Alpha-glucan oligosaccharide and synthetic method and application thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and particularly relates to alpha-glucan oligosaccharide as well as a synthesis method and application thereof. The alpha-dextran oligosaccharide disclosed by the invention is prepared by catalyzing cane sugar and maltose under the combined action of dextran sucrase I and dextran sucrase II; the alpha-glucan oligosaccharide contains alpha-1, 2 glucosidic bonds, and the degree of polymerization is mainly between 3 and 6. Two kinds of glucan sucrase-producing genetically engineered bacteria are constructed, sucrose and maltose are used as raw materials, and the sucrose and the maltose are catalyzed by the two kinds of glucan sucrase to generate the alpha-glucan oligosaccharide which contains alpha-1, 2 glucosidic bonds and has the polymerization degree of 3-6. The obtained converted product is high in purity and easy to separate, and the product quality can be further improved. The obtained product is high in digestion resistance, so that the application of the alpha-glucan oligosaccharide is expanded. The method can be applied to industrial production of alpha-glucan oligosaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to alpha-glucan oligosaccharide and a synthesis method and application thereof. Background Art

[0002] α-Glucan oligosaccharide, also known as isomalto-oligosaccharide (IMO), is a type of oligosaccharide, containing α-l,4 glycosidic bonds, α-l,6 glycosidic bonds, a small amount of α-l,3 glycosidic bonds and α-l,2 glycosidic bonds in the molecule. Natural IMO is mainly found in fermented foods such as miso and rice wine, or in trace amounts in starch crops such as sweet potatoes and cassava in a free state. Isomaltooligosaccharide has good physical and chemical properties and physiological functions, with a sweetness of 40%-50% of sucrose, a soft and beautiful sweetness, and a refreshing taste; it is difficult to be digested by gastric enzymes and can promote the proliferation of bifidobacteria in the human intestine. It also has anti-caries properties and is widely used in food, medicine, feed, cosmetics and other industries.

[0003] Currently, the commercially available IMO is mainly produced by enzymatic methods, with a degree of polymerization (DP) of about 2 to 6. The mainstream industrial preparation method of IMO is based on the conversion of starch using enzymes. With starch as the substrate, starch is liquefied and saccharified by α-amylase and β-amylase to produce a low-polymerization oligosaccharide mixture mainly composed of maltose, and then combined with the transglycosidation of α-glucosidase to produce a complex oligosaccharide mixture with α-1,6 glycosidic bonds. The yield of IMO prepared by this method is generally 50% to 55%, which is the commercially available IMO50, whose main components are isomaltose, isomaltotriose and panose. The proportion of isomaltose disaccharide in IMO produced by this method is very high (about 50%), and the trisaccharide and above components generally account for less than 20% of the total sugar content. These have been proven to have a high digestibility. Studies have shown that the probiotic function of IMO is mainly attributed to the high-polymerization components of trisaccharides and above.

[0004] Oligosaccharides containing α-l,2 glycosidic bonds are much more resistant to digestive enzymes than oligosaccharides linked by linear α-(1,6) glycosidic bonds. If the proportion of digestible sugars in the product can be reduced and the proportion of α-l,2 glycosidic bonds in the product can be increased, the product quality can be further improved, the digestive resistance can be improved, and the selectivity of the intestinal flora can be improved. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide an α-glucan oligosaccharide and a synthesis method and application thereof. The present invention constructs two genetically engineered bacteria that produce glucan sucrase, uses sucrose and maltose as raw materials, and uses two glucan sucrases to catalyze sucrose and maltose to generate α-glucan oligosaccharides with a degree of polymerization between 3 and 6 containing α-1,2 glycosidic bonds. The obtained conversion product has high purity and is easy to separate, which can further improve the product quality. The obtained product has high digestion resistance, thereby expanding the application of α-glucan oligosaccharides. The present invention can be applied to the industrial production of α-glucan oligosaccharides.

[0006] The purpose of the present invention and the solution to the technical problem are achieved by adopting the following technical solutions.

[0007] One aspect of the present invention provides an α-glucan oligosaccharide, wherein the α-glucan oligosaccharide is produced by the combined action of dextran sucrase I and dextran sucrase II to catalyze sucrose and maltose; the α-glucan oligosaccharide contains α-1,2 glycosidic bonds, and the degree of polymerization is mainly between 3 and 6.

[0008] In a preferred embodiment of the present invention, the dextranase I has an amino acid sequence as shown in any one of the following:

[0009] (1) the amino acid sequence shown in SEQ ID NO: 1;

[0010] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1), and having the same or similar function as the amino acid sequence described in (1); or

[0011] (3) An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence described in (1) or (2).

[0012] In a preferred embodiment of the present invention, the dextranase II has an amino acid sequence as shown in any one of the following:

[0013] (1) the amino acid sequence shown in SEQ ID NO: 4;

[0014] (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1), and having the same or similar function as the amino acid sequence described in (1); or

[0015] (3) An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence described in (1) or (2).

