CN109439640B

CN109439640B - Fumonisin degrading enzyme FumDSB and gene and application thereof

Info

Publication number: CN109439640B
Application number: CN201811171697.3A
Authority: CN
Inventors: 李中媛; 张同存; 赵军旗; 刘会会
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2018-10-09
Filing date: 2018-10-09
Publication date: 2021-07-20
Anticipated expiration: 2038-10-09
Also published as: CN109439640A

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to fumonisin degrading enzyme FumDSB, and a gene and application thereof. The invention provides a source Sphingomonadales Bacterium fumonisin degrading enzyme FumDSB, the amino acid sequence of which is shown in SEQ ID NO.1, and the invention provides a coding gene for coding the detoxifying enzyme. The fumonisin degrading enzyme can be applied to industries such as agriculture, feed, food and the like, and reduces the harm of fumonisin to the health of animals and human beings.

Description

Fumonisin degrading enzyme FumDSB and gene and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to fumonisin degrading enzyme FumDSB, and a gene and application thereof.

Background

Fumonisins (FB) are some water-soluble secondary metabolites produced by Fusarium spp under specific conditions. Fumonisins are diester compounds composed of polyhydric alcohols and tricarballylic acid, and the main active functional groups in the structure are primary amine, tricarboxylic acid, hydroxyl and aliphatic skeleton, which are closely related to the toxic effect of the primary amine, the tricarboxylic acid, the hydroxyl and the aliphatic skeleton. Research shows that fumonisins can cause renal tumor and liver cancer of rats, leukomalacia of horse (ELEM), pulmonary edema of pig (PPE) and the like, and are closely related to the pathogenesis of human esophageal cancer. To date, 28 fumonisins have been discovered and classified into four categories, FA, FB, FC and FP, and among the B series, fumonisins B1(FB1), B2(FB2) and B3(FB3) are most commonly found, with FB1 being present in natural amounts of about 70% to 80%, being the most toxic and the most polluting. The fumonisins can be removed by using physical and chemical methods, but the chemical method has large limitation, and although the content of FB1 can be reduced, a new chemical substance can be introduced to increase the potential safety hazard, and in addition, the European Union also prohibits the use of the chemical method for eliminating mycotoxins. Physical methods remove a certain amount of FB1, but require high conditions and are prone to change in food quality and flavor. The degradation of FB1 by the biodegradation method has the advantages of mild reaction, strong specificity, high specificity and wide application prospect.

The pollution of mycotoxins is increasing worldwide, which causes waste of valuable food resources and is carcinogenic to humans. Therefore, the development of the enzyme preparation for degrading the mycotoxin can well inhibit the generation of the toxin in the polluted food crops and recover the economic loss.

Disclosure of Invention

In order to degrade fumonisins by a biodegradation method, the invention provides a fumonisin degrading enzyme FumDSB and a gene and application thereof.

The invention aims to provide fumonisin degrading enzyme FumDSB.

The invention further aims to provide a gene of fumonisin degrading enzyme FumDSB.

The invention also aims to provide a recombinant expression vector containing fumonisin degrading enzyme FumDSB gene.

Still another object of the present invention is to provide a recombinant strain containing fumonisin degrading enzyme FumDSB gene.

Still another object of the present invention is to provide a method for preparing the fumonisin degrading enzyme FumDSB.

The invention also aims to provide application of the fumonisin degrading enzyme FumDSB.

According to the fumonisin degrading enzyme FumDSB of the specific embodiment of the invention, the amino acid sequence is shown in SEQ ID NO. 1:

MTLASLLLLAPAVAHAGPSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHAATGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

wherein, the enzyme gene codes 511 amino acids and a stop codon, the first 16 amino acids are signal peptides, therefore, the theoretical molecular weight of mature FB1 degrading enzyme FumDSB is 54 kDa.

