CN104099306A - Alpha transglucosidase and recombinant expression bacterial strain thereof - Google Patents

Alpha transglucosidase and recombinant expression bacterial strain thereof Download PDF

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CN104099306A
CN104099306A CN201310118904.XA CN201310118904A CN104099306A CN 104099306 A CN104099306 A CN 104099306A CN 201310118904 A CN201310118904 A CN 201310118904A CN 104099306 A CN104099306 A CN 104099306A
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alpha
transglucosidase
grape glycosidase
transfering grape
aspergillus niger
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王华明
訾祯祯
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Tianjin Institute of Industrial Biotechnology of CAS
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Tianjin Institute of Industrial Biotechnology of CAS
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01024—1,4-Alpha-glucan 6-alpha-glucosyltransferase (2.4.1.24)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

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Abstract

The invention relates to a novel alpha transglucosidase with an amino acid sequence of SEQ ID NO: 1. Aspergillus niger At3 for the recombinant expression of the alpha transglucosidase has a preservation number of CGMCC No.6923. The recombinant bacterial strain obtained by the invention can express alpha transglucosidase, which has enzyme activity much higher than that in wild mushrooms, and higher than that of the alpha transglucosidase expressed by the host of Aspergillus niger. At the same time, the recombinant alpha transglucosidase is secreted by the Aspergillus niger with food safety, so as enlarge the application field of alpha transglucosidase.

Description

α transfering grape glycosidase and recombinant strains thereof
Technical field
The invention belongs to the separated expression technology of enzyme field, be specifically related to a kind of novel α transfering grape glycosidase that derives from terreus (Aspergillus terreus), and this α transfering grape glycosidase at filamentous fungal host cell (such as the heterogenous expression in aspergillus niger (Aspergillus niger).
Background technology
α transfering grape glycosidase (α-transglucosidase E.C. 2.4.1.24) can cut α-1,4 glycosidic links from the non reducing end of oligosaccharides substrate, discharge glucose, the glucosyl residue maybe dissociating is transferred on another carbohydrate substrate with α-1,6 glycosidic links, thereby obtain non-fermentable oligomeric isomaltose (be called for short IMO, mainly comprise isomaltose, panose, Isomaltotriose etc.), glycolipid or glycopeptide etc.This enzyme had both had hydrolysis ability, can carry out the shift reaction of glucoside bond again single-mindedly, was one of indispensable enzyme preparation of producing oligomeric isomaltose.
α transfering grape glycosidase is widely distributed at occurring in nature, of a great variety, and character is different, is almost present in all organisms, aspect the mankind's glycogen degraded and the sugar metabolism of animal, plant and microorganism, has important physiological function.α transfering grape glycosidase is mainly used in produces IMO, and IMO is the multiplicaiton factor of human intestine's profitable strain bifidus bacillus, not digested after absorption, be also difficult for being utilized by the most putrefactive bacterium in large intestine, but can be utilized as the carbon source of bifidus bacillus.Therefore as a kind of functional food ingredient, IMO has been widely used in the manufacture of various food, occupies first of various functional oligoses.
At present industrial to have had the starch of take or maltose be raw material, and by the technique of Production by Enzymes oligomeric isomaltose, but existing production technique transformation efficiency is not high.In addition, although China's isomaltose output is very high, the consumption of α transfering grape glycosidase is very large, there is no suitability for industrialized production, and this enzyme is dependence on import still.Therefore, this area is needed badly and is obtained the higher oligomeric isomaltose of highly purified activity of conversion, improves the transformation efficiency of production technique; Also need badly and obtain corresponding highly active α transfering grape glycosidase and produce accordingly bacterial strain.
Summary of the invention
The object of this invention is to provide a kind of α transfering grape glycosidase and recombinant strains thereof, thereby make up the deficiencies in the prior art.
One aspect of the invention provides a kind of novel α transfering grape glycosidase, and its aminoacid sequence is SEQ ID NO:1.
Another aspect of the present invention be provided for the encoding gene of above-mentioned α transfering grape glycosidase, its nucleotides sequence is classified SEQ ID NO:2 as.