[0016] Another aspect of the present invention provides a method for preparing the α-glucan oligosaccharide as described above, the method comprising the following steps:

[0017] S1: constructing a dextran sucrase strain SD1022, and producing dextran sucrase I by the dextran sucrase strain SD1022;

[0018] constructing a dextran sucrase strain SD1021, and producing dextran sucrase II by the dextran sucrase strain SD1021;

[0019] S2: using sucrose and maltose as substrates, dextran sucrase I and dextran sucrase II jointly act to carry out an enzyme catalytic reaction to obtain the α-glucan oligosaccharide.

[0020] In a preferred embodiment of the present invention, in step S1, the construction method of the dextran sucrase strain SD1022 comprises:

[0021] The synthesized dextransucrase pUC57-1022 plasmid was used as a template to perform PCR amplification to obtain a dextransucrase 1022 gene fragment, which was ligated to a pBAD HisB vector and then transformed into competent Escherichia coli BW25113 cells to obtain a genetically engineered bacterium BW / pBAD-1022, namely, a dextransucrase strain SD1022.

[0022] In a preferred embodiment of the present invention, the primers used for PCR amplification include:

[0023] The nucleotide sequence of the upstream primer is shown in SEQ ID NO:2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:3.

[0024] In a preferred embodiment of the present invention, in step S1, the production process of dextransucrase I comprises: inoculating the dextransucrase strain SD1022 into an induction medium to induce the production of dextransucrase I whole cells.

[0025] In a preferred embodiment of the present invention, the production process of dextransucrase I comprises:

[0026] (i) inoculating the dextran sucrase strain SD1022 into a 2YT liquid culture medium, and culturing at 37° C. for 4 to 6 hours to obtain a culture solution;

[0027] (ii) The obtained culture solution is inoculated into a shake flask at an inoculation rate of 1-2%, and after culturing at 37°C and 200 rpm for 2-3 hours, the enzyme is induced with arabinose at a final concentration of 1-2 wt%. After the induction is completed, the obtained product is centrifuged and the cell product in the culture medium is collected, namely, the dextran sucrase I whole cell.

[0028] In a preferred embodiment of the present invention, the 2YT liquid culture medium in step (i) is composed of: molecular grade peptone 9-11 g / L, molecular grade yeast powder 4-6 g / L, NaCl 4-6 g / L, ampicillin 100 μg / mL, and pH 6.8-7.2.

[0029] In a preferred embodiment of the present invention, the induction enzyme production conditions in step (ii) are: the induction temperature is 25-30°C, the induction speed is 220 rpm, and the induction time is 14-18 h.

[0030] In a preferred embodiment of the present invention, in step S1, the construction method of the dextran sucrase strain SD1021 comprises:

[0031] The synthesized dextransucrase pUC57-1021 plasmid was used as a template to perform PCR amplification to obtain the dextransucrase 1021 gene fragment, which was ligated to the pBAD HisB vector and then transformed into Escherichia coli BW25113 competent cells to obtain the genetically engineered bacteria BW / pBAD-1021, i.e. the dextransucrase strain SD1021.

[0032] In a preferred embodiment of the present invention, the primers used for PCR amplification include:

[0033] The nucleotide sequence of the upstream primer is shown in SEQ ID NO:5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:6.

[0034] In a preferred embodiment of the present invention, in step S1, the production process of dextransucrase II comprises: inoculating the dextransucrase strain SD1021 into an induction medium to induce the production of dextransucrase II whole cells.

[0035] In a preferred embodiment of the present invention, the production process of dextransucrase II comprises:

[0036] (i) inoculating the dextran sucrase strain SD1021 into a 2YT liquid culture medium, and culturing at 37° C. for 4 to 6 hours to obtain a culture solution;

[0037] (ii) The obtained culture solution is inoculated into a shake flask at an inoculation rate of 1-2%, and after culturing at 37°C and 200 rpm for 2-3 hours, the enzyme is induced with arabinose at a final concentration of 1-2 wt%. After the induction is completed, the obtained product is centrifuged and the cell product in the culture medium is collected, namely, the dextran sucrase II whole cell.

[0038] In a preferred embodiment of the present invention, the 2YT liquid culture medium in step (i) is composed of: molecular grade peptone 9-11 g / L, molecular grade yeast powder 4-6 g / L, NaCl 4-6 g / L, ampicillin 100 μg / mL, and pH 6.8-7.2.

[0039] In a preferred embodiment of the present invention, the induction enzyme production conditions in step (ii) are: the induction temperature is 25-30°C, the induction speed is 220 rpm, and the induction time is 14-18 h.

[0040] In a preferred embodiment of the present invention, in step S2, the sucrose concentration is 300-600 g / L, the maltose concentration is 150-300 g / L, the dextran sucrase I activity is 500-1000 U / mL, and the dextran sucrase II activity is 1000-5000 U / mL;

[0041] The enzyme catalytic reaction conditions are: temperature: 25-37°C, time: 4-6h.

[0042] Another aspect of the present invention provides a use of the aforementioned α-glucan oligosaccharide in the preparation of prebiotics, skin care products, skin probiotics or anti-digestion products.