The amino acid sequence of the mature fumonisin degrading enzyme FumDSB is shown in SEQ ID NO. 2:

PSVVTTDAGRLRGAVEGELDVFRGVPFAAAPIGQLRWREPQRIAPWTDIRDASKFAPACMQSGVSIPGEPAPQISEDCLYLNIWAPRHSGRAKLPVMIFIHGGGWQNGATALPLYWGDRLAQQGAVVVSVSYRLGALGFLAHPELTAESPHHTSGNYGLLDQIAALNWVQRNIAAFGGDPANVTLFGQSAGSSSIAILMASPLAKGLFHRVIGQSGGFFEPLQLAPHYELALAEKQGVAFAHSLETSTLADLRSLPPQALLTKQAASVSHPVIEPWLLPRTPFEVFSVGQQHGADILVGYNAEEGRAFFDASSVTAANFGEQLRAELGDLPPAIMAAYPFASDVEAGQARVALERDLRFGWNMWTWAKLHAATGKNAVHAYYFTHKPPFPSDSVRRNWQASHFAELWYMFDHLGQEDWQWTKFDRQIARTMSRYWVNFARNGNPNGRGLPHWPAYRTDQPLVLQIGQPITPTPEPNTGSIGVIDAVFSAVRGDT

the invention provides a genome of a gene for coding fumonisin degrading enzyme FumDSB, and the nucleotide sequence of the genome is shown in SEQ ID NO. 3:

atgacgctggcttccttgcttctcctcgctccggcagttgcacatgccggacccagtgtcgtcaccaccgacgccggccggcttcgtggagcggtcgaaggcgagcttgacgtgttcagaggcgtaccttttgcggcagcgcccattggtcaacttcgttggcgcgagcctcagcggatcgcgccttggaccgatatacgggatgccagcaaatttgccccggcatgcatgcaaagcggtgtttcaatcccgggtgagccagcgccgcagatcagcgaagattgcctatacctcaatatttgggcgccgcggcattctggtcgggcgaagctgcccgtaatgattttcattcatggcggcggttggcagaatggtgcgaccgcgctgccactctattggggcgatcggttggcgcagcaaggggctgtcgtcgtttcggtgagctaccggcttggtgcgcttggcttcctcgcccatcccgaacttaccgccgaatcaccacatcacacgtcagggaactacggcctgctcgatcaaatcgccgccctcaattgggtgcagcgcaacatcgcggcgtttgggggtgacccggcaaatgttactctatttggtcagtcggcgggttcgagttcgatcgctatcctgatggcttcaccgttggccaagggcctgtttcatcgcgtcatcggccaaagtggcggttttttcgagccgctacaactggccccgcattacgaactcgcgctcgctgaaaaacagggagttgcctttgcacattccctagaaacttccacgcttgctgatctccggtcgctccccccgcaggccttgttgacgaaacaggccgccagcgtgtcgcatcctgtaatcgaaccatggctattgccgcgaaccccgtttgaggttttctcggtagggcagcaacacggtgcagacattctcgtcggatacaatgccgaagaaggccgagcattctttgacgcttcgtcggtcactgctgcgaacttcggtgaacagcttcgcgccgaattgggcgacctgcctcctgcaatcatggcggcctacccttttgccagcgatgtcgaggcggggcaggcgcgcgttgcgctggagcgggatctgcgtttcggctggaacatgtggacctgggcaaagctccacgccgcaaccggaaaaaacgcggtccatgcctattacttcacccacaaaccaccgttcccaagcgattcagttcgaagaaattggcaggcaagccacttcgccgaactgtggtacatgtttgaccatcttggccaagaagattggcaatggacaaagtttgatcgtcaaatcgctcgcaccatgtcgcgttactgggtaaattttgcccggaacggaaatcccaacgggcgaggtcttccgcactggcctgcctatcgtaccgaccaaccgcttgtgttgcagattggccaaccgataacacccacgccggagcccaataccgggtctattggagtgatcgacgcggttttttcagcagtacgaggcgacacctga

the fumonisin degrading enzyme FumDSB gene is obtained by a full-gene synthesis method, and the full-length 1536bp of the structural gene of the fumDSB is shown by a DNA full-sequence analysis result.

