CN114262701B - Exo-type inunold and preparation method and application thereof - Google Patents
Exo-type inunold and preparation method and application thereof Download PDFInfo
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- CN114262701B CN114262701B CN202111457065.5A CN202111457065A CN114262701B CN 114262701 B CN114262701 B CN 114262701B CN 202111457065 A CN202111457065 A CN 202111457065A CN 114262701 B CN114262701 B CN 114262701B
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- inulinase
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Abstract
The invention discloses an exo-type inulinase INUGALD, a preparation method and application thereof, and relates to the technical field of bioengineering. The amino acid sequence of the exo-type inulinase INUGALD is shown in SEQ ID NO: 2. The exo-type inulinase inulina provided by the invention takes the amino acid sequence (X57202.1) of the exo-type inulinase INU1 of Kluyveromyces marxianus (Kluyveromyces marxianus) as an original material, and performs site-directed mutagenesis transformation on the exo-type inulinase INU1 through artificial design transformation to obtain the exo-type inulina (SEQ ID NO: 2) with high activity, so that the production efficiency of fructose is obviously improved when the exo-type inulinase inulina is used for preparing fructose, and the exo-type inulinase inulina can be used for industrial production of fructose.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an exo-type inunold and a preparation method and application thereof.
Background
Fructose is an excellent monosaccharide, which has a good taste, high sweetness and low glycemic index, and is known as "healthy sugar". Fructose is the main edible sugar. Especially, with the change of consumption concept, the food and beverage industry has an increasing demand for fructose, and the use of fructose and fructose syrup has become a major product of edible sugar.
Currently, there are two main methods for industrial production of fructose: 1. hydrolysis by amylase to glucose, followed by conversion of glucose to fructose by glucose isomerase, a double enzyme process, but usually only a mixture of fructose and glucose (so-called fructose syrup) is obtained, and if pure or high fructose content is required, a complex purification process is required; 2. sucrose is hydrolyzed into glucose and fructose by an acid method and an enzyme method, and then fructose is separated from glucose by ion exclusion chromatography, whereas acid hydrolysis of sucrose has potential pollution, and fructose syrup mixtures prepared by converting glucose by isomerase usually have a fructose content of only 42%. If high purity fructose is desired, a subsequent complex separation and purification system is required.
Inulin (also called inulin) is an edible and storable polysaccharide which is inferior to starch, is widely used in the asteraceae plants, and is a renewable resource with abundant sources and low cost. It is mainly present in jerusalem artichoke and chicory, so it is named inulin. Inulin is a natural macromolecular polymer formed by polymerization of fructose monomers, which was first extracted from plants by german scientists in 1804. Inulin is a chain polysaccharide formed by connecting a plurality of fructosyl groups through beta-D-fructosyl bonds. Natural inulin is a mixture of fructose polymers of different lengths and degrees of polymerization, the Degree of Polymerization (DP) of which is approximately between 2 and 100. Inulin is therefore an ideal material for the production of fructose. Hydrolysis of inulin by exo-type synanthase to produce fructose is an efficient, green and advanced technique.
Exo-type inulase is distributed in plants and microorganisms, but the activity of exo-type inulase produced by microorganisms obtained from nature is low, so that the industrial application of the exo-type inulase is limited.
Disclosure of Invention
The invention mainly aims to provide an exo-type inulinase INUGALD, a preparation method and application thereof, and aims to provide the exo-type inulinase with high activity.
In order to achieve the aim, the invention provides an exo-type inulinase inunold, wherein the amino acid sequence of the exo-type inulinase inunold is shown as SEQ ID NO: 2.
In addition, the invention also provides an exo-type inulinase gene, which is used for encoding the exo-type inulinase INUGALD.
Alternatively, the nucleotide sequence of the exo-chrysnase gene is as set forth in ID NO: 1.
In addition, the invention also provides an expression cassette, which comprises the exoinulinase gene.
In addition, the invention also provides a recombinant expression vector, which comprises the exoinulinase gene.
In addition, the invention also provides a recombinant strain, wherein the recombinant strain comprises the exochrysase gene.
Alternatively, the host cell of the recombinant strain includes any one of E.coli, bacillus, aspergillus and Yeast.
Optionally, the yeast comprises kluyveromyces lactis.
In addition, the invention also provides a preparation method of the exo-type inulinase INUGALD, and the recombinant strain is cultured to obtain the exo-type inulinase.
In addition, the invention also provides an application of the exo-type inulinase INUGALD in preparing fructose.
