CN103667212B - Glucoamylase and application thereof - Google Patents
Glucoamylase and application thereof Download PDFInfo
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- CN103667212B CN103667212B CN201310717049.4A CN201310717049A CN103667212B CN 103667212 B CN103667212 B CN 103667212B CN 201310717049 A CN201310717049 A CN 201310717049A CN 103667212 B CN103667212 B CN 103667212B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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Abstract
The invention provides a glucoamylase of which the amino acid sequence is SEQ ID NO.1. The optimum acting temperature of the glucoamylase provided by the invention is 50 DEG C, greater than 60% of the enzyme activity can be maintained within the temperature range of 35-60 DEG C, the optimum acting pH value is 5.5, and greater than 90% of the enzyme activity can be maintained within the pH value range of 3.0-6.5. The glucoamylase can be widely applied to production of glucose; after being used within 5 hours, the DE value (Reducing Sugar Value) can be 95% at most, and the content of glucose in a reaction liquid is as high as 85%. The glucoamylase provided by the invention can be also applied to production of a sweetening agent or a fermentation method of organisms such as ethanol, citric acid, ascorbic acid, amino acid, antibiotics and the like.
Description
Technical field
The present invention relates to functional gene screening field, be specifically related to a kind of saccharifying enzyme and application thereof.
Background technology
Saccharifying enzyme, also known as glucoamylase (Glucoamylase), it can produce glucose starch from non reducing end hydrolyzing alpha-Isosorbide-5-Nitrae glucoside bond.The Substratspezifitaet of saccharifying enzyme is lower, and it, except cutting α-Isosorbide-5-Nitrae key from the non reducing end of starch chain, also slowly can cut α-1,6 glycosidic link.Therefore, what it can be very fast cuts glucose unit amylose starch successively from non reducing end, is running into 1, during 6 key, is first split by α-1,6 key, then by α-Isosorbide-5-Nitrae key segmentation, thus make amylopectin be hydrolyzed into glucose.
Saccharifying enzyme is of many uses, for doing the fermentation of the various microbiotic of fermention medium, organic acid, amino acid, VITAMIN with glucose, also can be used for the glucose producing all size.Allly must carry out the industrial of enzymic hydrolysis to starch, dextrin, all applicable.Saccharifying enzyme does not corrode equipment, use safety, and technique is simple, stable performance, utilizes saccharifying enzyme to be hydrolyzed to starch safer, also helps and realize production mechanization and carry out production strictly in line with rules and regulations.
Saccharifying enzyme conventional is at present all from aspergillus niger.From the mid-1970s, S.3.4309 the screening of Institute of Micro-biology of the Chinese Academy of Sciences obtains high-yield glucoamylase strains A.The domestic many large-scale microbiotic pharmaceutical factories of this bacterial strain, grain distillery, distillery have carried out industrial scale test, all succeed.The success of this project is that China's fermentation industry has saved a large amount of grain, creates huge economic benefit and social benefit.Increasing market pays close attention to high efficiency glucoamylase, and this makes one of cloning and expressing high efficiency glucoamylase focus becoming research.Protein expression system is aspergillus niger, and its yield of enzyme (protein content) is higher, but is subject to the impact of saccharifying enzyme nature, and the specific activity that aspergillus niger expresses saccharifying enzyme is lower, can not adapt to the demand in market.Therefore, develop excellent property, concern that saccharifying enzyme that specific activity is high is more and more subject to each side.
Summary of the invention
The object of this invention is to provide a kind of Novel saccharification enzyme and application thereof, the present invention obtains a kind of novel glucoamylase gene from penicillium funiculosum (Penicillium funiculosum) clone, and proceeded in Trichodermareesei (Trichoderma reesei), build the recombinant bacterial strain obtaining this saccharifying enzyme of high expression, significantly improve the output of saccharifying enzyme, be conducive to the widespread use of saccharifying enzyme.
One aspect of the present invention relates to a kind of saccharifying enzyme, it is characterized in that, described saccharifying enzyme comprises:
A) its aminoacid sequence is the enzyme of SEQ ID NO:1;
B) its aminoacid sequence and SEQ ID NO:1 similarity are higher than more than 95%, and have the enzyme of saccharifying enzyme function.
The encoding gene of described saccharifying enzyme, its a kind of nucleotide sequence is SEQ ID NO:2.
The present invention relates to the expression vector carrying above-mentioned glucoamylase gene on the other hand.
A kind of engineering strain, it carries above-mentioned expression vector.
Described engineering bacteria is Trichodermareesei (Trichoderma reesei).