The nucleotide sequence of the mature fumonisin degrading enzyme FumDPS coding gene is shown as SEQ ID NO. 4:

ggacccagtgtcgtcaccaccgacgccggccggcttcgtggagcggtcgaaggcgagcttgacgtgttcagaggcgtaccttttgcggcagcgcccattggtcaacttcgttggcgcgagcctcagcggatcgcgccttggaccgatatacgggatgccagcaaatttgccccggcatgcatgcaaagcggtgtttcaatcccgggtgagccagcgccgcagatcagcgaagattgcctatacctcaatatttgggcgccgcggcattctggtcgggcgaagctgcccgtaatgattttcattcatggcggcggttggcagaatggtgcgaccgcgctgccactctattggggcgatcggttggcgcagcaaggggctgtcgtcgtttcggtgagctaccggcttggtgcgcttggcttcctcgcccatcccgaacttaccgccgaatcaccacatcacacgtcagggaactacggcctgctcgatcaaatcgccgccctcaattgggtgcagcgcaacatcgcggcgtttgggggtgacccggcaaatgttactctatttggtcagtcggcgggttcgagttcgatcgctatcctgatggcttcaccgttggccaagggcctgtttcatcgcgtcatcggccaaagtggcggttttttcgagccgctacaactggccccgcattacgaactcgcgctcgctgaaaaacagggagttgcctttgcacattccctagaaacttccacgcttgctgatctccggtcgctccccccgcaggccttgttgacgaaacaggccgccagcgtgtcgcatcctgtaatcgaaccatggctattgccgcgaaccccgtttgaggttttctcggtagggcagcaacacggtgcagacattctcgtcggatacaatgccgaagaaggccgagcattctttgacgcttcgtcggtcactgctgcgaacttcggtgaacagcttcgcgccgaattgggcgacctgcctcctgcaatcatggcggcctacccttttgccagcgatgtcgaggcggggcaggcgcgcgttgcgctggagcgggatctgcgtttcggctggaacatgtggacctgggcaaagctccacgccgcaaccggaaaaaacgcggtccatgcctattacttcacccacaaaccaccgttcccaagcgattcagttcgaagaaattggcaggcaagccacttcgccgaactgtggtacatgtttgaccatcttggccaagaagattggcaatggacaaagtttgatcgtcaaatcgctcgcaccatgtcgcgttactgggtaaattttgcccggaacggaaatcccaacgggcgaggtcttccgcactggcctgcctatcgtaccgaccaaccgcttgtgttgcagattggccaaccgataacacccacgccggagcccaataccgggtctattggagtgatcgacgcggttttttcagcagtacgaggcgacacc

the fumonisin degrading enzyme FumDSB sequence and the amino acid sequence thereof are subjected to BLAST comparison in GenBank, and the consistency of the amino acid sequence and the amino acid sequence from sphingosine box bacterium MTA144 is 33 percent, which indicates that the fumonisin degrading enzyme FumDSB is a novel FB1 detoxification enzyme.

The invention also provides a recombinant vector containing the fumonisin degrading enzyme FumDSB, and a preferred recombinant vector is named as pET28 a-FumDSB. The fumonisin degrading enzyme FumDSB gene of the invention is inserted between the proper restriction enzyme cutting sites of the expression vector, so that the nucleotide sequence is operably connected with the expression regulation sequence. As a most preferred embodiment of the present invention, it is preferable that the detoxification enzyme gene of the present invention is inserted between EcoR I and Xhol restriction sites on the plasmid pET28a, and the nucleotide sequence is positioned and controlled downstream of the T7 promoter to obtain a recombinant large intestine expression plasmid pET28 a-FumDSB.

The invention also provides a recombinant strain containing the degrading enzyme FumDSB, and a preferable strain is Escherichia coli.

The invention also provides a method for preparing fumonisin degrading enzyme FumDSB, which comprises the following steps:

(1) transforming a host cell by using a recombinant vector containing a gene encoding fumonisin degrading enzyme FumDSB to obtain a recombinant strain;

(2) culturing the recombinant strain, and inducing fumonisin degrading enzyme FumDSB to express;

(3) separating and purifying the obtained fumonisin degrading enzyme FumDSB.

The invention also provides application of the fumonisin degrading enzyme FumDSB, in particular application of the fumonisin degrading enzyme FumDSB in degrading fumonisin B1.

The optimum temperature of fumonisin degrading enzyme FumDSB is 30 ℃, the optimum pH is 6.0, and the activity of the fumonisin degrading enzyme FumDSB is still maintained to be more than 60% within the range of pH 5.5-9. Under the optimal temperature and the optimal pH, the degradation rate of FB1 by fumDSB of the fumonisin degrading enzyme of the invention is 100%.