The exo-type inulinase inunold provided by the invention takes the amino acid sequence (X57202.1) of the exo-type inulinase INU1 of Kluyveromyces marxianus (Kluyveromyces marxianus) as an original material, and performs site-directed mutagenesis transformation on the exo-type inulinase INU1 through artificial design transformation to obtain the exo-type inulinase inunold with high activity, wherein the amino acid sequence of the exo-type inulinase inulold is shown as SEQ ID NO:2, when the catalyst is used for preparing fructose, the production efficiency of the fructose is obviously improved, and the catalyst can be used for industrial production of the fructose.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a three-dimensional structure of exo-type INU1 enzyme as a starting material in example 1 of the present invention;
FIG. 2 is a three-dimensional structure of exo-type inunold active center in example 1 of the present invention;
FIG. 3 is a map of the recombinant expression vector pGKLAC-inunold of example 3 of this invention;
FIG. 4 is a gel electrophoresis chart of recombinant expression vector pGKLAC-inunold of example 3 of the present invention after double enzyme digestion;
FIG. 5 shows SDS-PEGE results of each fermentation supernatant in example 5 of the present invention;
FIG. 6 shows the results of the enzyme activity test of each fermentation supernatant in example 5 of the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides an exo-type inulinase inulinate, the amino acid sequence of which is shown in SEQ ID NO: 2.
In the embodiment, the amino acid sequence (X57202.1) of the exo-type inula marxianus (Kluyveromyces marxianus) INU1 is taken as a starting material, and the novel exo-type inulinase inunold is obtained through artificial design, transformation and optimization, so that the activity of the original inulinase is improved, and when the exo-type inulinase inunold with high activity is used for preparing fructose, the production efficiency of the fructose is obviously improved, and the method can be used for industrial production of the fructose.
Specifically, the invention is based on the protein sequence of Hou Xuanju carbohydrase INU1, on the basis of statistical analysis of sequence characteristics, the relation between the sequence, the structure and the function of the protein is explored, and based on information of the protein structure, large-scale data statistics and deep learning, a novel exochrysanthemic enzyme INUGAL with improved sequence substrate/product catalytic efficiency is designed and obtained. Novel exo-type inulinase inunold amino acid sequence SEQ ID NO: 2. The sequence differences from the original INU1 sequence are mainly the mutation of aspartic acid at position 65 to leucine (D65L), glycine at position 241 to aspartic acid (G241D), methionine at position 306 to phenylalanine (M306F), tyrosine at position 326 to leucine (Y326L), and leucine at position 476 to proline (L476P). It should be noted that, the point mutation may be performed by various mutation methods conventional in the art, and will not be described herein.
The invention also provides an exotype inulinase gene, and the invention does not limit the specific nucleotide sequence of the exotype inulinase gene, as long as the exotype inulinase gene can code the exotype inulinase (SEQ ID NO: 2).
The completely new design and optimization of the exochrysase gene codons are completed based on the evolution information of the expression host, the preference of the expression host to codons, the evolution information of enzyme molecules and the like; optimizing the adaptive host cell. In this example, the nucleotide sequence of the exo-type chrysanthenase gene synthesized artificially is shown in SEQ ID NO:1, which is designated as exo-type inulinase gene inukold. After induced expression, the recombinant strain containing the exotype inulinase gene inunold can obtain the exotype inulinase inunold with high yield and the obtained exotype inulinase inunold has high activity.
The invention also provides an expression cassette, which comprises the exoinulinase gene inunold (SEQ ID NO: 1) as described above. Specifically, the lactose inducible promoter PLAC2, the exochrysin gene inunold and the lactose operon termination sequence TT are combined to form an expression cassette which is subjected to lactose induced expression.
It will be appreciated that the expression cassette includes a lactose promoter for driving expression of the exoinulase gene inunold described above. In the process of culturing the recombinant strain, when lactose is added into the culture medium, the lactose can be carbon nutrition produced by the recombinant strain and can induce and start the strong expression of the exochrysin gene inunold.
Preferably, the expression cassette further comprises a signal peptide sequence Alpha-mate factor located between the downstream of the promoter and the upstream of the exochrysase gene inugenol for directing the transmembrane transfer of the exochrysase out of the cell.
The invention also provides a recombinant expression vector which comprises the exoinulinase gene inunold (SEQ ID NO: 1) described above. Further, the recombinant expression vector comprises the expression cassette. In the case of determination of the nucleotide sequence of the exo-type inulinase gene inulinate and the amino acid sequence of the exo-type inulinase gene inulinate, the person skilled in the art is able to select an appropriate expression vector as well as other functional units. Wherein the expression vector may be a vector suitable for expression in a host such as bacteria, yeast, fungi, etc. In a preferred embodiment, the expression vector is any one of a pichia expression vector and a kluyveromyces lactis expression vector. More preferably, the expression vector is a kluyveromyces lactis expression vector.
