CN103740599B - Strain for producing alpha-transglucosidase and application thereof - Google Patents
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Abstract
The invention provides Asperillus niger Afm78 for producing alpha-transglucosidase with high yield. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology of Chinese Academy of Science in Datun Road, Chaoyang District in Beijing on November 12th, 2013, and the preservation number of the strain is CGMCC No.8470. According to the strain, Aspergillus niger CGMCC No.8245 is used as an original strain, the Asperillus niger Afm78 for producing alpha-transglucosidase with high yield is obtained by using an ultraviolet mutagenesis method, and the fermenting enzyme activity of the strain is as high as 6699U/mL, that is, is 54% higher than that of the original strain. The alpha-transglucosidase expressed by the Asperillus niger Afm78 can be widely applied to the field of food machining. By adding the alpha-transglucosidase provided by the invention in production of isomalto oligosaccharide, the glycoside transferring efficiency can be greater than 50%, the mass volume ratio of the isomalto oligosaccharide in an obtained product can be greater than 92%, and wide market prospect is achieved.
Description
Technical field
The invention belongs to microbe to screen technical field, be specifically related to a kind of production bacterial strain and application thereof of α-transfering grape glycosidase.
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 saccharide substrates with α-1,6 glycosidic links, thus obtain the oligomeric isomaltose (be called for short IMO, mainly comprise the oligose of more than isomaltose, panose, Isomaltotriose and tetrose) of non-fermented, glycolipid or glycopeptide etc.This enzyme both had hydrolysis ability, exclusively can carry out again the shift reaction of glucoside bond, was one of indispensable enzyme preparation producing oligomeric isomaltose.
α-transfering grape glycosidase is widely distributed at occurring in nature, of a great variety, distinct, be almost present in all organisms, in the sugar metabolism of the glycogen degradation of the mankind and animal, plant and microorganism, have important physiological function, this enzyme is mainly used in production oligomeric isomaltose.IMO is as functional oligose, not digested after absorption, also not easily utilize by the most putrefactive bacteriums in large intestine, but IMO is the multiplicaiton factor of human intestine's profitable strain bifidus bacillus, can be utilized as the carbon source of bifidus bacillus, have promote that intestinal beneficial flora rises in value, relax bowel, adjusting blood lipid, low sugariness, the unique effects such as low in calories.As a kind of functional food ingredient, IMO has been widely used in the manufacture of various food as milk-product, candy class, bakery product, beverage, drinks, meat product, infant or baby food etc.
Industrial at present had with starch or maltose for 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,
α-transfering grape glycosidase produce as oligomeric isomaltose in the consumption of zymin of most critical very large, but domesticly never realize suitability for industrialized production, this enzyme still dependence on import, these limiting factors all greatly constrain the production of domestic oligomeric isomaltose.Therefore, this area needs the production bacterial strain obtaining highly active α-transfering grape glycosidase and corresponding more stable, high yield α-transfering grape glycosidase thereof badly, to adapt to the needs of oligomeric isomaltose production technique, for suitability for industrialized production lays the foundation.
Summary of the invention
The present invention is for solving prior art problem, provide a kind of production bacterial strain of α-transfering grape glycosidase, this bacterial strain is for starting strain with aspergillus niger (Asperillus niger) CGMCC No.8245, by the method for ultraviolet mutagenesis, it is suddenlyd change, finishing screen chooses the aspergillus niger mutant strain that a plant height produces α-transfering grape glycosidase, thus is conducive to the widespread use promoting α-transfering grape glycosidase.
The invention provides the aspergillus niger Afm78(Asperillus nigerAfm78 of a kind of high yield α-transfering grape glycosidase), be stored on November 12nd, 2013 and be positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of Institute of Microorganism, Academia Sinica, bacterial strain deposit number is CGMCC No.8470.
For aspergillus niger (Asperillus niger) the Af7 strain as initiating mutagenic, be preserved on September 25th, 2013 and be positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.8245.
Aspergillus niger Afm78 of the present invention is for the production of α-transfering grape glycosidase.
The present invention with aspergillus niger CGMCC No.8245 for starting strain, ultraviolet mutagenesis method is utilized to obtain the mutant strain aspergillus niger Afm78(Asperillus niger Afm78 of a plant height product α-transfering grape glycosidase), its fermenting enzyme work is up to 6699U/mL, and than setting out, bacterium improves 54%.α-transfering grape glycosidase that described aspergillus niger Afm78 expresses, can be widely used in food processing field.Wherein in isomaltooligosaccharide syrup is produced, add α-transfering grape glycosidase of the present invention, can make to turn glycosides efficiency and improve more than 50%, in the product obtained, the mass volume ratio of dextrinosan reaches more than 92%, wide market.