The fumonisin B1 fumonisin degrading enzyme with excellent property can be obtained by applying the method of the invention, and the enzyme can be applied to agriculture, feed, food and other industries, so that the harm of fumonisin B1 to the health of animals and human beings is reduced.

Drawings

FIG. 1 is a SDS-PAGE purification of fumonisin degrading enzyme FumDSB, wherein M: protein marker; 1, fumonisin degrading enzyme FumDSB;

FIG. 2 shows the temperature optimum of fumonisin-degrading enzyme FumDSB;

FIG. 3 shows the optimum pH profile of fumonisin-degrading enzyme FumDSB.

Detailed Description

Test materials and reagents

1. Bacterial strain and carrier: an escherichia coli expression vector pET28a and an escherichia coli strain Bl21(DE 3);

2. culture medium:

coli culture LB (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0).

Example 1 obtaining FumDSB Gene of fumonisin-degrading enzyme

The gene fragment derived from Sphingomonadales Bacterium fumonisin degrading enzyme FumDSB is obtained by an artificial chemical synthesis method, and endonuclease site EcoR I is introduced into the 5 'end and endonuclease site Xho I is introduced into the 3' end.

Example 2 preparation of fumonisin degrading enzyme FumDSB

Carrying out double enzyme digestion (EcoR I + Xho I) on the expression vector pET28a, simultaneously carrying out double enzyme digestion (EcoR I + Xho I) on the gene FumDSB encoding mature fumonisin degrading enzyme, cutting out the gene fragment encoding the degrading enzyme, connecting the gene fragment with the expression vector pET28a, obtaining a recombinant plasmid pET28a-FumDSB containing the degrading enzyme FumDSB, transforming the recombinant plasmid pET 28-FumDSB into escherichia coli BL21(DE3), and obtaining a recombinant escherichia coli strain BL21(DE 3)/FumDSB.

A recombinant expression vector of the fumonisin degrading enzyme gene containing a signal peptide sequence was constructed in the same manner.

Taking BL21(DE3) strain containing recombinant plasmid, inoculating into 100mL LB culture solution, shaking and culturing at 37 deg.C and 220rpm for 2-3h, and then OD₆₀₀When the concentration is 0.6-0.8, adding 1mM IPTG, inducing at 25 deg.C for 20 hr, and centrifuging at 4 deg.C to collect thallus. The supernatant was collected by ultrasonication, purified by nickel column, and SDS-PAGE showed that the recombinant detoxification enzyme was expressed in E.coli, as shown in FIG. 1, lane 1 is purified fumDSB.

Example 3 determination of the Properties of the fumonisin degrading enzyme FumDSB

The high performance liquid chromatography is used for detecting the enzyme activity of fumonisin degrading enzyme, and the specific method comprises the following steps:

(1) FB1 standard stock: weighing 1mg of standard substance, dissolving with 10mL of acetonitrile and water (1: 1), preparing into standard solution with the concentration of 100ug/mL, and storing at-20 ℃.

(2) Preparation of a sample: 900ul of purified fumonisin degrading enzyme solution is taken and added into 100ul of FB1 standard stock solution, so that the final concentration of FB1 is 10ug/ml, the temperature is 37 ℃, the rpm is 220, and the culture is carried out for 20min in a dark place.

(3) Derivatization of the sample: taking 100ul of a sample to be detected, adding 400ul of 50% acetonitrile water and 500ul of OPA derivative solution, uniformly mixing for 30s, carrying out sample injection within 2min of derivatization, and filtering the membrane to be detected. The enzymatic activity of fumonisin degrading enzyme FumDSB was determined by comparison with the peak pattern of the standard of FB 1.

1. Determination of the optimum temperature of the fumonisin-degrading enzyme FumDSB

In a buffer system of citric acid-disodium hydrogen phosphate buffer solution (pH7.0) and at different temperatures, FB1 is used as a substrate, the final concentration is 10 mu g/ml, 100 mu L of the substrate is taken and 900 mu L of enzyme solution is added, the reaction is carried out for 20min, and then boiling water is carried out for 10min to inactivate the enzyme. After cooling to room temperature, the solution is passed through a membrane and subjected to HPLC detection.