The invention still further relates to the application of above-mentioned saccharifying enzyme.
The optimum temperature of saccharifying enzyme of the present invention is 50 DEG C, and the enzyme of more than 60% can be kept within the scope of 35 DEG C-60 DEG C to live; The suitableeest action pH is 5.5, and within the scope of pH3.0-6.5, the enzyme of more than 90% can be kept to live.This saccharifying enzyme can be widely used in the production of glucose, and during 5h, its DE value (reducing sugar value) reaches and is up to 95%, and in reaction solution, the content of glucose is up to 85%.Saccharifying enzyme of the present invention also can be used in the production of sweeting agent or for the production of in organic fermentation process, described organic compound is ethanol, citric acid, xitix, amino acid, microbiotic etc. such as.
Accompanying drawing explanation
Fig. 1: the expression vector plasmid map that the present invention builds.
Fig. 2: the SDS-PAGE electrophoretic analysis figure of Trichodermareesei engineering bacterium fermentation supernatant liquor, wherein arrow indication is recombinant expressed saccharifying enzyme.
Fig. 3: saccharifying enzyme operative temperature of the present invention-enzyme graphic representation alive relatively.
Fig. 4: saccharifying enzyme VL-2 graphic representation alive with VL-3 operative temperature-relative enzyme.
Fig. 5: saccharifying enzyme action pH of the present invention-enzyme graphic representation alive relatively.
Fig. 6: saccharifying enzyme VL-2 graphic representation alive with VL-3 action pH-relative enzyme.
Embodiment
The present invention has used routine techniques and the method for genetic engineering and biology field use, such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) method and described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general reference provide definition well known by persons skilled in the art and method.
Unless be separately construed as limiting in this article, whole technical term used herein and scientific terminology have usual the understood identical meanings of common counting personnel in field belonging to the present invention.DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY, 3nd Ed. (Singleton et al., 2006) and COLLINS DICTIONARY BIOLOGY (Hale et al., 2003) for technician provide the many terms used in the present invention generality explain.
Unless otherwise mentioned, nucleic acid writes from left to right by 5 ' to 3 ' direction; Amino acid writes from left to right by the direction of amino to carboxyl.
As used herein, term " restructuring ", when being used to refer to cell, nucleic acid, albumen or carrier, represents this cell, nucleic acid, albumen or carrier by importing heterologous nucleic acids or albumen or being modified by change natural acid or albumen.Therefore, such as, reconstitution cell expresses the gene never found in this cell of natural (non-recombinant) form, or express natural gene.
Term " protein " and " polypeptide " can exchange use in this article.Use traditional single-letter or the three-letter codes of amino-acid residue herein.
As used herein, term " gene " refers to the DNA fragmentation participating in producing polypeptide, comprises the region before and after coding region, and the insertion sequence (intron) between each encode fragment (exon).
Term " nucleic acid " comprises DNA, RNA, strand or double-strand, and their chemical modification object.
Term " nucleic acid " and " polynucleotide " can exchange use in this article.
Term " carrier " refers to the polynucleotide sequence being designed to nucleic acid be imported one or more cell types.Carrier comprises cloning vector, expression vector, shuttle vectors, plasmid, phagemid, sequence box and analogue.
Term " expression vector " represents the DNA construction comprising DNA sequence dna, and described DNA sequence dna is connected to the suitable control sequence that can affect this DNA and express in suitable host by steerable.This type of control sequence can comprise the sequence of the termination that the promotor of transcribing, the sequence optionally controlling ribosome bind site suitable on the operon sequence of transcribing, the mRNA that encodes, enhanser and control are transcribed and translated.
Have the polynucleotide of the sequence iden of a certain per-cent with another sequence or polypeptide refers to, when comparing this two sequences, the base of described per-cent or amino-acid residue are identical.
Because genetic code is degeneracy, so more than one codon can be used to specific amino acid of encoding, the present invention includes the polynucleotide of specific aminoacid sequence of encoding.
Term " host strain " or " host cell " refer to the suitable host of expression vector or DNA construction, and described expression vector or DNA construction comprise the polynucleotide of encoding lipase of the present invention.Specifically, host strain is preferably filamentous fungal cells.This host cell can be wild-type filamentous fungal host cells or genetically modified host cell.Term " host strain " or " host cell " refer to the nucleus protoplastis produced by filamentous fungal strains cell.