Accompanying drawing explanation
Fig. 1: mutant strain aspergillus niger Afm78 fermented supernatant fluid SDS-PAGE electrophoresis detection figure of the present invention, wherein swimming lane 1 is depicted as protein standard molecular weight marker, is from top to bottom 116.0kD, 66.2kD, 45.0kD, 35.0kD, 25.0kD, 18.4kDa and 14.4kD; Swimming lane 2 is depicted as protein expression situation in starting strain Asperillus nigerCGMCC No.8245 fermented supernatant fluid; Swimming lane 3 is depicted as protein expression situation in mutant strain aspergillus niger Afm78 fermented supernatant fluid, and the protein band at arrow indication 109kDa place is recombinant expressed α-transfering grape glycosidase;
The relative enzyme work of Fig. 2: α-transfering grape glycosidase and the graphic representation of pH;
The relative enzyme work of Fig. 3: α-transfering grape glycosidase and the graphic representation of temperature.
Embodiment
The present invention has used the routine techniques and method that use in genetic engineering and biology field.Such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and technology described in the book of reference such as CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).But this does not also mean that and limits the invention to the concrete grammar described in specific embodiment, experimental program and reagent, and those of ordinary skill in the art can select published technology to implement the scheme recorded in the embodiment of the present invention.
In the present invention, 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.
The oligomeric isomaltose recorded in specification sheets of the present invention and dextrinosan are used interchangeably, and all refer to one or more sugar being selected from lower group: isomaltose, Isomaltotriose, Isomaltotetraose or panose.In addition, should be understood that described term also comprises the mixture of above-mentioned sugar.
α transfering grape glycosidase described in the present invention, be also called alpha-D-glucose glycosides lytic enzyme, α-1 in maltose and malto-oligosaccharide molecular structure can be cut, 4 glycosidic links, and the α-1 that a free glucosyl residue out can be transferred in another glucose molecule or maltose or trisaccharide maltose equimolecular, on 6, its transglycosylation can by the α-1 in oligose, 4 glycosidic links change into α-1,6 glycosidic links or other forms of link, thus the oligomeric isomaltose or sugar ester, the glycopeptide etc. that obtain non-fermented.
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).
Nucleic acid comprises DNA, RNA, strand or double-strand, and their chemical modification object; Nucleic acid and polynucleotide can exchange use in this manual.
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 coding for alpha-transfering grape glycosidase 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.
Filamentous fungus refers to that the Eumycotina biology of all filamentous form is (see INTRODUCTORYMYCOLOGY, 4th Ed. (Alexopoulos, 2007) and AINSWORTH AND BISBYDICTIONARY OF THE FUNGI, 10th Ed. (Kirk et al., 2008)).The feature of these fungies is the vegetative myceliums with the cell walls be made up of chitin, Mierocrystalline cellulose and other complicated polysaccharide.Filamentous fungus of the present invention is different from yeast on morphology, physiology and genetics.Nourishing and growing of filamentous fungus has been come by the extension of mycelia, and carbon metablism is obligate aerobic.In the present invention, filamentous fungal parent cell can make, but is not limited to, Aspergillus certain (Aspergillus sp.) (such as excellent aspergillus (A.clavatus), Aspergillus fumigatus (A.fumigatus), Aspergillus awamori (A.awamori), flavus (A.flavus), terreus (A.terreus) and aspergillus oryzae (A.oryzae)), Penicillium certain (Penicillium sp.) (such as Penicllium chrysogenum (P.chrysogenum)), Xin Satuo Pseudomonas certain (Neosartorya sp.) (such as Fei Xixinsatuo bacterium (N.fischeri)), gliocladium germ belongs to certain (Gliocladium sp.) (such as Gliocladium roseum (G.roseum)), Trichoderma certain (Trichoderma sp.) (such as Trichodermareesei (T.reesei), viride (T.viride), healthy and free from worry wood mould (T.koningii), trichoderma harziarum (T.harzianum)), Humicola certain (Humicola sp.) (such as Humicola insolens (H.insolens) and grey humicola lanuginosa (H.grisea)), the gold mould genus of spore certain (Chrysosporium sp.), Fusarium certain (Fusarium sp.), Neurospora sp belongs to certain (Neurospora sp.), the cell of Hypocrea certain (Hypocrea sp.) and Emericella certain (Emericella sp.).
Aspergillus involved in the present invention or Aspergillus certain referred to before or be classified as any fungi of Aspergillus at present.