[0043] By means of the above technical solution, the present invention has at least the following advantages:

[0044] The present invention provides a method for enzymatically synthesizing α-glucosidic oligosaccharides containing α-1,2 glycosidic bonds. The present invention constructs two strains producing dextran sucrase I and dextran sucrase II and produces dextran sucrase I whole cells and dextran sucrase II whole cells, and catalyzes sucrose and maltose to generate α-glucosidic oligosaccharides containing α-1,2 glycosidic bonds and a degree of polymerization mainly between 3 and 6 through the combined action of the two enzymes. The obtained conversion product has the characteristics of high purity and easy separation, and the quality of α-glucosidic oligosaccharides can be further improved. The obtained α-glucosidic oligosaccharides have the advantage of high digestion resistance and can be used in the preparation of digestion-resistant products, thereby expanding the application of α-glucosidic oligosaccharides. The method of the present invention can be applied to the industrial production of α-glucosidic oligosaccharides.

[0045] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a liquid chromatogram of the product prepared according to the method of Example 3 of the present invention;

[0047] Figure 2 Composition analysis of the product prepared according to the method of Example 3 of the present invention;

[0048] Figure 3 This is a liquid chromatogram of the product prepared according to the method of Example 4 of the present invention;

[0049] Figure 4 Composition analysis of the product prepared according to the method of Example 4 of the present invention;

[0050] Figure 5 The liquid chromatography composition of the product yeast after desugaring prepared according to the method of Example 4 of the present invention;

[0051] Figure 6 This is a liquid chromatogram of the product prepared according to the method of Example 5 of the present invention;

[0052] Figure 7 Composition analysis of the product prepared according to the method of Example 5 of the present invention;

[0053] Figure 8 The NMR spectra of the products prepared according to the methods of Examples 3 and 4 of the present invention;

[0054] Fig. 9 This is the NMR spectrum of commercially available IMO50;

[0055] Fig.10 This is an in vitro digestibility test of the products prepared according to the methods of Examples 3 and 4 of the present invention and commercially available IMO50. DETAILED DESCRIPTION

[0056] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Unless otherwise specified, the experimental methods in the following examples without specifying detailed conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0058] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0059] Unless otherwise specified, the percentage concentrations referred to in the present invention all refer to final concentrations, which refer to the percentage of the added component in the system after the addition of the component.

[0060] The temperature parameters in the present invention, if not specifically limited, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument.

[0061] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0063] 1. Enzyme activity determination

[0064] Definition of enzyme activity: The amount of enzyme that decomposes sucrose to produce 1um reducing sugar within 1 minute at 37°C is 1U (pH 7).

[0065] Assay method: 50 mM pH 7.0 Tris-HCl, 200 mM sucrose, 1 mM CaCl 2 Add 0.2-6OD / mL of the crushed crude enzyme solution (adjust the concentration of the crude enzyme solution according to the color after boiling, it should not be black after boiling), react at 37℃ for 10min, take 0.5mL of the reaction solution and add 0.5mL of DNS reagent, boil for 5min, cool, add 4mL of water, and measure the absorbance at 540nm.

[0066] 2. Product component detection--Liquid chromatography

[0067] Instrument: Shimadzu GC-2014

[0068] Chromatographic column: Agilent Hi-Plex Na (7.7×300mm); detector: evaporative light scattering detector (ELSD); evaporator tube temperature: 80℃; nebulizer temperature: 80℃; gas flow rate: 1.6SLM; column temperature: 80℃; flow rate: 0.25mL / min; mobile phase: water; elution program: water, isocratic elution; acquisition time: 35min; injection volume: 5uL.

[0069] Example 1: Production of dextransucrase I whole cells by dextransucrase strain SD1022

[0070] 1. Construction of dextran sucrase strain SD1022

[0071] The amino acid sequence shown in SEQ ID NO: 1 was synthesized, and the synthesized dextran sucrase pUC57-1022 plasmid was used as a template, and the primers 1022-F and 1022-R were used for PCR amplification to obtain the dextran sucrase 1022 target gene fragment, the DNA full length was 4113 bp, the start codon was ATG, the stop codon was TAA, the sequence had no introns, had a complete open reading frame, and encoded 1371 amino acids. After the obtained target gene fragment was recovered by gel, it was connected to the pBAD HisB vector using the ClonExpress II OneStep Cloning Kit of Novozymes to obtain a recombinant plasmid. Subsequently, the obtained recombinant plasmid was transformed into Escherichia coli Fast-T1 competent cells, and positive clones were screened on LB plates containing ampicillin, and sequenced and verified using primers PBAD-F (SEQ ID NO: 7: 5'-aggaggaattaaccatgggg-3'). The correctly sequenced plasmid was transferred into competent cells of Escherichia coli BW25113 to obtain the genetically engineered bacteria BW / pBAD-1022, which is the dextran sucrase strain SD1022.

[0072] The primer sequences used in the above PCR amplification are:

[0073] Upstream primer 1022-F:

[0074] SEQ ID NO: 2: 5'-gtaacaggaggaattaaccatggGCatggaaaaagcccctgatt-3';

[0075] Downstream primer 1022-R:

[0076] SEQ ID NO: 3: 5'-gctgcagaccgagctcaccgaattcttaaccttttgccatcgtac-3'.