α transfering grape glycosidase of the present invention amplifies from terreus.
The present invention is also provided for expressing the Aspergillus strain of above-mentioned α transfering grape glycosidase;
A kind of aspergillus niger of express alpha transfering grape glycosidase (Asperillus niger) At3, on November 30th, 2012, be stored in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, strain number is CGMCC No.6923.
α transfering grape glycosidase of the present invention and recombinant aspergillus niger bacterial strain are for the production of oligomeric isomaltose.
The resulting restructuring bacterial classification of the present invention can express alpha transfering grape glycosidase, and its enzyme activity is much higher than its enzyme in wild mushroom lives, and higher than the enzyme activity of the α transfering grape glycosidase of Host Strains aspergillus niger oneself expression.Meanwhile, gained recombinant alpha transfering grape glycosidase is by food safety bacterium aspergillus niger secretion gained, has expanded the Application Areas of α transfering grape glycosidase.
Accompanying drawing explanation
Fig. 1: the genetic map of pGm plasmid used in the present invention;
Fig. 2: the fermented liquid protein SDS-PAGE gel figure of aspergillus niger (Asperillus niger) At3, the expression that has shown aspergillus niger transformant At3, and the protein expression situation of wild mushroom and Host Strains aspergillus niger G1, wherein swimming lane 1 is depicted as protein standard molecular weight marker, be from top to bottom 116.0 kD, 66.2 kD, 45.0 kD, 35.0kD, 25.0kD and 18.4kD; Swimming lane 2 is depicted as the protein expression situation of aspergillus niger host G1 after fermentation 5d; Swimming lane 3 is depicted as the α transfering grape glycosidase expression of At3, at 107kDa place, can see clear protein band; Swimming lane 4 is depicted as the protein expression situation of wild mushroom.
Embodiment
The present invention has used routine techniques and the method in genetic engineering and biology field use.MOLECULAR CLONING:A LABORATORY MANUAL for example, the technology of recording in the book of reference such as 3nd Ed. (Sambrook, 2001) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).But this does not also mean that described any concrete grammar, experimental program and reagent, the scheme that those of ordinary skill in the art can select published technology to implement to record in the embodiment of the present invention of limiting the invention to.
Oligomeric isomaltose and the dextrinosan in specification sheets of the present invention, recorded are used interchangeably, and all refer to be selected from one or more sugar of lower group: isomaltose, Isomaltotriose, Isomaltotetraose or panose.In addition, should be understood that described term also comprises the mixture of above-mentioned sugar.
The α transfering grape glycosidase that the present invention records, be called again alpha-D-glucose glycosides lytic enzyme, can cut α-1 in maltose and malto-oligosaccharide molecular structure, 4 glycosidic links, and a free glucosyl residue out can be transferred to α-1 in another glucose molecule or maltose or trisaccharide maltose equimolecular, on 6, its transglycosylation can be by α-1 in oligose, 4 glycosidic links change into α-1,6 glycosidic links or other forms of link, thus non-fermentable oligomeric isomaltose or sugar ester, glycopeptide etc. obtained.
Gene refers to participate in producing the DNA fragmentation of polypeptide, region before comprising coding region and afterwards, and the insertion sequence (intron) between each encode fragment (exon).
Nucleic acid comprises DNA, RNA, strand or two strands, and their chemical modification object; Nucleic acid and polynucleotide can exchange use in this manual.
The analysis of the expression of enzymes in the present invention and the method for measuring of enzyme activity are as follows:
In order to evaluate the expression of α transfering grape glycosidase, can analyze at protein level or nucleic acid level.Adaptable analytical procedure comprises the in situ hybridization that Northern trace, Dot blot (DNA or RNA analyze), Southern trace, radioautograph, RT-PCR (ThermoScript II polymerase chain reaction) and the probe (based on nucleic acid coding sequence) that contains suitable mark carry out.In addition, genetic expression can be passed through immunological method, such as the immunohistochemical staining of cell, tissue slice or the immunity test of tissue culture medium (TCM).For example by Western trace or ELISA, assess.Such immunity test can be for evaluating the expression of α transfering grape glycosidase (such as At3) qualitatively and quantitatively.The details of these class methods is well known by persons skilled in the art, and can business obtain for implementing many reagent of these class methods.In some embodiments, the expression of α transfering grape glycosidase (such as At3) is analyzed by SDS-PAGE.