The analysis of the expression of enzymes in the present invention and the measuring method of enzyme activity as follows:
In order to evaluate the expression of α-transfering grape glycosidase, can analyze at protein level or nucleic acid level.The in situ hybridization that adaptable analytical procedure comprises Northern trace, Dot blot (DNA or RNA analysis), Southern trace, radioautograph, RT-PCR (ThermoScript II polymerase chain reaction) and carries out containing the probe (based on nucleic acid coding sequence) suitably marked.In addition, genetic expression can pass through immunological method, the such as immunohistochemical staining of cell, tissue slice or the immunity test of tissue culture medium (TCM).Such as assessed by western blot or ELISA.Such immunity test may be used for the expression evaluating α-transfering grape glycosidase (such as Afm78) qualitatively and quantitatively.The details of these class methods is well known by persons skilled in the art, and can business obtain for the many reagent implementing these class methods.In some embodiments, the expression of α-transfering grape glycosidase (such as Afm78) is analyzed by SDS-PAGE.
Embodiment 1: ultraviolet mutagenesis and screening
Starting strain: aspergillus niger (Asperillus niger) CGMCC No.8245 is by entering in aspergillus niger Host Strains G1 by the α-transfering grape glycosidase gene transformation deriving from Aspergillus fumigatus (Aspergillus fumigatus), builds the aspergillus niger engineering bacteria of the energy heterogenous expression α-transfering grape glycosidase obtained.
1.1 ultraviolet mutagenesis methods
(1) starting strain Asperillus niger CGMCC No.8245 is inoculated on CMA inclined-plane activates, at 37 DEG C, cultivate 4d;
(2) obtain spore suspension with the fresh inclined-plane that the 0.1%Tween-80 washing that 3ml is aseptic activates, absorption 10ul is placed on blood counting chamber and counts, and is about 10 according to count results dilution spore suspension to spore number
6individual/about ml;
(3) spore suspension drawn after 5ml dilution is placed in 9cm culture dish, under the condition of ultraviolet lamp 30w, irradiation distance 22cm, irradiation time 4min, carry out ultraviolet mutagenesis;
(4) after uv irradiating, the spore suspension after mutagenesis is diluted 100 times, draw the spore suspension coating CMA after dilution dull and stereotyped, each CMA flat board is coated with 100ul, and be about coated with 100 dull and stereotyped left and right, at 37 DEG C, lucifuge cultivates 40h;
(5), after cultivating 40h, the individual morphology that picking flat board grows is little, mycelia is dense and the sudden change bacterium colony of consolidation, and on another CMA flat board, carry out line purifying, streak plate cultivates 40h at 37 DEG C;
(5) choose single bacterium colony that about 100 streak plates grow, be seeded to CMA inclined-plane, cultivate more than 4 days at 37 DEG C;
CMA is dull and stereotyped: 20g glucose, 20g Fructus Hordei Germinatus extract, 1g peptone, 15g agar, adds dlH
2o to final volume 1000mL, autoclaving.
The screening of 1.2 mutant strains
1, primary dcreening operation
According to the growing state of mutagenic strain on CMA inclined-plane, select the close mutagenic strain of growing way and carry out fermentation expression in batches, compare with starting strain simultaneously.Fermentation employing one step shake flask culture: be inoculated in respectively by the spore suspension of aspergillus niger mutagenic strain in 20mL TSB fermention medium, each bacterial strain connects 1 bottle, at 30 DEG C, cultivates 5d under the condition of 200rpm; By gained fermented liquid 8 layers of filtered through gauze, filtrate is centrifugal 10min under 14000 × g condition, collects supernatant liquor; By supernatant liquor in concentration be 12% SDS-PAGE glue on carry out electrophoresis, by the expression amount of observation and comparison α-transfering grape glycosidase recombinant protein band, pick out the mutant strain that 16 strains improve than starting strain expression efficiency.
2, multiple sieve
The 16 plant mutant bacterial strains picked out are carried out fermentation expression again, compares with starting strain simultaneously.Fermentation employing two step shake flask culture: the spore suspension of aspergillus niger mutagenic strain is inoculated in 20ml CLS fermention medium, at 30 DEG C, cultivates 48h under the condition of 200rpm; Then by 10% inoculum size, draw 2ml CSL nutrient solution thalline and be inoculated in 20mL TSB fermention medium, at 30 DEG C, under the condition of 200rpm, cultivate 5d; By gained fermented liquid 8 layers of filtered through gauze, filtrate is centrifugal 10min under 14000 × g condition, collects supernatant liquor; Use Econo-Pac10DG Columns(BOI-RAD) desalination is carried out except sugar process to supernatant liquor; Then carried out 12%SDS-PAGE detection analyze and measure enzyme work; Finally pick out a strain mutant strain that α-transfering grape glycosidase expression amount is the highest, called after aspergillus niger Afm78(Aspergillus nigerAfm78), and China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica is preserved on November 12nd, 2013, bacterial strain deposit number is CGMCC No.8470.