[0077] The PCR amplification system was: 5×SF Buffer 10uL, dNTP Mix (10mM each) 1uL, template 1μL, primers (10uM) 2uL each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novezan Biotechnology Co., Ltd., product catalog P501) 1uL, distilled water 33uL, and the total volume was 50uL.

[0078] PCR amplification conditions were as follows: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 2 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

[0079] SEQ ID NO:1

[0080] <h2 style=";text-align:left;direction:ltr">MPANAPDKQS VTNAPVVPPK HDTDQQDDSL EKQQVLEPSV NSNIPKKQTNQQLAVVTAPANSAPANSAPQTKTTA EISAGTELDT MPNVKHVDGK VYFYGDDGQPKKNFTTIIDG KPYYFDKDTG ALSNNDKQYVSELFSIGNKH NAVYNTSSDNFTQLEGHLTA SSWYRPKDIL KNGKRWAPST VTDFRPLLMAWWPDKSTQVTYLNYMKDQGL LSGTHHFSDN ENMRTLTAAA MQAQVNIEKK IGQLGNTDWLKTAMTQYIDAQPNWNIDSEA KGDDHLQGGA LLYTNSDMSP KANSDYRKLSRTPKNQKGQI ADKYKQGGFE LLLANDVDNSNPVVQAEQLN AFDIYNADEL KADKVYTPYNIPASYALLLT NKDTIPRVYYGDMFTDDGQY MAKQSPYYQA IDALLKARIK YAAGGQTMKMNYFPDEQSVMTSVRYGKGAM TASDSGNQET RYQGIGLVVN NRPDLKLSDK DEVKMDMGAAHKNQDYRPVLLTTKSGLKVY STDANAPVVR TDANGQLTFK ADMVYGVNDPQVSGYIAAWV PVGASENQDA RTKSETTQSTDGSVYHSNAA LDSQVIYEGFSNFQDFPTTP DEFTNIKIAQ NVNLFKDWGI TSFEMAPQYRASSDKSFLDAIVQNGYAFTD RYDIGYNTPT KYGTADNLLD ALRALHGQGIQAINDWVPDQIYNLPDEQLVTAIRTDGSGD HTYGSVIDHT LYASKTVGGG IYQQQYGGAFLEQLKTQYPQ LFQQKQISTD QPMNPDIQIKSWEAKYFNGSNIQGRGAWYVLKDWGTQQYF NVSDAQTFLP KQLLGEKAKT GFVTRGKETSFYSTSGYQAKSAFICDNGNW YYFDDKGKMV VGNQVINGIN YYFLPNGIEL QDAYLVHDGMYYYYNNIGKQLHNTYYQDKQ KNFHYFFEDG HMAQGIVTIIQSDGTPVTQYFDENGKQQKG VAVKGSDGHL HYFDGASGNMLFKSWGRLAD GSWLYVDEKGNAVTGKQTIN NQTVYFNDDG RQIKNNFKEL ADGSWLYLNNKGVAVTGEQIINGQTLYFGN DGRQFKGTTH INATGESRYY DPDSGNMITD RFERVGDNQWAYFGYDGVAVTGDRIIKGQK LYFNQNGIQM KGHLRLENGI MRYYDADTGELVRNRFVLLSDGSWVYFGQD GVPVTGVQVINGQTLYFDAD GRQVKGQQRVIGNQRYWMDK DSGEMKKITY.

[0081] The steps for transformation of E. coli Fast-T1 competent cells are as follows:

[0082] (1) Take a tube containing 100 μL of E. coli Fast-T1 competent cells stored at -80°C;

[0083] (2) Add the recombinant plasmid to the thawed competent cells, gently flicking with your fingers during the addition process;

[0084] (3) placing the product of step (2) on ice for 20 to 30 minutes, then placing it in a 42°C water bath for 45 seconds for heat shock, and immediately taking it out after the heat shock is completed and placing it back on ice for 2 to 5 minutes;

[0085] (4) Add 800 μL of 2YT liquid culture medium and culture in a 37°C shaking incubator (purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., model ZWY-2102C) with a shaking frequency of 200 rpm for 40 min;

[0086] (5) Centrifuging the transformed bacterial solution from step (4) at 4000 rpm for 2 min, spreading the solution on a 2YT solid culture medium plate, and culturing the plate at 37° C. for 10 to 12 h;

[0087] (6) Select a single colony and place it in a resistant liquid 2YT medium containing 100 μg / mL ampicillin, culture it at 37°C for 4 to 6 hours, and then verify it.

[0088] The correctly sequenced plasmid was transferred into competent cells of Escherichia coli BW25113 to obtain the genetically engineered bacteria BW / pBAD-1022, which is the dextran sucrase strain SD1022.