The determination of activity of α transfering grape glycosidase (such as At3) adopts HPLC method, both can use starch for raw material, also can use maltose for raw material, according to the amount of the oligose such as the isomaltose generating, Pan Tang, judges enzyme height alive.A kind of preferred measuring method comprises: take starch as raw material (concentration 10% ~ 40%), through high temperature α-amylase (0.05% ~ 0.5%), 90 ℃ ~ 115 ℃ liquefaction, arrive DE10 ~ 20, boil 5 minutes ~ 15 minutes enzymes that go out, be cooled to 60 ℃, adjust pH to 4 ~ 7, add Pullulanase (0.05% ~ 0.5%), α fungal amylase (0.05% ~ 0.5%) and Maltogenase (0.05% ~ 0.5%), then add α transfering grape glycosidase (0.05% ~ 0.5%) to be incubated conversion (30 ℃ ~ 60 ℃ of temperature; 24 ~ 72 hours time), thereby obtain the oligomeric isomaltose slurry of high-content, gained syrup is carried out to HPLC analysis.Another kind of preferred measuring method comprises: the maltose (concentration 10% ~ 40%) of take is substrate, adds α transfering grape glycosidase (0.05% ~ 0.5%), is then incubated conversion (30 ℃ ~ 60 ℃ of temperature; 24 ~ 72 hours time), obtain the oligomeric isomaltose slurry of high-content, gained syrup is carried out to HPLC analysis.In some embodiments, the activity of α transfering grape glycosidase (such as At3) selects maltose to measure as substrate.
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1 clone α transfering grape glycosidase Gene A t3
According to the specification sheets of manufacturers, use fungal genomic DNA to extract test kit (Omega) and extract genomic dna from the overnight culture of terreus.According to the sequences Design PCR primer that is numbered XM_001210809 on NCBI.For cloning the forward primer At3-F sequence of the At3 gene of terreus, be 5 '-ccaTTACGTAATGGTTGACATCACCGACCTTCTGG, reverse primer At3-R sequence is 5 '-CTGCTCTAGACTACCACTCCAGGACCCAGTCCTT, and this gene is increased out from terreus genomic dna with Phusion archaeal dna polymerase (Thermo scientific).
Amplification condition:
The first step: 98 ℃ keep 3min.
Second step: 98 ℃ keep 10s, then 72 ℃ keep 75s, and this step repeats 30 times.
The 3rd step: 72 ℃ keep 10min.
Use gel-purified test kit (Fermentas) by above-mentioned PCR product purification.With restriction enzyme SnaBI and XbaI (Fermentas), sublimed PCR product being carried out to enzyme cuts; Meanwhile, with restriction enzyme SnaBI and XbaI to plasmid pGm(genetic map as Fig. 1) carry out enzyme and cut.Use gel-purified test kit that enzyme is cut to product purification, and with T4 DNA ligase (Fermentas), above-mentioned two enzymes are cut to product connection.Connection product is transformed into Trans5 α intestinal bacteria (Transgen), with penbritin, selects.For guaranteeing accurately, to some clones check order (Invitrogen).
According to the specification sheets of manufacturers, use amount in plasmid to prepare test kit (Axygen) plasmid purification from the correct escherichia coli cloning of sequencing result.2 plasmids of gained are respectively pGm-At3, and the nucleotides sequence of the gene that result amplifies is classified SEQ ID NO:2 as, and the amino acid of its translation (albumen) sequence is SEQ ID NO:1, and sequence alignment result shows that it is a new allelotrope.
The protoplast fusion of embodiment 2:PEG mediation transforms aspergillus niger
Draw aspergillus niger G1 spore suspension in the dull and stereotyped center (9 cm culture dish) of CMA, treat that bacterium colony covers with whole culture dish, cut the cultivation of 1/4 size based in 200 mL CMA liquid nutrient mediums, at 30 ℃, the condition of 200 rpm, cultivate 14 ~ 16 h.