α-transfering grape glycosidase enzyme activity determination method
Adopt People's Republic of China (PRC) light industry standard QB2525-2001, α transfering grape glycosidase is acted on substrate Alpha-Methyl-D-Glucose glycosides and generate glucose, the glucose generated carries out color reaction with the 4-AA (4-Aminoantipyrin) containing glucose oxidase, peroxidase and phenol reagent and carrys out quantitative assay.
Concrete measuring method comprises: draw 2% Alpha-Methyl-D-Glucose glycosides substrate solution 1ml and 0.02mol/L acetic acid-sodium acetate buffer solution (pH5.0) 1ml and add in test tube (15mm × 150mm), in the constant water bath box of (50 ± 0.5) DEG C, be incubated 10min.Add sample enzyme liquid 0.5ml, mixing, in the constant water bath box of (50 ± 0.5) DEG C accurately after insulation 60min, test tube is transferred in boiling water bath and heats 5min, then cool fast with flowing water.After cooling, inhale this solution 0.1ml in test tube, and add 4-AA-phenol developer 3ml, mixing.The water bath with thermostatic control of this test tube being put into (40 ± 0.5) DEG C is incubated 20min, measure the absorbancy at 500nm place, the enzyme activity (U/ml) of α transfering grape glycosidase is calculated according to absorbance, namely under this test conditions, in reaction mixture 2.5ml, the 60min enzyme amount produced needed for 1 μ g glucose is defined as a α transfering grape glycosidase unit of activity.
TSB fermention medium: 12g NaNO
3, 0.5g KCl, 1.5g KH
2pO
4, 2.05gMgSO
47H
2o, 3.5g NaH
2pO
4h
2o, 45g Tryptose soy meat soup, 70g Trisodium Citrate, 1g tween 80,1mL trace elements (see below), adds dlH
2o, to final volume 700mL, adds 40% maltose that the 300mL filtering with microporous membrane of 0.22 μm is degerming after autoclaving.
Trace elements: at 250mL dlH
21g FeSO is added in O
47H
2o, 8.8g ZnSO
4.
7h
2o, 0.4g CuSO
45H
2o, 0.15g MnSO
44H
2o, 0.1g Na
2b
4o
710H
2o, 50mg (NH
4)
6mo
7o
244H
2the dense HCl of O, 0.2mL, uses dlH after dissolving completely
2o is settled to 1L, degerming with the filtering with microporous membrane of 0.22 μm.
TSB fermention medium: 12g NaNO
3, 0.5g KCl, 1.5g KH
2pO
4, 2.05gMgSO
47H
2o, 3.5g NaH
2pO
4h
2o, 45g Tryptose soy meat soup, 70g Trisodium Citrate, 1g tween 80,1mL trace elements (see below), adds dlH
2o, to final volume 700mL, adds 40% maltose that the 300mL filtering with microporous membrane of 0.22 μm is degerming after autoclaving.
Trace elements: at 250mL dlH
21g FeSO is added in O
47H
2o, 8.8g ZnSO
4.
7h
2o, 0.4g CuSO
45H
2o, 0.15g MnSO
44H
2o, 0.1g Na
2b
4o
710H
2o, 50mg (NH
4)
6mo
7o
244H
2the dense HCl of O, 0.2mL, uses dlH after dissolving completely
2o is settled to 1L, degerming with the filtering with microporous membrane of 0.22 μm.
CSL fermention medium: 100g corn steep liquor, 1g NaH
2pO
4h
2o, 0.5g MgSO
4, 100g maltose, 10g glucose, 50g fructose, adds dlH
2o to 900mL, is settled to 1L, autoclaving after adjusting pH5.8 with Solid NaOH pellets.