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1 gene cloning and expression vector construction
The extraction of 1.1 STb gene
By penicillium funiculosum (purchased from China General Microbiological culture presevation administrative center, strain number 3.3791) incubated overnight, get appropriate thalline and be placed in centrifuge tube, the centrifugal 5min of 13000rpm, abandons supernatant; Add 400 μ l extraction buffers (100mM Tris-HCl, 100mM EDTA, 250mM NaCl, 1%SDS); Then add 100mg quartz sand or granulated glass sphere, beat instrument thermal agitation about 2min on pearl; After 65 DEG C of water-bath 20min, add 200 μ l10M NH
4aC, ice bath 10min; The centrifugal 10min of 13000rpm, gets supernatant; Add the dehydrated alcohol of 2 times of volumes, place 30min for-20 DEG C; The centrifugal 10min of 13000rpm, abandons supernatant; By 70% washing with alcohol 2 times; Dry, add water dissolution, in-20 DEG C of preservations.
1.2 gene clone
With the genome DNA extracted in 1.1 for template carries out pcr amplification, primer sequence is as follows:
Upstream primer: ATGACCGTTACAACTGCGCTCG
Downstream primer: TTATTGCCACGAGTCGTTAATG
Pcr amplification condition is 94 DEG C of 3min; 94 DEG C of 30S; 58 DEG C of 30S, 72 DEG C 2min30 circulation; 72 DEG C of 7min.Utilize gel to reclaim test kit and reclaim pcr amplification product.After connecting carrier T, deliver to Huada Gene Research Center, Beijing and carry out sequencing analysis.Translated according to three disjunctor codon principles by the sequencing result of amplified production, the aminoacid sequence obtaining described amplified production coding is SEQ ID NO:1.
Find through NCBI Blast sequence alignment, the albumen that SEQ ID NO:1 encodes belongs to glycoside hydrolase the 15th family, and the highest with the saccharifying enzyme amino acid sequence similarity of Talaromyces marneffei, being 92%, is a new allelotrope.
Be optimized according to the codon preference of Trichodermareesei by the aminoacid sequence SEQ ID NO:1 of described saccharifying enzyme, its coding nucleotide sequence is SEQ ID NO:2, is synthesized by Shanghai Sheng Gong biotechnology limited-liability company.Add KpnI restriction enzyme site in initiator codon ATG front end, add XbaI enzyme cutting site in terminator codon TAA rear end, for the structure of expression vector; The nucleotide sequence of synthesis is connected to pUC57 carrier, called after pU-Glu9298.
Plasmid pU-Glu9298 is carried out Kpn I and Xba I double digestion, reclaim required object band and glucoamylase gene.Equally, Kpn I and Xba I double digestion are also carried out to the mould expression plasmid pTG of wood; Glucoamylase gene and expression vector carried out 22 DEG C with T4 ligase enzyme to be connected and to spend the night; Finally, connection product is imported escherichia coli DH5a; Corresponding positive colony expression plasmid called after pKVL-glu9298, plasmid map as shown in Figure 1.
The structure of embodiment 2 Trichodermareesei engineering strain
Inoculation Trichodermareesei mycelia was in PDA grow on plates 4 days; Cut the bacterium colony that diameter is about 3cm and be placed in about 60ml YEG(0.5% yeast powder, 1% glucose) liquid nutrient medium, 30 DEG C, 200rpm shaking culture is spent the night; Multilayer filtered through gauze collects mycelia; Mycelia is placed in and fills 20ml lyase liquid (0.2g/10ml, 0.7M NaCl dissolves, Sigma L1412) enzymolysis 2 hours; Take out enzymolysis solution, jiggle, fall in three layers of sterilizing lens wiping paper and filter, collect filtrate, 3000rpm, centrifugal 10min; Abandon supernatant, add 5ml solution 2 and suspend, then 3000rpm, centrifugal 10min; Add appropriate solution 2 suspend packing (200 μ l/ manage, 10
8individual/ml).
Get pKVL-glu9298DNA to join in 200 μ l protoplastiss, then add 50 μ l25%PEG solution and mix gently, ice bath 20min; Then add 2ml25%PEG, mix gently, room temperature leaves standstill 5min, is cooled to the upper strata semisolid medium (0.1%MgSO of 45-55 DEG C after protoplastis is added to about 50ml fusing
4, 1%KH
2pO4,0.6% (NH
4)
2sO
4, 1% glucose, 18.3% sorbyl alcohol, 0.35% agarose), pour into containing 100 μ g/ml Totomycin subfoundation culture medium flat plate (2% glucose, 0.5% (NH4) after mixing gently
2sO
4, 1.5%KH
2pO
4, 0.06%MgSO
4, 0.06%CaCl
2, 1.5% agar), 30 DEG C of dark culturing a couple of days grow to transformant.