In a specific embodiment, the recombinant expression vector is prepared by the steps of:
s10, respectively introducing Nde I and EcoR I enzyme cutting sites at two ends of an exotype inulinase gene inulinate fragment, double-enzyme cutting the exotype inulinase gene inulinate by Nde I and EcoR I, and connecting the enzyme-cut inulinase gene fragment to a glue-recovered lactic acid Kluyveromyces expression vector pGKLAC after the same enzyme cutting at 16 ℃ overnight under the action of T4 ligase to obtain a recombinant expression vector named pGKLAC-inulinate.
The invention also provides a recombinant strain, which comprises the exo-type chrysanthemic enzyme gene inunold (SEQ ID NO: 1) as described above. Further, the recombinant strain comprises a recombinant expression vector as described above. The expression product of the recombinant strain is the exo-type inulinase INUGALD (SEQ ID NO: 2). Wherein, the host cell of the recombinant strain comprises any one of escherichia coli, bacillus, aspergillus and saccharomycetes. Preferably, the host cell of the recombinant strain is kluyveromyces lactis in yeast.
In a specific embodiment, the recombinant strain is prepared by the steps of:
step S20, linearizing the recombinant expression vector pGKLAC-inunold prepared in the step S10 by using restriction enzymes;
s21, converting the linearized recombinant expression vector into Kluyveromyces lactis by adopting an electrotransport converter;
step S22, screening single colonies growing in a flat plate containing acetamide;
and step S23, after streaking and purification, the positive transformant is verified to be accurate by PCR, and the yeast recombinant strain containing the exonuclease inunold gene is obtained.
The invention also provides a preparation method of the exo-type inumuld, which comprises the following steps: culturing the recombinant strain as described above to obtain exo-type inumuld. Specifically, the recombinant strain containing the exo-type inulinase gene inulinate is cultured in a lactose-containing YPGL culture solution, and the exo-type inulinase inulinate is gradually induced to be expressed and secreted into the culture solution in the growth process of the strain, and the culture solution can be used as liquid containing the exo-type inulinase and can be further refined.
The invention also provides an application of the exo-type inunold in preparing fructose. Specifically, exo-type inunold is adopted to contact with inulin, and fructose is obtained through hydrolysis of the inulin by the exo-type inunold.
Further, the exo-type inulinase inunold is obtained by encoding the exo-type inulinase gene inunold. Because the exo-type inulinase gene inulinate synthesized by artificial design has the characteristics of high yield and high activity, compared with the traditional fructose preparation method, the method for obtaining the fructose is more efficient and can be suitable for industrial production of the fructose.
Preferably, the hydrolysis parameters of exo-type inumuld on inulin are: the pH value is 5.0, the temperature is 50 ℃, the concentration of the inulin is 15%, the ratio of exo-type inulin to the inulin is 5000U/g (i.e. the mass ratio of enzyme activity unit to substrate is 5000U/g), the hydrolysis time is 4 hours, and the fructose syrup with high purity can be efficiently and conveniently obtained under the hydrolysis parameters.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1 obtaining exo-type inunold inulinase
Taking the amino acid sequence (X57202.1) of the exo-type INU1 of Kluyveromyces marxianus (Kluyveromyces marxianus) as a starting material, wherein the nucleotide sequence of the starting exo-type INU1 gene (named as exo-type INU gene INU 1) consists of the amino acid sequence shown in SEQ ID NO:3, the amino acid sequence of exo-type INU1 is represented by SEQ ID NO: 4. It will be appreciated that the final TGA in the nucleotide sequence (SEQ ID NO: 3) is the stop codon.
Based on a protein sequence database of candidate exochrysase INU1, the invention carries out statistical analysis on sequence characteristics and constructs a hidden Markov model of the enzyme; searching potential candidate mutation sites through a hidden Markov model; exploring the relationship between the sequence-structure-function of a protein; based on information of protein structure and large-scale data statistics and deep learning, with the help of TrRosetta and the like, a novel exo-type inulinase inunold with improved sequence substrate/product catalytic efficiency is designed and obtained, and the amino acid sequence SEQ ID NO of the novel exo-type inulinase inunold: 2, the sequence differs from the original INU1 mainly in that aspartic acid at position 65 is mutated to leucine (D65L), glycine at position 241 is mutated to aspartic acid (G241D), methionine at position 306 is mutated to phenylalanine (M306F), tyrosine at position 326 is mutated to leucine (Y326L), and leucine at position 476 is mutated to proline (L476P).
