CN106148367A - Marine low temperature alpha amylase gene cloning and expression - Google Patents

Abstract

The invention belongs to the fields of enzyme engineering and genetic engineering, and specifically relates to the cloning and expression of a marine low-temperature α-amylase gene which still has activity and stability at low temperatures. A marine low-temperature α-amylase gene, its sequence is the base sequence shown in SEQ ID NO.1, the gene sequence is 1302bp in length, with ATG as the start codon, TGA as the stop codon, and GC base The content is 35%. The present invention can quickly and accurately obtain the low-temperature α-amylase gene whose activity optimum temperature is 15-25°C through the Bacillus thuringiensis strain, and the obtained gene is a complete reading frame, and realizes the rapid and high-efficiency expression of the low-temperature amylase gene , a large amount of marine low-temperature amylase can be obtained, which provides a basis for the research and development of low-temperature amylase.

Description

Cloning and expression of marine low temperature α-amylase gene

technical field

The invention belongs to the fields of enzyme engineering and genetic engineering, and specifically relates to the cloning and expression of a marine low-temperature alpha-amylase gene which still has activity and stability at low temperatures.

Background technique

Amylase is a general term for a class of enzymes that can degrade starch glycosidic bonds. According to different hydrolysis methods, it is mainly divided into α-amylase, autoamylase, glucoamylase, isoamylase and pullulanase.

The application of starch as a raw material in industry, the main way is to hydrolyze starch into glucose, maltose or oligosaccharide syrup, which is then used as raw material for industrial production such as alcohol, organic acid, amino acid, etc. or directly as a product Used in other industrial fields, amylase is one of the most widely used enzyme preparations. Its main uses include: food, medicine, washing, papermaking, textile and other fields. Amylase can decompose starchy dirt, degrade it, and achieve removal Purpose.

At present, the commercial amylases are medium and high temperature enzymes. The industrial use of starch sugar production mainly adopts the method of enzymatic hydrolysis. The enzymatic sugar production process generally adopts a two-step method. The first step is to use α-amylase to hydrolyze starch into low molecular weight. The second step is to use glucoamylase to hydrolyze the dextrin into glucose. The function of α-amylase is to cut off the α-1,4) ketone bond in the middle of the starch molecular chain. However, it is active at 0-20°C. Low, thus limiting the application of amylase in food, feed, textile and washing industries, the Japanese scholar Wada Gongshang has discussed in detail the conditions for low-temperature amylase as a detergent. Therefore, the research and development of low-temperature amylase has practical significance.

Contents of the invention

One of the objectives of the present invention is to provide a marine low-temperature alpha-amylase gene that still has activity and stability at low temperatures.

The second object of the present invention is to provide the encoded protein of the gene.

The third object of the present invention is to provide a method for cloning the gene.

To achieve above object, the present invention realizes by following scheme:

A marine low-temperature α-amylase gene, its sequence is the base sequence shown in SEQ ID NO.1, the gene sequence is 1302bp in length, with ATG as the start codon, TGA as the stop codon, and GC base The content is 35%.

The protein sequence of the marine low-temperature α-amylase gene is the amino acid sequence shown in SEQ ID NO.2, which is a protein sequence consisting of 433 amino acids, and its molecular relative mass is predicted to be 50.29 kDa.

The cloning method of described marine low-temperature α-amylase gene, comprises the following steps:

(1) Using the Bacillus thuringiensis genomic DNA as a template, the full-length sequence of the CDS region of the alpha amylase gene was obtained by PCR amplification;

(2) According to the acquired alpha amylase gene, perform NCBI comparison with the known sequence to obtain the homologous sequence, and design and synthesize the following primers according to the homologous sequence:

CTF035Fw1: 5'-CGACATATGCGTTTCATTCG-3'

CTF035Rw1: 5'-GCTAGGAAGAAGACTTTCTC-3'

CTF035Fw2: 5'-GTGATGTTAGAAAACCTAAC-3'

CTF035Rw2: 5'-GTATAAGAGTATTCTCTAAC-3';

(3), use HS DNA Polymerase (Code No.DR010S) was used for PCR amplification to obtain cloned gene fragments, and finally sequenced and analyzed to obtain the required gene cloned fragments.

The conditions of the PCR amplification are: 98° C. for 10 seconds, 50° C. for 10 seconds, 72° C. for 2 minutes, 30 cycles, and 72° C. for 5 minutes for one cycle.

The concrete steps of the method for expressing the above-mentioned marine low temperature alpha-amylase gene are:

(1), PCR amplified alpha amylase gene 1299bp, added TGA terminator at the 3' end, added NdeI/Hind III restriction sites on both sides, cloned into pCold I vector to form a recombinant plasmid.