Transformant is inoculated in YEG(0.5% yeast powder, 1% glucose) liquid nutrient medium in overnight incubation cultivate, get appropriate thalline and be placed in centrifuge tube, the centrifugal 5min of 13000rpm, abandons supernatant; Add 400 μ l extraction buffers (100mM Tris-HCl, 100mM EDTA, 250mM NaCl, 1%SDS); Then add 100mg quartz sand or granulated glass sphere, beat instrument thermal agitation about 2min on pearl; After 65 DEG C of water-bath 20min, add 200 μ l10M NH
4aC, ice bath 10min; The centrifugal 10min of 13000rpm, gets supernatant; Add the dehydrated alcohol of 2 times of volumes, place 30min for-20 DEG C; The centrifugal 10min of 13000rpm, abandons supernatant; By 70% washing with alcohol 2 times; Dry, add water dissolution, in-20 DEG C of preservations.
With said extracted transformant genomic dna for template, following primer amplification goal gene is utilized to verify transformant.
Upstream primer: ATGACCGTTACAACTGCGCTCG;
Downstream primer: TTGCCACGAGTCGTTAATGCAAG,
Pcr amplification condition is 94 DEG C of 3min; 94 DEG C of 30S; 58 DEG C of 30S, 72 DEG C 90S30 circulation; 72 DEG C of 7min.Utilize gel to reclaim test kit reclaim pcr amplification product and carry out sequence verification.Positive engineering bacteria called after Trichodermareesei K-VL9298(Trichoderma reesei K-VL9298 by obtaining).
Embodiment 3 is fermented and zymologic property measures
Above-mentioned Trichodermareesei engineering bacteria K-VL9298 is inoculated in MM fermention medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH
4)
2sO
4, 0.09%MgSO
4, 2%KH
2pO
4, 0.04%CaCl
2, 0.018% tween-80,0.018% trace element, 0.018% polypropylene glycol-2000), cultivate 48 hours for 28 DEG C, then 25 DEG C of lactose-induced cultivations 48 hours, get fermented supernatant fluid, carry out SDS-PAGE analysis.As shown in Figure 2, wherein arrow indication place is recombinant expressed saccharifying enzyme to result, illustrates that glucoamylase gene of the present invention is expressed in Trichodermareesei engineering bacteria K-VL9298, utilizes Coomassie Brilliant Blue to measure its expressing quantity and is about 0.32g/L.
Glucoamylase enzyme measuring method alive
First, second two 50mL colorimetric cylinders, add the 2% Zulkovsky starch solution of 25mL and the 0.1M acetic acid-sodium acetate buffer solution (pH4.6) of 5mL respectively; Preheating 5 ~ 10min in the water bath with thermostatic control of 40 ± 0.2 DEG C.In first pipe, add enzyme prepare liquid 2.0mL and shake up and clock immediately; After reaction 1h, manage 20% sodium hydroxide solution respectively adding 0.2mL immediately in first, second two, shake up immediately and two pipes are taken out rapid water cooling; Then in second pipe, add enzyme prepare liquid 2.0mL (in contrast).Get each 5mL of above-mentioned reaction solution in two pipes and put into iodine flask, accurately add 0.1N iodine liquid 10mL, then add 0.1N sodium hydroxide solution 15mL (limit edged rocks), open in dark place 15min, adds 2N sulfuric acid 2mL; Finally extremely colourless by 0.05N sodium thiosulfate solution titrated is terminal.
Enzyme X=(A-B) × N × 90.05 × n alive
Wherein: X---enzyme activity unit, μ/g (mL);
A---blank test consumes the milliliter number of hypo solution;
B---sample consumes the milliliter number of hypo solution;
The equivalent concentration of N---hypo solution;
N---extension rate;
90.05---the suitable glucose in milligrams number of 1mL1N Sulfothiorine;
The enzyme measuring above-mentioned shake flask fermentation supernatant liquor is according to the method described above lived as 527U/mL, illustrates that glucoamylase gene of the present invention obtains high expression in Trichodermareesei engineering bacteria.
Embodiment 4 characterization analysis
In order to analyze the zymologic property of the Novel saccharification enzyme that the present invention obtains further, detect the Substrate concentration of this enzyme, the two kinds of saccharifying enzyme related in two patents of invention (application number is respectively 201210491553.2 and 201210500500.2) of simultaneously applying for for 2012 with applicant respectively compare.