[0089] The transformation steps of E. coli BW25113 competent cells are as follows:

[0090] (1) Take a tube containing 50-200 μL of E. coli BW25113 competent cells stored at -80°C and thaw it on ice for 20-30 min;

[0091] (2) Take 2-3 μL of the verified plasmid solution and add it to the thawed competent cells, gently flicking with your fingers during the addition process;

[0092] (3) placing the product of step (2) on ice for 20 to 30 minutes, then placing it in a 42°C water bath for 90 seconds for heat shock, and immediately taking it out after the heat shock is completed and placing it back on ice for 2 to 5 minutes;

[0093] (4) Add 800 μL of 2YT liquid culture medium and culture in a 37°C shaking incubator (purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., model ZWY-2102C) with a shaking frequency of 200 rpm for 40 min; (5) Take 50 μL of the transformed bacterial solution in step (4) and spread it on a 2YT solid culture medium plate and culture it at 37°C for 10 to 12 h;

[0094] (6) Select a single colony and place it in liquid 2YT medium containing 100 μg / mL ampicillin and culture at 37°C for 6 to 8 h;

[0095] (7) Take an equal volume of bacterial solution and an equal volume of sterile 80% (volume) glycerol solution, mix them in a bacteria-preserving tube, and store them in a -80°C refrigerator for later use.

[0096] 2. Production of dextran sucrase I whole cell from dextran sucrase strain SD1022

[0097] The obtained genetically engineered bacteria, dextran sucrase strain SD1022, was inoculated into 2YT liquid culture medium (components: molecular grade peptone 10g / L, molecular grade yeast powder 5g / L, NaCl 5g / L, ampicillin 100μg / mL, pH 6.8-7.2), cultured at 37°C for 4h to obtain culture solution. Subsequently, the obtained culture solution was inoculated into a shake flask at an inoculation amount of 1%, cultured at 37°C, 200rpm for 2h; then, arabinose with a final concentration of 1% (mass) was used to induce enzyme production, the induction temperature was 25°C, the induction speed was 220rpm, and the induction time was 18h. After the induction, the obtained product was centrifuged and the cell product in the culture medium was collected, which was the whole cell producing dextran sucrase I.

[0098] Example 2: Production of dextransucrase II whole cells by dextransucrase strain SD1021

[0099] 1. Construction of dextran sucrase strain SD1021

[0100] The amino acid sequence shown in SEQ ID NO:4 was synthesized. The synthetic dextran sucrase pUC57-1021 plasmid was used as a template, and the primers 1021-F and 1021-R were used for PCR amplification to obtain the dextran sucrase 1021 target gene fragment, with a DNA full length of 5634 bp, a start codon of ATG, and a stop codon of TAA. The sequence had no introns, had a complete open reading frame, and encoded 1078 amino acids. The obtained target gene fragment was recovered by gel and connected to the pBAD HisB vector using the ClonExpress II OneStep Cloning Kit of Novozymes to obtain a recombinant plasmid. The obtained recombinant plasmid was then transformed into Escherichia coli Fast-T1 competent cells, and positive clones were screened on LB plates containing ampicillin, and sequenced and verified using primers PBAD-F (SEQ ID NO:7: 5'-aggaggaattaaccatgggg-3'). The correctly sequenced plasmid was transferred into competent cells of Escherichia coli BW25113 to obtain the genetically engineered bacteria BW / pBAD-1021, which is the dextran sucrase strain SD1021.

[0101] The primer sequences used in the above PCR amplification are:

[0102] Upstream primer 1021-F:

[0103] SEQ ID NO: 5: 5'-gccgcgcggcagcctcgaggcacaagcaggtcactatatc-3';

[0104] Downstream primer 1021-R:

[0105] SEQ ID NO: 6: 5'-gaccgagctcaccgaattcttaaatttgaggtaatgttg-3'.

[0106] The PCR amplification system was: 5×SF Buffer 10uL, dNTP Mix (10mM each) 1uL, template 1μL, primers (10uM) 2uL each, Phanta Super-Fidelity DNA Polymerase (Nanjing Novezan Biotechnology Co., Ltd., product catalog P501) 1uL, distilled water 33uL, and the total volume was 50uL.

[0107] Amplification conditions were as follows: pre-denaturation at 95°C for 2 minutes (1 cycle); denaturation at 95°C for 10 seconds, annealing at 55°C for 20 seconds, extension at 72°C for 2 minutes (30 cycles); extension at 72°C for 10 minutes (1 cycle).