(2) Transfer the recombinant plasmid into BL21T1R competent cells, induce and express the positive clones.

Beneficial effects of the present invention: the present invention can quickly and accurately obtain the low-temperature α-amylase gene whose activity optimum temperature is 15-25°C through the Bacillus thuringiensis strain, and the obtained gene is a complete reading frame, and realizes fast High-efficiency expression of low-temperature amylase gene can obtain a large amount of marine low-temperature amylase, providing a basis for research and development of low-temperature amylase.

Description of drawings

Fig. 1 is the influence of temperature on α-amylase activity;

Figure 2 is the effect of temperature on the stability of α-amylase.

detailed description

The present invention will be further described now in conjunction with embodiment.

A marine low-temperature α-amylase gene, its sequence is the base sequence shown in SEQ ID NO.1, the gene sequence is 1302bp in length, with ATG as the start codon, TGA as the stop codon, and GC base The content is 35%.

The protein sequence of the marine low-temperature α-amylase gene is the amino acid sequence shown in SEQ ID NO.2, which is a protein sequence consisting of 433 amino acids, and its molecular relative mass is predicted to be 50.29 kDa.

The cloning method of described marine low-temperature α-amylase gene, comprises the following steps:

(1) Using the Bacillus thuringiensis genomic DNA as a template, the full-length sequence of the CDS region of the alpha amylase gene was obtained by PCR amplification;

(2) According to the obtained alpha amylase gene known sequence, NCBI alignment was performed to obtain the homologous sequence, and the following primers were designed and synthesized according to the homologous sequence:

CTF035Fw1: 5'-CGACATATGCGTTTCATTCG-3'

CTF035Rw1: 5'-GCTAGGAAGAAGACTTTCTC-3'

CTF035Fw2: 5'-GTGATGTTAGAAAACCTAAC-3'

CTF035Rw2: 5'-GTATAAGAGTATTCTCTAAC-3';

(3), use HS DNA Polymerase (Code No.DR010S) was used for PCR amplification to obtain cloned gene fragments and PCR product purification, and finally sequenced and analyzed to obtain the desired gene cloned fragments.

The conditions of the PCR amplification are: 98° C. for 10 seconds, 50° C. for 10 seconds, 72° C. for 2 minutes, 30 cycles, and 72° C. for 5 minutes for one cycle. Then PCR amplification was used to obtain the gene fragment and the PCR product was purified, and finally the desired gene clone fragment was obtained by sequencing analysis.

The concrete steps of the method for expressing the above-mentioned marine low temperature alpha-amylase gene are:

(1), PCR amplified alpha amylase gene 1299bp, added TGA terminator at the 3' end, added NdeI/Hind III restriction sites on both sides, cloned into pCold I vector to form a recombinant plasmid.

(2), transfer the recombinant plasmid into BL21T1R competent cells, induce positive clones, and express:

①Transformation: Transfer 0.5ul of the recombinant plasmid into BL21T1R; use LB/antibiotic Amp (100ug/ml) plate, 10ul of transformation solution to coat, incubate at 37°C O/N for 23h, and do the same with Control pCold I;

②Cultivation and induction: Pick a single colony into 2ml LB/Amp (100ug/ml) medium, culture at 37°C O/N for 23h. Add 5ml LB/Amp (100ug/ml) medium to the Glass tube, add 100ul of seed culture solution, culture at 37°C until the OD ₆₀₀ nm value is about 0.6, 15°C for 15min, add 100mM IPTG (isopropyl-β- D-thiogalactopyranoside) 50ul (final 1mM IPTG) for induction, and cultured at 15°C for 22h.

③Protein extraction: After the collection of bacteria, the 1.0OD equivalent bacteria were added to 160ulPBS suspension, then ultrasonically crushed, and the broken bacteria were centrifuged.

④ Extract electrophoresis: take 8ul of each extract (total protein, supernatant, precipitate) (equivalent to 0.05OD), add 2ul 5×SDS Loading Buffer, heat at 95°C for 10 minutes, perform SDS-PAGE electrophoresis, and finally get the ocean Low temperature alpha-amylase.

The activity and stability of marine low-temperature α-amylase were tested as follows by using different temperatures:

1. Effect of temperature on low temperature α-amylase activity

The specific implementation steps are:

(1) Under sterile conditions, transfer the bacterium on the slant to 3 rings in the seed culture medium, shake the shaker at 20°C and 140r/min for 20 hours, and use it as the seed liquid; then transfer the seed liquid with a pipette Received into the fermentation medium, 20°C, 140r/min shaking culture. Regular sampling was performed to measure the enzyme activity in the fermentation broth. Two parallel experiments were performed for each experiment, and the results were averaged.