As shown in FIG. 2, the optimum temperature of fumonisin degrading enzyme FumDSB of the present invention was 30 ℃.

2. Determination of the optimum pH of the fumonisin-degrading enzyme FumDSB

Different buffer solutions were selected: 100mM citric acid-disodium hydrogen phosphate (pH 3.0-8.0), 100mM Tris-HCl (pH8-9), 100mM glycine-NaOH (pH 9.0-12.0), and the enzymatic reaction was carried out under the above-mentioned different pH buffers to determine the optimum pH thereof.

As shown in FIG. 3, the optimum pH of fumonisin-degrading enzyme FumDSB of the present invention was 6.0.

Meanwhile, the temperature stability and the pH stability of the fumonisin degrading enzyme FumDSB are measured, and the result shows that the fumonisin degrading enzyme FumDSB has better temperature stability and pH stability.

Sequence listing

<110> Tianjin science and technology university

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ggacccagtg tcgtcaccac cgacgccggc cggcttcgtg gagcggtcga aggcgagctt 60

gacgtgttca gaggcgtacc ttttgcggca gcgcccattg gtcaacttcg ttggcgcgag 120

cctcagcgga tcgcgccttg gaccgatata cgggatgcca gcaaatttgc cccggcatgc 180

atgcaaagcg gtgtttcaat cccgggtgag ccagcgccgc agatcagcga agattgccta 240

tacctcaata tttgggcgcc gcggcattct ggtcgggcga agctgcccgt aatgattttc 300

attcatggcg gcggttggca gaatggtgcg accgcgctgc cactctattg gggcgatcgg 360

ttggcgcagc aaggggctgt cgtcgtttcg gtgagctacc ggcttggtgc gcttggcttc 420

ctcgcccatc ccgaacttac cgccgaatca ccacatcaca cgtcagggaa ctacggcctg 480

ctcgatcaaa tcgccgccct caattgggtg cagcgcaaca tcgcggcgtt tgggggtgac 540

ccggcaaatg ttactctatt tggtcagtcg gcgggttcga gttcgatcgc tatcctgatg 600

gcttcaccgt tggccaaggg cctgtttcat cgcgtcatcg gccaaagtgg cggttttttc 660

gagccgctac aactggcccc gcattacgaa ctcgcgctcg ctgaaaaaca gggagttgcc 720

tttgcacatt ccctagaaac ttccacgctt gctgatctcc ggtcgctccc cccgcaggcc 780

ttgttgacga aacaggccgc cagcgtgtcg catcctgtaa tcgaaccatg gctattgccg 840

cgaaccccgt ttgaggtttt ctcggtaggg cagcaacacg gtgcagacat tctcgtcgga 900

tacaatgccg aagaaggccg agcattcttt gacgcttcgt cggtcactgc tgcgaacttc 960

ggtgaacagc ttcgcgccga attgggcgac ctgcctcctg caatcatggc ggcctaccct 1020

tttgccagcg atgtcgaggc ggggcaggcg cgcgttgcgc tggagcggga tctgcgtttc 1080

ggctggaaca tgtggacctg ggcaaagctc cacgccgcaa ccggaaaaaa cgcggtccat 1140

gcctattact tcacccacaa accaccgttc ccaagcgatt cagttcgaag aaattggcag 1200

gcaagccact tcgccgaact gtggtacatg tttgaccatc ttggccaaga agattggcaa 1260

tggacaaagt ttgatcgtca aatcgctcgc accatgtcgc gttactgggt aaattttgcc 1320

cggaacggaa atcccaacgg gcgaggtctt ccgcactggc ctgcctatcg taccgaccaa 1380

ccgcttgtgt tgcagattgg ccaaccgata acacccacgc cggagcccaa taccgggtct 1440

attggagtga tcgacgcggt tttttcagca gtacgaggcg acacc 1485

Claims

1. The fumonisin degrading enzyme FumDSB with an amino acid sequence shown as SEQ ID number 1 or SEQ ID number 2 is applied to degrading fumonisin B1.

2. The fumonisin degrading enzyme FumDSB encoding gene is applied to degrading fumonisin B1, wherein the fumonisin degrading enzyme FumDSB encoding gene encodes protein with an amino acid sequence shown as SEQ ID number 1 or SEQ ID number 2.