By the saccharifying enzyme of saccharifying enzyme called after described in patent of invention 201210491553.2 VL-2;
By the saccharifying enzyme of saccharifying enzyme called after described in patent of invention 201210500500.2 VL-3;
4.1 optimum temperature analyses
Under pH5.0 condition, the enzyme measuring Trichodermareesei engineering bacteria K-VL9298 fermented supernatant fluid of the present invention respectively under 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C conditions is lived, live as 100% with the highest enzyme, calculate relative enzyme and live.As shown in Figure 3, the optimum temperature of saccharifying enzyme of the present invention is 50 DEG C to result, and the enzyme of more than 60% can be kept within the scope of 35 DEG C-60 DEG C to live.
The enzyme detecting saccharifying enzyme VL-2 and saccharifying enzyme VL-3 according to above-mentioned same method is respectively lived, and lives as 100% with the highest enzyme, calculates relative enzyme and lives.As shown in Figure 4, the optimum temperature of saccharifying enzyme VL-2 and VL-3 is 70 DEG C to result, can keep the enzymic activity of more than 60% at 55 DEG C-75 DEG C.
4.2 the suitableeest action pH analyses
Under 50 DEG C of conditions, Trichodermareesei engineering bacteria K-VL9298 fermented supernatant fluid of the present invention is diluted respectively with the damping fluid that pH is 2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0, mensuration enzyme is lived, live as 100% with the highest enzyme, calculate relative enzyme and live.As shown in Figure 5, the suitableeest action pH of the present invention is 5.5 to result, and within the scope of pH3.0-6.5, the enzyme of more than 90% can be kept to live.
The enzyme detecting saccharifying enzyme VL-2 and saccharifying enzyme VL-3 according to above-mentioned same method is respectively lived, and lives as 100% with the highest enzyme, calculates relative enzyme and lives.As shown in Figure 6, the suitableeest action pH of saccharifying enzyme VL-2 is 5.0 to result, and the suitableeest action pH of saccharifying enzyme VL-3 is 5.5, and the enzyme running water of more than 80% can be kept within the scope of pH3.5-6.0 to put down.
To sum up, compare with saccharifying enzyme VL-2 and VL-3, the Novel saccharification enzyme operative temperature that the present invention obtains is lower slightly, and belong to middle temperature saccharifying enzyme, action pH scope is more wide in range, and therefore, the Application Areas of Novel saccharification enzyme of the present invention is more wide in range.
The application of embodiment 5 Novel saccharification enzyme of the present invention
Saccharifying enzyme from the non-reducing end efficient-decomposition α of sugar chain-Isosorbide-5-Nitrae-glycosidic link, can be widely used in the production of glucose.Aborning, first utilize amylase by starch liquefacation, the pH of liquefying starch is about 5.5, is cooled to 55 DEG C, then adds saccharifying enzyme and carries out follow-up saccharifying.
Get 10ml liquefying starch, add 1.5ml restructuring saccharifying enzyme (1000U/mL) of the present invention; After 55 DEG C of effect 3h, the DE value (reducing sugar value) of assaying reaction liquid, reaches 91%; During 5h, its DE value reaches and is up to 95%, and in reaction solution, the content of glucose is 85%.Result shows, saccharifying enzyme specificity of the present invention is strong, and energy efficient-decomposition α-Isosorbide-5-Nitrae-glycosidic link, in product, the content of glucose is up to 85%.
Restructuring saccharifying enzyme of the present invention also can be used in the production of sweeting agent or for the production of in organic fermentation process, described organic compound is ethanol, citric acid, xitix, amino acid, microbiotic etc. such as.
Claims (7)
1. a saccharifying enzyme, is characterized in that, the aminoacid sequence of described saccharifying enzyme is SEQ ID NO:1.
2. a gene, is characterized in that, described genes encoding saccharifying enzyme according to claim 1.
3. gene as claimed in claim 2, its nucleotides sequence is classified as SEQ ID NO:2.
4. a recombinant expression vector, is characterized in that, described recombinant expression vector carries gene according to claim 2.
5. an engineering bacteria, is characterized in that, described engineering bacteria is the bacterial strain carrying recombinant expression vector according to claim 4.
6. engineering bacteria as claimed in claim 5, it is characterized in that, described bacterial strain is Trichodermareesei.
7. the application of saccharifying enzyme according to claim 1 in glucose production.
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| CN103013956A (en) * | 2012-11-30 | 2013-04-03 | 青岛蔚蓝生物集团有限公司 | Saccharifying enzyme and recombinant expression strain thereof |
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| CN103013956A (en) * | 2012-11-30 | 2013-04-03 | 青岛蔚蓝生物集团有限公司 | Saccharifying enzyme and recombinant expression strain thereof |
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