FIG. 1 shows the three-dimensional structure of exo-type inula enzyme INU1 as a starting material, and it can be seen from FIG. 1 that the exo-type inula enzyme INU1 surrounds the 5 critical amino acids Asp65, gly241, met306, tyr326 and Leu476 of the active center.
FIG. 2 shows the three-dimensional structure of the active center of exo-type inulinase INUGAL, and it can be seen from FIG. 2 that the redesigned exo-type inulinase INUGAL surrounds the 5 key amino acids Leu65, asp241, phe306, leu326 and Phe476 of the active center.
Example 2 obtaining of exo-type inulinase Gene inunold
Based on the evolution information of the expression host, the preference of the expression host to codons, the evolution information of enzyme molecules, the sequence information of enzyme genes and the like, the brand-new design and optimization of the exo-type chrysanthemic enzyme Gene inunold codons are completed based on Gene Designer and the like; optimizing the adaptive host cell. The nucleotide sequence SEQ ID NO. 1 of the exotype chrysanthemic enzyme gene inugold with high activity and high yield is obtained through manual optimization, design and synthesis. It will be appreciated that the last TAA of nucleotide sequence SEQ ID NO. 1 is the stop codon.
EXAMPLE 3 construction of recombinant expression vectors
(1) The Nde I and EcoR I restriction sites are respectively introduced at two ends of the segment of the exo-type inulinase gene inulinac (SEQ ID NO: 1) synthesized in example 1, the Nde I and EcoR I restriction sites are used for double restriction of the exo-type inulinase gene inulinac, the restriction enzyme is used for overnight at the temperature of 16 ℃ under the action of T4 ligase, the restriction enzyme gene segment is connected to a kluyveromyces lactis expression vector pGKLAC recovered by glue after the same restriction enzyme, and a recombinant expression vector is obtained and named pGKLAC-inulinac, and the graph of the pGKLAC-inulinac recombinant expression vector is shown in figure 3. The exo-type inulinase gene inunold is driven to express by a lactose inducer and is stopped to express by a lactose TT terminator.
(2) The recombinant expression vector of the exo-type chrysanthemic gene inu1 serving as a starting material is constructed in the same manner as in the step (1) and is named pGKLAC-inu1.
The plasmid pGKLAC-inutold vector was extracted and double digested with Nde I and EcoR I was performed, and the results of the execution are shown in FIG. 4 (in FIG. 4: M is DNA Marker,1 represents the pGKLAC-inutold vector without digestion, 2 represents the double digested pAO-INU vector). As can be seen from FIG. 4, there is a band at 1600bp position, indicating that the exoinulinase gene inunold was successfully ligated to the expression vector, i.e., the recombinant expression vector was successfully constructed.
EXAMPLE 4 construction of recombinant strains
(1) The recombinant expression vector pGKLAC-inunold prepared in example 3 above was linearized with restriction enzymes under the following conditions: each 200L volume contained 4. Mu.g of plasmid, 10U of Sac II enzyme, which was linearized at 37℃for 2h and the pellet was dried for further use.
(2) And (3) converting the linearized recombinant expression vector into the kluyveromyces lactis by adopting an electrotransformation instrument, wherein the condition parameters of the electrotransformation are V=1500V, PC=200Ω and C=25μF.
(3) In the flat plate containing acetamide, the single colony is cultured in an incubator at 28 ℃ for 2-3 d to select the single colony.
(4) After streaking and purification, the positive transformant is verified to be accurate by PCR, and the yeast recombinant strain containing the exonuclease inunold gene is obtained.
(5) The recombinant strain of the exo-type chrysanthenase gene inu1 is constructed by adopting the same mode of the steps (1), (2) and (3) to obtain the recombinant strain containing the exo-type chrysanthenase gene inu1.
Through identification, the obtained cell of the recombinant strain of the Kluyveromyces lactis containing the exonuclease chrysanthemumase gene inugold is white and spherical, the temperature is 28-30 ℃ in the growth process, and lactose is the main carbon source.