[0108] SEQ ID NO:4

[0109] MRQKETITRK KLYKSGKSWV AAATAFAVMG VSAVTTVSAD TQTPVGTTQSQQDLTGQRGQDKPTTKEVID KKEPVPQVSA QNAGDLSADA KTTKADDKQDTQPTNAQLPD QGNKQTNSNSDKGVKESTTAPVKTTDVPSK SVTPETNTSINGGQYVEKDG QFVYIDQSGK QVSGLQNIEG HTQYFDPKTGYQTKGELKNIDDNAYYFDKN SGNGRTFTKI SNGSYSEKDG MWQYVDSHDK QPVKGLYDVEGNLQYFDLNSTGNQAKHQIRS VDGVTYYFDA DSGNATAFKA VTNGRYAEQTTKDKDGNETS YWAYLDNQGN AIKGLNDVNGEIQYFDEHTG EQLKGHTATLDGTTYYFEGN KGNLVSVVNT APTGQYKING DNVYYLDNNNEAIKGLYGINGNLNYFDLAT GIQLKGQAKN IDGIGYYFDK DTGNGSYQYT LMAPSNKNDYTQHNVVNNLSESNFKNLVDG FLTAETWYRP AQILSHGTDW VASTDKDFRPLITVWWPNKD IQVNYLRLMQ NEGVLNQSAVYDLNTDQLLL NEAAQQAQIGIEKKISQTGN TDWLNNVLFT THDGQPSFIK QQYLWNSDSEYHTGPFQGGYLKYQNSDLTP NVNSKYRNAD NSLDFLLAND VDNSNPIVQA EDLNWLYYLLNFGSITTQGKENNSNFDSIR IDAVDFVSND LIQRTYDYLR AAYGVDKEANAHSLVE AGLDAGTTTI HQDALIESDIREAMKKSLTN GPGSNISLSNLIQDKEGDKL IADRANNSTE NVAIPNYSII HAHDKDIQDKVGAAITDATGADWTNFTPEQ LQKGLSLYYE DQRKIEKKYN QYNIPSAYAL LLTNKDTVPRVYYGDMYQDDGQYMQKQSLY FDTITALMEA RKQFVAGGQT INVDDNGVLTSVRFGKGAMT ANDIGTNETR TQGIGVVIANDPSLKLSKDSKVTLHMGAAHRNQNYRALLL TTDNGIDSYS SSKNAPVIKT DDNGDLVFSNQDINDQLNTKVHGFLNSEVS GYLSAWVPLD ATEQQDARTL PSEKSVNDGK VLHSNAALDSNLIYEAFSNFQPMPTNRNEY TNVVIADKAD TFKSWGITSF EMAPQYRSSQDKTFLDSTID NGYAFTDRYD LGFEKPTKYGNDEDLRQAIK QLHSSGMQVMADVVANQIYN LPGKEVASTN RVDWNGNNLS TPFGTQMYVVNTVGGGKYQNKYGGEFLDKL KAAYPDIFRS KNYEYDVKNY GGNGTGSVYY TVDSKTRAELDTDTKIKEWSAKYMNGTNVL GLGMGYVLKD WQTGQYFNVS NQNMKFLLPSDLISNDITVQ LGVPVTDKKI IFDPASAYNMYSNLPEDMQV MDYQDDKKSTPSIKPLSSYN NKQVQVTRQY TDSKGVSWNL ITFAGGDLQGQKLWVDSRALTMTPFKTMNQ ISFISYANRN DGLFLNAPYQ VKGYQLAGMS NQYKGQQVTIAGVANVSGKDWSLISFNGTQ YWIDSQALNT NFTHDMNQKV FVNTTSNLDGLFLNAPYRQP GYKLAGLAKN YNNQTVTVSQQYFDDQGTVW SQVVLGGQTVWVDNHALAQM QVRDTNQQLY VNSNGRNDGL FLNAPYRGQGSQLIGMTADYNGQHVQVTKQ GQDAYGAQWR LITLNNQQVW VDSRALSTTI MQAMNDDMYVNSSQRTDGLWLNAPYTMSGA KWAGDTRSAN GRYVHISKAY SNEVGNTYYLTNLNGQSTWI DKRAFTATFD QVVALNATIVARQRPDGMFK TAPYGEAGAQFVDYVTNYNQ QTVPVTKQHS DAQGNQWYLA TVNGTQYWIDQRSFSPVVTKVVDYQAKIVP RTTRDGVFSG APYGEVNAKL VNMATAYQNQ VVHATGEYTNASGITWSQFALSGQEDKLWIDKRALQA。

[0110] The transformation of E. coli Fast-T1 competent cells and the transformation of E. coli BW25113 competent cells were carried out according to the operations described in Example 1, which will not be repeated here.

[0111] 2. Production of dextran sucrase II whole cell from dextran sucrase strain SD1021

[0112] The obtained genetically engineered bacteria, dextran sucrase strain SD1021, was inoculated into 2YT liquid culture medium (components: molecular grade peptone 10g / L, molecular grade yeast powder 5g / L, NaCl 5g / L, ampicillin 100μg / mL, pH 6.8-7.2), cultured at 37°C for 4h to obtain culture solution. Subsequently, the obtained culture solution was inoculated into a shaking bottle at an inoculation amount of 1%, cultured at 37°C, 200rpm for 2h; then, the enzyme was induced with arabinose at a final concentration of 1% (mass), the induction temperature was 25°C, the induction speed was 220rpm, and the induction time was 18h. After the induction, the obtained product was centrifuged and the cell product in the culture medium was collected, which was the whole cell producing dextran sucrase II.

[0113] Example 3: Production of α-glucan oligosaccharides catalyzed by dextransucrase I whole cells

[0114] 300 g / L sucrose, 300 g / L maltose, 2 mM CaCl 2 , and whole cells producing dextran sucrase I (the measured dextran sucrase I activity was 500 U / mL) were mixed and reacted at 37°C for 6 hours, then boiled for 10 minutes to terminate the reaction, the obtained product was centrifuged, and the supernatant was diluted for liquid chromatography detection.