(2) The fermentation broth was centrifuged at 4° C. and 8000 r/min for 15 minutes, and the supernatant was used as the crude enzyme solution.

(3) Take an appropriate amount of crude enzyme solution at different temperatures (5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C ℃, 70℃, 75℃, 80℃) for 30 minutes, and then measure the relative enzyme activity of the crude enzyme solution.

According to the above experiments, the activity of low-temperature α-amylase is shown in Figure 1. The abscissa in Figure 1 indicates the action temperature, and the ordinate indicates the relative enzyme activity. It can be seen from the figure that the optimum temperature for the enzyme action is 15-25°C. At 15°C and 30°C, the enzyme activity retained more than 80% and 60% respectively, which shows that the enzyme has certain low-temperature characteristics and can maintain high enzyme activity at lower temperatures;

2. The specific implementation steps of the influence of temperature on the stability of α-amylase:

(1) Under sterile conditions, transfer the bacterium on the slant to 3 rings in the seed culture medium, shake the shaker at 20°C and 140r/min for 20 hours, and use it as the seed liquid; then transfer the seed liquid with a pipette Received into the fermentation medium, 20°C, 140r/min shaking culture. Regular sampling was performed to measure the enzyme activity in the fermentation broth. Two parallel experiments were performed for each experiment, and the results were averaged.

(2) The fermentation broth was centrifuged at 4° C. and 8000 r/min for 15 minutes, and the supernatant was used as the crude enzyme solution.

(3) Take an appropriate amount of crude enzyme solution and measure the relative enzyme activity every 10 minutes under different temperatures (10°C, 20°C, 30°C, 40°C, 50°C) for 6 consecutive measurements.

According to the above experiments, a schematic diagram of the stability of α-amylase is obtained, as shown in Figure 2. The abscissa of Figure 2 represents the action time, and the ordinate represents the relative enzyme activity. This graph shows the stability of this low-temperature enzyme at different temperatures , the enzyme is stable below 20°C; 60% of the enzyme activity is still retained at 30°C for 1 hour, which shows that the enzyme has certain low-temperature characteristics and belongs to low-temperature α-amylase, which has the potential to be used in starch liquefaction under low temperature conditions processing industry.

Although specific examples have been described herein, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes which are within the scope of this invention.