Embodiment 2: the shake flask fermentation checking of mutant strain aspergillus niger Afm78
By mutant strain aspergillus niger Afm78(CGMCC No.8470) and starting strain Simultaneous vaccination to CMA flat board, cultivate 4-5d, get its separately spore suspension be inoculated in 20ml CLS fermention medium respectively, at 30 DEG C, under the condition of 200rpm, cultivate 48h; Then by 10% inoculum size, draw 2ml CSL nutrient solution thalline and be inoculated in 20mL TSB fermention medium respectively, at 30 DEG C, under the condition of 200rpm, cultivate 5d, collect the fermented supernatant fluid of Afm78 and starting strain respectively; Use Econo-Pac10DG Columns(BOI-RAD) desalination is carried out except sugar process to supernatant liquor, then the analysis of 12%SDS-PAGE electrophoresis detection is carried out, result as shown in Figure 1, two strain bacterium all have obvious protein band at 109kDa place, illustrate that mutant strain aspergillus niger Afm78 that the present invention obtains also can recombinant expressed α-transfering grape glycosidase; Enzyme activity determination result shows, and the fermenting enzyme of starting strain Asperillus niger CGMCC No.8245 is lived as 4347U/mL, and the fermenting enzyme work of mutant strain aspergillus niger Afm78 is up to 6699U/mL, and than setting out, bacterium improves 54%.And after mutant strain goes down to posterity, the efficiency not significantly change of recombinant expressed α-transfering grape glycosidase, illustrates that the inherited character of the bacterial strain after sudden change keeps stablizing.
Embodiment 3: characterization analysis
With pH value be 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0, the fermented supernatant fluid of set out described in the damping fluid of 11.0 dilution embodiment 2 bacterium and mutant bacteria, measure its enzyme respectively to live, live as 100% with the highest enzyme, calculate relative enzyme and live, do the relative enzyme of effect pH-curve alive.As shown in Figure 2, α-transfering grape glycosidase that mutant strain aspergillus niger Afm78 of the present invention expresses is compared with the bacterium that sets out, and action pH-relative enzyme curve alive does not change, and Optimun pH is 5.0 for result.
Respectively at 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C, measure to set out described in embodiment 2 bacterium and mutant bacteria fermented supernatant fluid enzyme under pH5.0 condition and live, live as 100% with the highest enzyme, calculate relative enzyme and live, do the enzyme of temperature-relatively and to live curve.As shown in Figure 3, α-transfering grape glycosidase that mutant strain aspergillus niger Afm78 of the present invention expresses is compared with the bacterium that sets out, and operative temperature-relative enzyme curve alive does not change, and optimum temperature is 60 DEG C for result.
The above results shows, α-transfering grape glycosidase zymologic property generation Inactivating mutations that the sudden change of mutant strain aspergillus niger Afm78 that the present invention obtains does not cause it to express, and is that expression efficiency is improved.
Embodiment 4: α-transfering grape glycosidase is in the application of field of food
As a kind of functional food ingredient, isomaltooligosaccharide syrup has been widely used in the manufacture of various food as milk-product, candy class, bakery product, beverage, drinks, meat product, infant or baby food etc.
Industrial production isomaltooligosaccharide syrup, take starch as raw material, add water and make 30% powder slurry, be 6.0-7.0 in pH value, temperature is under 90 ~ 120 DEG C of conditions, after thermotolerance α-amylaseliquefied, be 5.0 in pH value, temperature is under the condition of 60 DEG C, with beta-amylase, Pullulanase and fungal alpha-amylase act on simultaneously, after changing into maltose, α-the transfering grape glycosidase adding mutant strain aspergillus niger Afm78 of the present invention expression carries out turning glycosides reaction and generates isomaltose, Isomaltotriose, tetrose and pentasaccharides etc. are containing α-1, the IMO of 6 keys and panose.Result shows, α-transfering grape glycosidase of the present invention can make its transformation efficiency significantly improve more than 50%.
By above-mentioned turn glycosides reaction product through activated carbon decolorizing, ion exchange resin desalination, being concentrated to solid substance is 60-70%, obtains common iso-maltose syrup, and wherein dextrinosan mass volume ratio is 45% ~ 55%, and glucose quality volume ratio is about 35%-40%; Again by the glucose yeast fermentation in iso-maltose syrup or membrane filtration removing, just can obtain containing dextrinosan mass volume ratio is the product of more than 92%.
The above results shows can be widely used in the production of isomaltooligosaccharide syrup, wide market by α-transfering grape glycosidase that mutant strain aspergillus niger Afm78 of the present invention expresses.
Claims (2)
1. an Aspergillus niger strain, is characterized in that, the deposit number of described Aspergillus niger strain is CGMCCNo.8470.
2. Aspergillus niger strain according to claim 1 is producing the application in α-transfering grape glycosidase.
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| 高产α-转移葡萄糖苷酶菌株的诱变选育研究;毕金峰 等;《生物技术》;20050630;第15卷(第3期);第32页左栏第1段倒数第1-4行 * |
| 黑曲霉α-葡萄糖转苷酶产生菌的诱变选育与低聚异麦芽糖生产的研究;管立忠;《中国优秀学位论文全文数据库 基础科学辑》;20111215;摘要第1-2段 * |
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