[0115] 30 g / L of commercially available active dry yeast was directly added to the supernatant obtained after centrifugation to digest and remove fructose and unreacted disaccharides, and the mixture was reacted at 30°C and 200 r / min on a shaker for 10 hours. The mixture was then boiled and sterilized and centrifuged. Liquid phase detection results showed that there were no residual monosaccharides and disaccharides. The obtained supernatant was freeze-dried to obtain the α-glucan oligosaccharide product.

[0116] The liquid chromatogram and product composition analysis results are shown in Figure 1 and 2 As shown. Figure 1 As shown in the figure, the degree of polymerization of oligosaccharides is mainly concentrated in 4-sugar and 5-sugar, with some 3-sugar and 6-sugar, and a small amount of 7-9-sugar. The concentration of each component is calculated based on the peak area. The calculation formula is: Concentration of each component = total sugar amount * peak area ratio of each component. The result is as follows Figure 2 shown.

[0117] Example 4: Production of α-glucan oligosaccharides catalyzed by dextransucrase I producing whole cells and dextransucrase II producing whole cells

[0118] 600 g / L sucrose, 300 g / L maltose, 2 mM CaCl 2 , dextran sucrase I producing whole cells (determined dextran sucrase I activity was 500U / mL) and dextran sucrase II producing whole cells (determined dextran sucrase II activity was 5000U / mL) were mixed and reacted at 37°C for 6h, then boiled for 10min to terminate the reaction, the obtained product was centrifuged, and the supernatant was diluted for liquid chromatography detection. The results are as follows Figure 3 and 4 shown.

[0119] 30 g / L of commercially available active dry yeast was added to the supernatant obtained above to digest and remove glucose, fructose and unreacted disaccharides, and the mixture was reacted at 30°C and 200 r / min on a shaker for 10 h. The mixture was then boiled and sterilized and centrifuged. The supernatant was freeze-dried to obtain the α-glucan oligosaccharide product.

[0120] In this example, the liquid phase test results after yeast removal of residual sugar are as follows Figure 5 As shown, no substrate 2 sugar and monosaccharide remain.

[0121] The liquid chromatogram and product composition analysis results are shown in Figure 3 and 4 As shown. Figure 3 As shown in the figure, the polymerization degree of the obtained α-glucan oligosaccharide product is mainly concentrated in 5-sugar, 6-sugar and 3-sugar, and there are also some 4-sugar mixed with 7-sugar, and a small amount of 8-9-sugar. The concentration of each component is calculated based on the peak area. The calculation formula is: Concentration of each component = total sugar amount * peak area ratio of each component. The result is as follows Figure 4 shown.

[0122] Example 5: Production of α-glucan oligosaccharides catalyzed by dextransucrase I producing whole cells and dextransucrase II producing whole cells

[0123] 400 g / L sucrose, 300 g / L maltose, 2 mM CaCl 2 , dextran sucrase I producing whole cells (determined dextran sucrase I activity was 500 U / mL) and dextran sucrase II producing whole cells (determined dextran sucrase II activity was 5000 U / mL) were mixed and reacted at 37°C for 6 h, then boiled for 10 min to terminate the reaction, the obtained product was centrifuged, the supernatant was diluted and detected by liquid chromatography, and the obtained spectrum is shown in Figure 6 .

[0124] In addition, the concentration results of each component calculated according to the method in Example 4 are as follows Figure 7 As shown. Figure 6 and 7 As shown, after the proportion of maltose increases, the molecular weight of oligosaccharides is mainly DP3, followed by DP4 and DP5, and the overall molecular weight is smaller.

[0125] Example 6: Nuclear magnetic spectrum analysis

[0126] Weigh 20 mg of freeze-dried α-glucan oligosaccharide products of Example 3 and Example 4 and commercially available isomaltooligosaccharide IMO50, respectively, and dissolve them in 500 μL of heavy water, wherein the heavy water contains 0.3 g / L trimethylsilyl phosphate (TMSP) as an internal standard for calibrating chemical shifts. Use a fully digital nuclear magnetic resonance spectrometer AVANCE III to perform hydrogen spectrum analysis on the product at 400 MHz at ambient temperature. The bond type ratio of the product is analyzed by integrating the peak areas of the signal peaks at δ 5.00 and 5.39. The results are shown in Figure 8 and Fig. 9 .

[0127] like Figure 8 As shown in the figure, the test results show that the α-glucan oligosaccharide of Example 3 contains only α-1,4 glycosidic bonds and α-1,6 glycosidic bonds, and does not contain α-1,2 glycosidic bonds. The proportion of α-1,6 glycosidic bonds in the α-glucan oligosaccharide of Example 4 is slightly reduced, and a certain proportion of α-1,2 glycosidic bonds are generated. Fig. 9 As shown, the commercially available IMO50 also has the same bond structure as Example 3, and contains only α-1,4 glycosidic bonds and α-1,6 glycosidic bonds.