EXAMPLE 5 preparation of exo-type inunold
The recombinant strains prepared in the step (4) and the step (5) of example 4 were inoculated into 250mL volumetric flasks containing 50mL of YPD medium, respectively, and cultured at a constant temperature of 28℃and 180r/min by shaking. When OD is 600 About 3.0 to 6.0, the culture medium was inoculated into a medium YPLac containing 100mL (YPLac medium was composed of yeast powder 10g, peptone 20g, lactose 20g per 1000mL of liquid), the fermentation broth was taken at various time points (12/24/36/48 h, 0h as starting point of fermentation at the time of inoculation), centrifuged at 10000rpm for 5min, and the supernatants (exo-type inunold and exo-type INU1, respectively) were taken.
The results of SDS-PAGE detection of the respective fermentation supernatants are shown in FIG. 5. As can be seen from FIG. 5, the protein content in the exotype inulinase gene inunold recombinant strain obtained by artificial design optimization is obviously higher than that of the recombinant strain containing the inulinase gene inu1.
The results of measuring the enzyme activities of the respective fermentation supernatants are shown in FIG. 6 (in the figure, the ordinate represents the enzyme activities in U/mL, and the abscissa represents different time points). As can be seen from FIG. 6, the activity of the exo-type inulase INUGALD modified by the optimized design is obviously higher than that of the original exo-type inulase gene INU1, specifically, at 48h, the enzyme activity reaches 21500U/mL, and the original gene is only 6150U/mL, and the activity is improved by about 3.5 times.
In conclusion, the recombinant strain constructed by the optimized exoenzyme inulinase gene inulinate can obtain the high-yield and high-activity exoenzyme inulinate after fermentation culture.
EXAMPLE 6 preparation of fructose
The exo-type inulinase inunold constructed by culturing the recombinant strain containing the exo-enzyme inulinase gene inunold in example 5 is contacted with inulin, and fructose is obtained by hydrolysis of inulin.
Optimizing a reaction system:
1. effects of inulin concentration on hydrolysis; the hydrolysis rate of inulin is obviously affected by the concentration of the substrate by detecting the hydrolysis products under the conditions that the pH value is 5.0, the temperature is 50 ℃, the volume ratio of the enzyme solution to the inulin substrate is 1:1, the hydrolysis time is 0.5h, the concentration of the substrate is 8%,10%,15%,30% and 50% respectively, and when the concentration of the substrate is 15%, the hydrolysis rate is close to 100%, the concentration of the substrate is higher than 15%, and the hydrolysis rate is obviously reduced. 15% inulin was used for the following experiments.
2. The influence of the mass ratio of the enzyme activity unit to the inulin on the inulin hydrolysis rate; under the conditions of pH5.0, temperature 50 ℃, concentration of a reaction inulin substrate 15% and hydrolysis time 0.5h, a series of different exo-type inulinase enzyme activity units and inulin substrate weight ratios (U/g) are set, and the methods are as follows: 25000. 12000, 10500, 7500, 5000 and 4000, and the hydrolysis rate of inulin at each ratio was examined. The results showed that with increasing exo-type inulinase, the rate of hydrolysis of inulin increased. Meanwhile, when the enzyme amount was changed from 5000 to 12000, the hydrolysis rate thereof was very similar. Considering that the hydrolysis time was set to 0.5 hours, a ratio of 5000U/g was selected for the subsequent experiments.
3. Effect of hydrolysis time on inulin hydrolysis rate. The effect of hydrolysis time on the rate of inulin hydrolysis was investigated at pH5.0, temperature 50℃and substrate concentration 15% and enzyme/inulin ratio 10000U/g. When the reaction time is 2 hours, about 95% of the inulin is hydrolyzed, and after the time is prolonged to more than 4 hours, the inulin is almost 100% hydrolyzed to monosaccharides.