With aseptic Miracloth filter cloth, collect mycelium, and clean once by solution A, the mycelium cleaning is transferred to 40 mL protoplast formation solution under aseptic condition, under 30 ℃, the condition of 200 rpm, temperature is bathed 1 ~ 2 h, with microscopic examination, detects protoplast formation progress.
With the above-mentioned temperature body lotion of aseptic Miracloth filter-cloth filtering body, gained filtrate is protoplastis solution.Protoplastis solution is sub-packed in the aseptic disposable centrifuge tube of two 50 mL, and the volume of every pipe is settled to 45 mL by solution B, under 4000 rpm conditions, centrifugal 8 min are to obtain precipitation abandoning supernatant.By 20 mL solution B, twice of washing and precipitating again.Pellet resuspended, in 10 mL solution B, and is counted protoplastis with blood counting chamber.By protoplastis recentrifuge abandoning supernatant, according to blood counting chamber count results, add the resuspended precipitation of appropriate solution B, make protoplastis number 1 * 10 7individual/mL.
On ice, the above-mentioned protoplastis solution of 100 μ L is added in the aseptic 15 mL centrifuge tubes of precooling to 1 pipe for each conversion reaction.Add 10 μ g DNA.Add 12.5 μ L solution C, after gentleness mixes, place again 20 min on ice.
MMSA top-agar test tube is melted and remain on 55 ℃.From ice, shift out above-mentioned 15 mL centrifuge tubes, and Xiang Guanzhong adds 1 mL solution C and 2 mL solution B, gentleness mixes each pipe, and gained mixture is protoplastis mixture.In each in 3 top-agar test tubes, add the above-mentioned protoplastis mixture of 1 mL, and topple over immediately with MMSA flat board on, and flat board is cultivated at 30 ℃ to 7 ~ 10 d.
Solution A: 2.5 mL 1M K 2hPO 4, 2.5 mL 1M KH 2pO 4, 48.156 g MgSO 4, add dlH 2o is to final volume 500 mL, with the filtering with microporous membrane degerming of 0.22 μ m.
Solution B: 5 mL 1M Tris (pH 7.5), 2.77 g CaCl 2, 109.32 g sorbyl alcohols, add dlH 2o is to final volume 500 mL, with the filtering with microporous membrane degerming of 0.22 μ m.
Solution C: 250 g PEG 4000,2.77 g CaCl 2, 5 mL 1M Tris (pH 7.5), add dlH 2o is to final volume 500 mL, with the filtering with microporous membrane degerming of 0.22 μ m.
Protoplast formation solution: 0.6 g lyase (Lysing Enzyme from Trichoderma harzianum, Sigma) is dissolved in 40 mL solution A, with the filtering with microporous membrane degerming of 0.22 μ m.
MMSA is dull and stereotyped: 0.59 g ethanamide (Sigma), 3.4 g CsCl (Sigma), 0.52 g KCl, 1.52 g KH 2pO 4, 218.5 g sorbyl alcohols, 1 ml trace elements (see below), 20 g agar, add dlH 2o, to final volume 972.5 mL, adds 25 mL 40% glucose and 2.5 mL 20% MgSO with the filtering with microporous membrane degerming of 0.22 μ m after high pressure steam sterilization 4.
MMSA top-agar test tube: 0.59 g ethanamide (Sigma), 3.4 g CsCl (Sigma), 0.52 g KCl, 1.52 g KH 2pO 4, 218.5 g sorbyl alcohols, 1 ml trace elements (see below), 10 g low melting-point agaroses, add dlH 2o, to final volume 972.5 mL, after high pressure steam sterilization, when substratum does not solidify, adds 25 mL 40% glucose and 2.5 mL 20% MgSO with the filtering with microporous membrane degerming of 0.22 μ m 4, be sub-packed in immediately afterwards in sterile test tube every pipe 10 mL.