[0128] Example 6: Digestion resistance test

[0129] Accurately take 0.20g of the α-glucan oligosaccharide samples obtained in Examples 3 and 4 and commercially available IMO50 and place them in a conical flask filled with 20mL of sodium acetate buffer (pH 5.2, 0.5mol / L), add 5mL of mixed enzyme solution (290U / mL porcine pancreatic α-amylase and 20U / mL amyloglucosidase), place in a 37°C constant temperature shaker, shake thoroughly at 190r / min, and use liquid chromatography to determine the glucose content in the hydrolysis solution at 0, 20, 60, 90, 120, 150, and 180min, and calculate the hydrolysis rate:

[0130] H=[(Gt-G0) / M]×0.9×100%

[0131] Wherein: H is the hydrolysis rate, %; Gt is the glucose content at different times, mg; G0 is the initial glucose content, mg; M is the mass of the sample, mg; 0.9 is the glucose conversion coefficient.

[0132] The results are as follows Fig.10As shown, the in vitro digestibility of IMO50 was the highest at 59%, that of Example 3 was 48%, and that of Example 4 was only 17%. The possible reason for this is that the obtained α-glucan oligosaccharides contain 1,2 glycosidic bonds that resist digestion.

[0133] At present, α-glucan oligosaccharides are mainly processed and produced from starch raw materials, involving steps such as liquefaction and saccharification of raw materials and the use of α-glucosidase to perform transglycosylation reaction to prepare isomaltooligosaccharides. However, the yield of isomaltooligosaccharides produced by this method is low, and the subsequent product separation cost is high. For example, CN104131051A discloses a method for preparing isomaltooligosaccharides, but the product is mainly low-polymerization sugars with poor digestibility, and the reaction time is as long as more than 30 hours, which is difficult to meet high quality requirements.

[0134] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An α-glucan oligosaccharide, characterized in that: The alpha-glucan oligosaccharide is produced by the combined action of dextran sucrase I and dextran sucrase II to catalyze sucrose and maltose; the alpha-glucan oligosaccharide contains alpha-1,2 glycosidic bonds, and the degree of polymerization is mainly between 3 and 6.

2. The α-glucan oligosaccharide according to claim 1, characterized in that The dextranase I has an amino acid sequence as shown in any of the following: (1) the amino acid sequence shown in SEQ ID NO: 1; (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1), and having the same or similar function as the amino acid sequence described in (1); or (3) An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence described in (1) or (2).

3. The α-glucan oligosaccharide according to claim 1, characterized in that The dextranase II has an amino acid sequence as shown in any of the following: (1) the amino acid sequence shown in SEQ ID NO:4; (2) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (1), and having the same or similar function as the amino acid sequence described in (1); or (3) An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence described in (1) or (2).

4. A method for preparing α-glucan oligosaccharide according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1: constructing a dextran sucrase strain SD1022, and producing dextran sucrase I by the dextran sucrase strain SD1022; constructing a dextran sucrase strain SD1021, and producing dextran sucrase II by the dextran sucrase strain SD1021; S2: using sucrose and maltose as substrates, dextran sucrase I and dextran sucrase II jointly act to carry out an enzyme catalytic reaction to obtain the α-glucan oligosaccharide.

5. The preparation method according to claim 4, characterized in that: In step S1, the construction method of the dextran sucrase strain SD1022 comprises: The synthesized dextransucrase pUC57-1022 plasmid was used as a template to perform PCR amplification to obtain a dextransucrase 1022 gene fragment, which was ligated to a pBAD HisB vector and then transformed into competent Escherichia coli BW25113 cells to obtain a genetically engineered bacterium BW / pBAD-1022, namely, a dextransucrase strain SD1022.

6. The preparation method according to claim 4, characterized in that: In step S1, the production process of dextran sucrase I comprises: inoculating the dextran sucrase strain SD1022 into an induction medium to induce the production of dextran sucrase I; The induction medium contains arabinose with a final concentration of 1-2 wt%.

7. The preparation method according to claim 4, characterized in that: In step S1, the construction method of the dextran sucrase strain SD1021 comprises: The synthesized dextransucrase pUC57-1021 plasmid was used as a template to perform PCR amplification to obtain the dextransucrase 1021 gene fragment, which was ligated to the pBAD HisB vector and then transformed into Escherichia coli BW25113 competent cells to obtain the genetically engineered bacteria BW / pBAD-1021, i.e. the dextransucrase strain SD1021.

8. The preparation method according to claim 4, characterized in that: In step S1, the production process of dextran sucrase II comprises: inoculating the dextran sucrase strain SD1021 into an induction medium to induce the production of dextran sucrase II; The induction medium contains arabinose with a final concentration of 1-2 wt%.

9. The preparation method according to claim 4, characterized in that: In step S2, the sucrose concentration is 300-600 g / L, the maltose concentration is 150-300 g / L, the dextran sucrase I activity is 500-1000 U / mL, and the dextran sucrase II activity is 1000-5000 U / mL; The enzyme catalytic reaction conditions are: temperature: 25-37°C, time: 4-6h.

10. Use of the α-glucan oligosaccharide according to any one of claims 1 to 3 in the preparation of prebiotics, skin care products, skin probiotics or anti-digestion products.

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