4. According to the above optimization test, the optimal hydrolysis parameter of inulin is pH5.0, the temperature is 50 ℃, the concentration of inulin is 15%, the enzyme/inulin ratio is 5000U/g, and the hydrolysis time is 4 hours. After 4 hours of hydrolysis, the mixture of fructose polymers with different chain lengths was completely hydrolyzed and the product contained only two components, with a fructose recovery of 95% and other substances glucose of at most 5%.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Jin Ketian, science and technology Co., ltd
<120> exo-type inunold, preparation method and application thereof
<130> 20211124
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1668
<212> DNA
<213> Synthesis
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ccaactgatc catggagatc ttctatgtct ttggttagac aatttacttt gaaggatttt 1080
tctactaacc caaactctgc tgatgttgtt ttgaactctc aaccagtttt gaactacgat 1140
gctttgagaa agaacggtac tacttactct attactaact acactgttac ttctgaaaac 1200
ggtaagaaga ttaagttgga taacccatct ggttctttgg aatttcattt ggaatacgtt 1260
tttaacggtt ctccagatat taagtctaac gtttttgctg atttgtcttt gtactttaag 1320
ggtaacaacg atgataacga atacttgaga ttgggttacg aaactaacgg tggtgctttt 1380
tttttggata gaggtcatac taagattcca tttgttaagg aaaacccatt ttttactcat 1440
caattggctg ttactaaccc agtttctaac tacactacta acgtttttga tgtttacggt 1500
gttattgata agaacattat tgaattgtac tttgataacg gtaacgttgt ttctactaac 1560
actttttttt tttctactaa caacgttatt ggtgaaattg atattaagtc tccatacgat 1620
aaggcttaca ctattaactc ttttaacgtt actcaattta acgtttaa 1668
<210> 2
<211> 555
<212> PRT
<213> Synthesis
<400> 2
Met Lys Phe Ala Tyr Ser Leu Leu Leu Pro Leu Ala Gly Val Ser Ala
1 5 10 15
Ser Val Ile Asn Tyr Lys Arg Asp Gly Asp Ser Lys Ala Ile Thr Asn
20 25 30
Thr Thr Phe Ser Leu Asn Arg Pro Ser Val His Phe Thr Pro Ser His
35 40 45
Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr Asp Ala Lys Glu Glu
50 55 60
Leu Trp His Leu Tyr Tyr Gln Tyr Asn Pro Ala Ala Thr Ile Trp Gly
65 70 75 80
Thr Pro Leu Tyr Trp Gly His Ala Val Ser Lys Asp Leu Thr Ser Trp
85 90 95
Thr Asp Tyr Gly Ala Ser Leu Gly Pro Gly Ser Asp Asp Ala Gly Ala
100 105 110
Phe Ser Gly Ser Met Val Ile Asp Tyr Asn Asn Thr Ser Gly Phe Phe
115 120 125
Asn Ser Ser Val Asp Pro Arg Gln Arg Ala Val Ala Val Trp Thr Leu
130 135 140
Ser Lys Gly Pro Ser Gln Ala Gln His Ile Ser Tyr Ser Leu Asp Gly
145 150 155 160
Gly Tyr Thr Phe Glu His Tyr Thr Asp Asn Ala Val Leu Asp Ile Asn
165 170 175
Ser Ser Asn Phe Arg Asp Pro Lys Val Phe Trp His Glu Gly Glu Asn
180 185 190
Gly Glu Asp Gly Arg Trp Ile Met Ala Val Ala Glu Ser Gln Val Phe
195 200 205
Ser Val Leu Phe Tyr Ser Ser Pro Asn Leu Lys Asn Trp Thr Leu Glu
210 215 220
Ser Asn Phe Thr His His Gly Trp Thr Gly Thr Gln Tyr Glu Cys Pro
225 230 235 240
Asp Leu Val Lys Val Pro Tyr Asp Ser Val Val Asp Ser Ser Asn Ser
245 250 255
Ser Asp Ser Lys Pro Asp Ser Ala Trp Val Leu Phe Val Ser Ile Asn
260 265 270
Pro Gly Gly Pro Leu Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp
275 280 285
Phe Asn Gly Thr His Phe Thr Pro Ile Asp Gly Gln Thr Arg Phe Leu