Trace elements: at 250 mL dlH 2in O, add 1 g FeSO 47H 2o, 8.8 g ZnSO 4. 7h 2o, 0.4 g CuSO 45H 2o, 0.15 g MnSO 44H 2o, 0.1 g Na 2b 4o 710H 2o, 50 mg (NH 4) 6mo 7o 244H 2o, the dense HCl of 0.2 mL, uses dlH after dissolving completely 2o is settled to 1 L, with the filtering with microporous membrane degerming of 0.22 μ m.
CMA is dull and stereotyped: 20 g glucose, and 20 g Fructus Hordei Germinatus extracts, 1 g peptone, 15 g agar, add dlH 2o is to final volume 1000 mL, autoclaving.
CMA liquid nutrient medium: 20 g glucose, 20 g Fructus Hordei Germinatus extracts, 1 g peptone, adds dlH 2o is to final volume 1000 mL, autoclaving.
Until growing on flat board after bacterium colony, select 25 bacterium colonies, according to the specification sheets of manufacturers, use fungal genomic DNA to extract test kit (Omega) and extract genomic dna from its overnight culture.By experimental procedure described in embodiment 1, the aspergillus niger transformant of At3 is carried out to PCR checking, gained PCR product is checked order.The spore suspension of gained positive colony is inoculated in 30mL TSB fermention medium, at 30 ℃, under the condition of 200 rpm, cultivates 5d, 8 layers of filtered through gauze for gained fermented liquid, and filtrate is centrifugal 10 min under 14000 * g condition, collect supernatant liquor.Gained supernatant liquor is enzyme liquid.On the SDS-PAGE glue that is 12% in concentration by enzyme liquid, carry out electrophoresis, to there is the positive colony of obvious band to carry out HPLC analysis at desired location place.According to HPLC, analyze collection of illustrative plates, choose enzyme activity soprano and further study as the recombinant bacterium of At3 transfering grape glycosidase.The aspergillus niger of choosing (Asperillus niger) At3, on November 30th, 2012, be stored in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) that is positioned at Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, strain number is CGMCC No.6923.
Embodiment 3: aspergillus niger (Asperillus niger) fermentation of At3 and the expression of enzyme
The spore suspension of expressing aspergillus niger (CGMCC No.6923) transformant of At3 α transfering grape glycosidase is inoculated in 30mL TSB fermention medium, at 30 ℃, under the condition of 200 rpm, cultivates 5 d.8 layers of filtered through gauze for gained fermented liquid, filtrate is centrifugal 10 min under 14000 * g condition, collect supernatant liquor.Gained supernatant liquor is At3 α transfering grape glycosidase enzyme liquid.On the SDS-PAGE glue that is 12% in concentration by enzyme liquid, carry out electrophoresis (Fig. 2); Electrophoresis result is presented at 107kDa place can see obvious band, and wild mushroom does not have visible protein band in desired location, and Host Strains G1 has extremely faint band, but expression amount is obviously much smaller than the expression amount of recombinant bacterium.
Acetic acid-sodium-acetate buffer of preparation pH4.6, At3 enzyme liquid is diluted to suitable concentration with above-mentioned damping fluid.Take 10% maltose as substrate, add appropriate At3 dilution enzyme liquid, then 50 ℃ of insulations, transform more than 24 hours, obtain converted product oligomeric isomaltose slurry.By 10 times of product syrup dilutions, get 1ml dilution syrup after the disposable filtering with microporous membrane of 0.22 μ m, carry out HPLC analysis.The demonstration of HPLC result, the content of substrate maltose has obvious minimizing, and generation and its output that this process is attended by isomaltose and Isomaltotriose significantly increase, and also have a little panose to produce simultaneously.This experimental result was with previously expected result was consistent, proved that terreus α transfering grape glycosidase of the present invention is transformed in aspergillus niger to express, and truly had good transfering grape glycosidase active, can produce oligomeric isomaltose for catalysis maltose.
TSB fermention medium: 12 g NaNO 3, 0.5 g KCl, 1.5 g KH 2pO 4, 2.05 g MgSO 47H 2o, 3.5 g NaH 2pO 4h 2o, 45 g Trypsin soybean broths, 70 g Trisodium Citrates, 1 g tween 80,1 mL trace elements (see below), adds dlH 2o, to final volume 700 mL, adds 40% maltose of the filtering with microporous membrane degerming of 0.22 μ m for 300 mL after autoclaving.