<110> Dalian University

<120> Cloning and expression of marine low temperature α-amylase gene

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1302

<212>DNA

<213> Synthetic

<400> 1

atgcgtgtgg gaaaaatacg gactaggaaa ctattcattt gtttttgctt agctgttgtt 60

ttgtttgtac caatacatac ttttgcagac gaaaaaagag agtggcgaga cgaagttatc 120

tattccatta tgattgatcg cttcaataat ggagaaccga aaaatgacaa acagttagaa 180

gttggtaatt tagaagggta tcaaggtggg gatataagag gaattataaa aaggttggat 240

tacataaaag aaatgggttt tactaccgtt atgctttcgc cgctttttga aagtggaaag 300

tacgatggag tagacgtgcg aaattttaaa aaggtaaatg aacatttcgg ggcagacaat 360

gatgtgaaag aacttgtaag agaagctcat gcaaaaggaa tgaaagttgt atttcaattt 420

ccgcttggag aaaacgaaca acaggtaatt gatgcaatga aatggtggat aaaagaagtt 480

gatttagatg gaagttatgt tatacatagt gaaaaaaagc cgcgttcctt ttgggataac 540

gcgcaaaaag atatgcaagt gataaagaaa gattttcgta ttatgacaaa ggaggatagt 600

gaatacaacg aaaaaatagt agaatcgttt tccaaagcgg atgtatcggt gaaatcgtta 660

tacgatgtga gtaaaaaaga agggggaattt gttacgtttt tagatgatca agaaacaaaa 720

agatatgcgc gtattgcaaa agaaaatatg tattatccgc catcacgttt aaaactggcg 780

cttacatatt tgttaacatc acctggaatt ccgaattttt attacggaac tgaaattgca 840

ttagatggag gaggtgtacc tgataataga cgattaatgg attttaagtc agacgaaaaa 900

tttatgcagc acataacaaa actaggtgaa cttagacagg ctagaccgtc tttacgacgc 960

ggtacgtttg agctactgta tgataagagc gggatgagca tactaaaacg aaaatataaa 1020

aatgaagtaa ctttagtagc aattaataat acgaaagaaa cacaaaaagt ctccttacct 1080

gcaagtacga ttggtgaaaa acaagagtta agaggattgt tagaggatga aattataaga 1140

gaggaaaatg gggaatttta tctcgtttta aagcgtgaag aatcaaacgt gtataaagtt 1200

aacggagaaa caggtgtgaa ttggttatt atctccttaa tcgttggtgt gaatgtatta 1260

tttattgcgt ttttaattgc agttaaaagg aaacgtagat ga 1302

<210> 2

<211> 433

<212> PRT

<213> Synthetic

<400> 2

MRVGKIRTRK LFICFCLAVV LFVPIHTFAD EKREWRDEVI YSIMIDRFNN GEPKNDKQLE 60

VGNLEGYQGG DIRGIIKRLD YIKEMGFTTV MLSPLFESGK YDGVDVRNFK KVNEHFGADN 120

DVKELVREAH AKGMKVVFQF PLGENEQQVI DAMKWWIKEV DLDGSYVIHS EKKPRSFWDN 180

AQKDMQVIKK DFRIMTKEDS EYNEKIVESF SKADVSVKSL YDVSKKEGEF VTFLDDQETK 240

RYARIAKENM YYPPSRLKLA LTYLLTSPGI PNFYYGTEIA LDGGGVPDNR RLMDFKSDEK 300

FMQHITKLGE LRQARPSLRR GTFELLYDKS GMSILKRKYK NEVTLVAINN TKETQKVSLP 360

ASTIGEKQEL RGLLEDEIIR EENGEFYLVL KREESNVYKV NGETGVNWLF ISLIVGVNVL 420

FIAFLIAVKR KRR 433

<210> 3

<211> 20

<212>DNA

<213> Synthetic

<400> 3

cgacatatgc gtttcattcg 20

<210> 4

<211> 845

<212>DNA

<213> Synthetic

<400> 4

gctaggaaga agactttctc 20

<210> 5

<211> 20

<212>DNA

<213> Synthetic

<400> 5

gtgatgttag aaaacctaac 20

<210> 6

<211> 20

<212>DNA

<213> Synthetic

<400> 6

gtataagagt attctctaac 20

Claims (4)

1. a marine low temperature alpha-amylase gene, it is characterised in that its sequence is the base sequence shown in SEQ ID NO.1, This gene order total length 1302bp, with ATG as start codon, with TGA as termination codon, GC base contents is 35%.

Marine low temperature alpha-amylase gene the most according to claim 1, it is characterised in that described marine low temperature alphalise starch The protein sequence of enzyme gene is the aminoacid sequence shown in SEQ ID NO.2, and this albumen is the albumen sequence of 433 Amino acid profiles Row, it was predicted that its molecule relative mass is 50.29kDa.

Marine low temperature alpha-amylase gene the most according to claim 1, it is characterised in that described marine low temperature alphalise starch The cloning process of enzyme gene, comprise the following steps into:

(1), with Bacillus thuringiensis Genes group DNA as template, alpha amylase gene C DS district is obtained with PCR amplification complete Long sequence；

(2), according to the alpha amylase gene obtained, carry out NCBI comparison with known array, obtain homologous sequence, and According to the homologous sequence design following primer of synthesis:

CTF035Fw1:5 '-CGACATATGCGTTTCATTCG-3 '

CTF035Rw1:5 '-GCTAGGAAGAAGACTTTCTC-3 '

CTF035Fw2:5 '-GTGATGTTAGAAAACCTAAC-3 '

CTF035Rw2:5 '-GTATAAGAGTATTCTCTAAC-3 '；

(3), useHS DNA Polymerase (Code No.DR010S) carries out PCR amplification and obtains clone's base Because of fragment, the cloned genomic fragment needed for the acquisition of last sequencing analysis；

Wherein, the condition of described PCR amplification is: 98 DEG C 10 seconds, 50 DEG C 10 seconds, 72 DEG C 2 minutes, 30 circulations, 72 DEG C 5 minutes One circulation.

Marine low temperature alpha-amylase gene the most according to claim 1, it is characterised in that express marine low temperature alpha-amylase Concretely comprising the following steps of the method for gene:

(1), PCR expands alpha amylase gene 1299bp, 3 ' end interpolation TGA terminators, both sides interpolation NdeI/Hind III digestion site, is cloned into pCold I carrier and forms recombiant plasmid；

(2), by recombiant plasmid plasmid proceed to, in BL21T1R competent cell, positive colony be induced, express.