290 295 300
Asp Phe Gly Lys Asp Tyr Tyr Ala Leu Gln Thr Phe Phe Asn Thr Pro
305 310 315 320
Asn Glu Lys Asp Val Leu Gly Ile Ala Trp Ala Ser Asn Trp Gln Tyr
325 330 335
Ala Gln Gln Ala Pro Thr Asp Pro Trp Arg Ser Ser Met Ser Leu Val
340 345 350
Arg Gln Phe Thr Leu Lys Asp Phe Ser Thr Asn Pro Asn Ser Ala Asp
355 360 365
Val Val Leu Asn Ser Gln Pro Val Leu Asn Tyr Asp Ala Leu Arg Lys
370 375 380
Asn Gly Thr Thr Tyr Ser Ile Thr Asn Tyr Thr Val Thr Ser Glu Asn
385 390 395 400
Gly Lys Lys Ile Lys Leu Asp Asn Pro Ser Gly Ser Leu Glu Phe His
405 410 415
Leu Glu Tyr Val Phe Asn Gly Ser Pro Asp Ile Lys Ser Asn Val Phe
420 425 430
Ala Asp Leu Ser Leu Tyr Phe Lys Gly Asn Asn Asp Asp Asn Glu Tyr
435 440 445
Leu Arg Leu Gly Tyr Glu Thr Asn Gly Gly Ala Phe Phe Leu Asp Arg
450 455 460
Gly His Thr Lys Ile Pro Phe Val Lys Glu Asn Pro Phe Phe Thr His
465 470 475 480
Gln Leu Ala Val Thr Asn Pro Val Ser Asn Tyr Thr Thr Asn Val Phe
485 490 495
Asp Val Tyr Gly Val Ile Asp Lys Asn Ile Ile Glu Leu Tyr Phe Asp
500 505 510
Asn Gly Asn Val Val Ser Thr Asn Thr Phe Phe Phe Ser Thr Asn Asn
515 520 525
Val Ile Gly Glu Ile Asp Ile Lys Ser Pro Tyr Asp Lys Ala Tyr Thr
530 535 540
Ile Asn Ser Phe Asn Val Thr Gln Phe Asn Val
545 550 555
<210> 3
<211> 1668
<212> DNA
<213> Kluyveromyces marxianus
<400> 3
atgaagttcg catactccct cttgcttcca ttggcaggag tcagtgcttc agtgatcaat 60
tacaagagag atggtgacag caaggccatc actaacacca cttttagttt gaacagacct 120
tctgtgcatt tcactccatc ccatggttgg atgaacgatc caaatggttt gtggtacgat 180
gccaaggaag aagactggca tttgtactac cagtacaacc cagcagccac gatctggggt 240
actccattgt actggggtca cgctgtttcc aaggatttga cttcctggac agattacggt 300
gcttctttgg gcccaggttc cgacgacgct ggtgcgttca gtggtagtat ggttatcgat 360
tataacaata cttctggttt cttcaacagc tctgtggacc caagacaaag agcagttgca 420
gtctggactt tgtctaaggg cccaagccaa gcccaacaca tcagttactc attggacggt 480
ggttacacct tcgagcacta caccgacaac gccgtgttgg acatcaacag ctccaacttc 540
agagacccta aggtgttctg gcacgagggc gagaacggcg aagatggtcg ttggatcatg 600
gccgttgctg aatcgcaagt gttctctgtg ttgttctact cttctccaaa cttgaaaaac 660
tggaccttgg aatccaactt cacccaccac ggctggactg gtacccaata cgaatgtcca 720
ggtctagtta aggttccata cgacagtgtt gttgactctt cgaactcctc cgactccaag 780
ccagactccg catgggtctt gtttgtctct atcaaccctg gtggtccatt gggtggttcc 840
gttacccaat actttgttgg tgacttcaac ggtactcact tcactccaat cgacggccaa 900
accagattcc tagacatggg taaggactac tacgcactac aaactttctt caacactcca 960
aacgagaagg acgtctacgg tatcgcatgg gcttctaact ggcaatacgc ccaacaagcc 1020
ccaactgacc catggcgttc atctatgagt ttggttagac aattcacatt gaaagacttc 1080
agcacaaacc ctaactccgc tgatgtcgtc ttgaacagtc aaccagtctt gaactatgat 1140
gcattgagaa agaacggtac cacttacagt atcacaaact acaccgtcac ctccgaaaac 1200
ggcaagaaga tcaagctaga caacccatcc ggttctcttg aattccatct tgaatacgtg 1260
tttaacggct ccccagatat caagagcaac gtgttcgctg atctttcctt gtacttcaag 1320
ggtaacaacg acgacaacga atacttgaga ttgggttacg aaaccaacgg tggtgccttc 1380
ttcttggacc gtggccacac caagattcct ttcgtgaagg agaacttgtt cttcacccac 1440
caattggcag ttaccaaccc agtttccaac tacaccacaa acgtcttcga cgtttacggt 1500