Trace elements: at 250 mL dlH 2in O, add 1 g FeSO 47H 2o, 8.8 g ZnSO 4. 7h 2o, 0.4 g CuSO 45H 2o, 0.15 g MnSO 44H 2o, 0.1 g Na 2b 4o 710H 2o, 50 mg (NH 4) 6mo 7o 244H 2o, the dense HCl of 0.2 mL, uses dlH after dissolving completely 2o is settled to 1 L, with the filtering with microporous membrane degerming of 0.22 μ m.
The application example of embodiment 4 express alpha transfering grape glycosidases
Industrial production oligomeric isomaltose slurry, take starch as raw material, add water and make 30% powder slurry, in pH value, be 6.0, temperature is under 90 ~ 120 ℃ of conditions, after thermotolerance α-amylaseliquefied, in pH value, be 5.0, temperature is under the condition of 60 ℃, with beta-amylase, Pullulanase and fungal alpha-amylase act on simultaneously, change into after maltose, the strain number that adds embodiment 3 is that the expressed α-transfering grape glycosidase of aspergillus niger of CGMCC No.6923 turns glycosides reaction generation isomaltose, Isomaltotriose, tetrose and pentasaccharides etc. are containing α-1, the IMO of 6 keys and panose.Add α-transfering grape glycosidase to make the obvious raising of its transformation efficiency can reach more than 40% ~ 50%, again through activated carbon decolorizing, ion exchange resin desalination, being concentrated to solid substance is 75%, obtain common iso-maltose syrup, containing dextrinosan, be wherein 40% ~ 50%, glucose is 40%, then glucose is removed with yeast fermentation or membrane filtration, just can obtain containing the product that dextrinosan is 90%.The recombinant bacterial strain that the above results shows institute of the present invention preservation is express alpha transfering grape glycosidase efficiently, and its enzyme activity is much higher than its enzyme in wild mushroom lives, and higher than the enzyme activity of the α transfering grape glycosidase of Host Strains aspergillus niger oneself expression.

Claims (5)

1. a α transfering grape glycosidase, is characterized in that, the aminoacid sequence of described α transfering grape glycosidase is SEQ ID NO:1.
2. the application of α transfering grape glycosidase claimed in claim 1 in producing oligomeric isomaltose.
3. for the gene of the α transfering grape glycosidase claimed in claim 1 of encoding, its nucleotides sequence is classified SEQ ID NO:2 as.
3, for the Aspergillus strain of recombinant expressed α transfering grape glycosidase claimed in claim 1.
4. Aspergillus strain as claimed in claim 3, is aspergillus niger (Asperillus niger) At3, and its deposit number is CGMCC No.6923.
5. the application of Aspergillus strain claimed in claim 4 in producing oligomeric isomaltose.
CN201310118904.XA 2013-04-08 2013-04-08 Alpha transglucosidase and recombinant expression bacterial strain thereof Pending CN104099306A (en)

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CN104878054A (en) * 2015-02-12 2015-09-02 广西南宁智天生物科技有限公司 Carrier-free immobilization aspergillus niger production method of isomaltose hypgather
CN104877911A (en) * 2015-02-12 2015-09-02 广西南宁智天生物科技有限公司 Aspergillus niger and application in production of isomaltose hypgather

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BIRREN,B.等: "Aspergillus terreus NIH2624 alpha-glucosidase precursor (ATEG_00723) partial mRNA", 《NCBI REFERENCE SEQUENCE: XM_001210809.1》, 31 March 2008 (2008-03-31) *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104878054A (en) * 2015-02-12 2015-09-02 广西南宁智天生物科技有限公司 Carrier-free immobilization aspergillus niger production method of isomaltose hypgather
CN104877911A (en) * 2015-02-12 2015-09-02 广西南宁智天生物科技有限公司 Aspergillus niger and application in production of isomaltose hypgather
CN104878054B (en) * 2015-02-12 2018-02-13 广西南宁智天生物科技有限公司 A kind of method of carrier-free immobilization aspergillus niger production oligoisomaltose
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