territory Fn3D (Gln504-Asn558)with chitinase binding domain CBD (Thr582- asn646)three parts are formed; Conserved sequence is " DGVDLDWE ", containing a signal peptide sequence SP be made up of 34 amino-acid residues in N-terminal structural domain (Met1-Ala34).
2. a mutant enzyme W50A of bacillus thuringiensis chitinase Chi9602, is characterized in that: described coding gene sequence chiw50a is as shown in SEQ ID NO.2; Described mutant enzyme mrna length is 2031bp.
3. a preparation method of the mutant enzyme W50A of bacillus thuringiensis chitinase Chi9602, is characterized in that adopting following steps:
(1) homology modeling and molecular docking are carried out to chitinase Chi9602 gene order, choose 50 tryptophanes as mutational site;
(2) adopt Overlap extension PCR to carry out rite-directed mutagenesis, jump reaction is divided into two steps.The first step, the gene order at amplification two ends, mutational site, is had two groups of PCR primer of overlapping fragments each other; Two groups of PCR primer are carried out next round pcr amplification as template by second step, and obtain the object fragment needed, described object fragment does not comprise N end signal peptide sequence; Design four primers as follows:
Primer P1:5'-CAG aGATCTgATTCACCAAAGCAAAGT-3'
Bgl?II
Primer P2:5'-CAG cTGCAGcTAGTTTTCGCTAATGACG-3'
Pst?I
Primer P3:5'-TGGGTACTTTCCTTCGGCGGGCGTTTACGGACGTAA-3'
Primer P4:5'-GCCGAAGGAAAGTACCCAACAATTTTTTGACTTTGC-3'
With the STb gene of Bacillus thuringiensis subsp.tenebronis 9602 bacterial strain for pcr template, use primer P1 and P2 to complete first round PCR to react, after the chi9602 fragment Bgl II obtained increasing and Pst I carries out double digestion, be connected on the expression vector pTrcHis B after same Bgl II and the recovery of Pst I double digestion.By this plasmid called after pMB332;
With the pMB332 containing chi9602 fragment for template, adopt overlap extension pcr first round PCR to react, complete the rite-directed mutagenesis of the 50th amino acids, obtain chitinase mutant enzyme gene W50A; Obtain after mutant nucleotide sequence reclaims, use Bgl II and Pst I to carry out double digestion, be connected to same Bgl II and Pst I double digestion reclaim after expression vector pTrcHis B on, by this plasmid called after pMB581;
(3) after checking order to plasmid sequence, compare with chi9602 gene, pMB581 plasmid is the prokaryotic expression recombinant vectors containing mutant enzyme gene, shown in its mutant enzyme called after ChiW50A, its aminoacid sequence SEQ ID NO.1;
(4) with the recombinant vectors transformed host cell comprising said mutation enzyme gene, prepare recombinant bacterial strain, wherein in recombinant vectors, prokaryotic expression carrier is pTrcHis B, and host cell is intestinal bacteria Escherichia coli TOP10;
(5) cultivate recombinant bacterial strain, induced mutation enzyme ChiW50A expresses;
(6) the mutant enzyme ChiW50A after expression is reclaimed and adopts affinitive layer purification to prepare mutant enzyme ChiW50A.
4. a purposes of the mutant enzyme W50A of bacillus thuringiensis chitinase Chi9602, is applied to the biological pest control of plant, it is characterized in that: (1) has obvious restraining effect for Rhizoctonia solani Kuhn; (2) brilliant mixture is composite with bacillus thuringiensis YBT-1520 spore kills bollworm and has synergism; (3) with the composite nematicide of the brilliant mixture of bacillus thuringiensis YBT-020 spore, there is notable synergistic effect.
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CN107832577A (en) * 2017-10-30 2018-03-23 中国农业大学 A kind of method for screening the inhibitor of chitinase OfCht I
CN109486794A (en) * 2018-12-06 2019-03-19 江南大学 A kind of chitinase mutant that enzyme activity improves
CN110894499A (en) * 2019-11-29 2020-03-20 湖北文理学院 Gene site-directed mutagenesis method
CN114350640A (en) * 2019-12-13 2022-04-15 中国科学院天津工业生物技术研究所 Construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant
CN114807095A (en) * 2022-05-05 2022-07-29 湖北大学 Chitinase mutant and application thereof
CN115725549A (en) * 2022-08-10 2023-03-03 中国海洋大学 Chitinase mutants

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CN107832577A (en) * 2017-10-30 2018-03-23 中国农业大学 A kind of method for screening the inhibitor of chitinase OfCht I
CN107832577B (en) * 2017-10-30 2021-07-13 中国农业大学 Method for screening chitinase OfChtI inhibitor
CN109486794A (en) * 2018-12-06 2019-03-19 江南大学 A kind of chitinase mutant that enzyme activity improves
CN109486794B (en) * 2018-12-06 2020-06-09 江南大学 Chitinase mutant with improved enzyme activity
CN110894499A (en) * 2019-11-29 2020-03-20 湖北文理学院 Gene site-directed mutagenesis method
CN114350640A (en) * 2019-12-13 2022-04-15 中国科学院天津工业生物技术研究所 Construction of recombinant bacterium for efficiently expressing chitinase and screening of high-enzyme-activity mutant
CN114350640B (en) * 2019-12-13 2023-09-08 中国科学院天津工业生物技术研究所 Construction of recombinant bacterium for expressing chitinase and screening of high-enzyme activity mutant
CN114807095A (en) * 2022-05-05 2022-07-29 湖北大学 Chitinase mutant and application thereof
CN114807095B (en) * 2022-05-05 2023-09-19 湖北大学 Chitinase mutant and application thereof
CN115725549A (en) * 2022-08-10 2023-03-03 中国海洋大学 Chitinase mutants

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