gtcattgaca agaacatcat cgaattgtac ttcgataacg gtaacgtcgt ctccaccaac 1560
actttcttct tctctaccaa caacgttatt ggtgaaattg acatcaagtc gccatacgac 1620
aaggcttaca ccattaactc atttaacgtt acccaattta acgtttga 1668
<210> 4
<211> 555
<212> PRT
<213> Kluyveromyces marxianus
<400> 4
Met Lys Phe Ala Tyr Ser Leu Leu Leu Pro Leu Ala Gly Val Ser Ala
1 5 10 15
Ser Val Ile Asn Tyr Lys Arg Asp Gly Asp Ser Lys Ala Ile Thr Asn
20 25 30
Thr Thr Phe Ser Leu Asn Arg Pro Ser Val His Phe Thr Pro Ser His
35 40 45
Gly Trp Met Asn Asp Pro Asn Gly Leu Trp Tyr Asp Ala Lys Glu Glu
50 55 60
Asp Trp His Leu Tyr Tyr Gln Tyr Asn Pro Ala Ala Thr Ile Trp Gly
65 70 75 80
Thr Pro Leu Tyr Trp Gly His Ala Val Ser Lys Asp Leu Thr Ser Trp
85 90 95
Thr Asp Tyr Gly Ala Ser Leu Gly Pro Gly Ser Asp Asp Ala Gly Ala
100 105 110
Phe Ser Gly Ser Met Val Ile Asp Tyr Asn Asn Thr Ser Gly Phe Phe
115 120 125
Asn Ser Ser Val Asp Pro Arg Gln Arg Ala Val Ala Val Trp Thr Leu
130 135 140
Ser Lys Gly Pro Ser Gln Ala Gln His Ile Ser Tyr Ser Leu Asp Gly
145 150 155 160
Gly Tyr Thr Phe Glu His Tyr Thr Asp Asn Ala Val Leu Asp Ile Asn
165 170 175
Ser Ser Asn Phe Arg Asp Pro Lys Val Phe Trp His Glu Gly Glu Asn
180 185 190
Gly Glu Asp Gly Arg Trp Ile Met Ala Val Ala Glu Ser Gln Val Phe
195 200 205
Ser Val Leu Phe Tyr Ser Ser Pro Asn Leu Lys Asn Trp Thr Leu Glu
210 215 220
Ser Asn Phe Thr His His Gly Trp Thr Gly Thr Gln Tyr Glu Cys Pro
225 230 235 240
Gly Leu Val Lys Val Pro Tyr Asp Ser Val Val Asp Ser Ser Asn Ser
245 250 255
Ser Asp Ser Lys Pro Asp Ser Ala Trp Val Leu Phe Val Ser Ile Asn
260 265 270
Pro Gly Gly Pro Leu Gly Gly Ser Val Thr Gln Tyr Phe Val Gly Asp
275 280 285
Phe Asn Gly Thr His Phe Thr Pro Ile Asp Gly Gln Thr Arg Phe Leu
290 295 300
Asp Met Gly Lys Asp Tyr Tyr Ala Leu Gln Thr Phe Phe Asn Thr Pro
305 310 315 320
Asn Glu Lys Asp Val Tyr Gly Ile Ala Trp Ala Ser Asn Trp Gln Tyr
325 330 335
Ala Gln Gln Ala Pro Thr Asp Pro Trp Arg Ser Ser Met Ser Leu Val
340 345 350
Arg Gln Phe Thr Leu Lys Asp Phe Ser Thr Asn Pro Asn Ser Ala Asp
355 360 365
Val Val Leu Asn Ser Gln Pro Val Leu Asn Tyr Asp Ala Leu Arg Lys
370 375 380
Asn Gly Thr Thr Tyr Ser Ile Thr Asn Tyr Thr Val Thr Ser Glu Asn
385 390 395 400
Gly Lys Lys Ile Lys Leu Asp Asn Pro Ser Gly Ser Leu Glu Phe His
405 410 415
Leu Glu Tyr Val Phe Asn Gly Ser Pro Asp Ile Lys Ser Asn Val Phe
420 425 430
Ala Asp Leu Ser Leu Tyr Phe Lys Gly Asn Asn Asp Asp Asn Glu Tyr
435 440 445
Leu Arg Leu Gly Tyr Glu Thr Asn Gly Gly Ala Phe Phe Leu Asp Arg
450 455 460
Gly His Thr Lys Ile Pro Phe Val Lys Glu Asn Leu Phe Phe Thr His
465 470 475 480
Gln Leu Ala Val Thr Asn Pro Val Ser Asn Tyr Thr Thr Asn Val Phe
485 490 495
Asp Val Tyr Gly Val Ile Asp Lys Asn Ile Ile Glu Leu Tyr Phe Asp
500 505 510
Asn Gly Asn Val Val Ser Thr Asn Thr Phe Phe Phe Ser Thr Asn Asn
515 520 525
Val Ile Gly Glu Ile Asp Ile Lys Ser Pro Tyr Asp Lys Ala Tyr Thr
530 535 540
Ile Asn Ser Phe Asn Val Thr Gln Phe Asn Val
545 550 555
Claims (10)
1. An exo-type inulinase inulina, which is characterized in that the amino acid sequence of the exo-type inulinase inulina is shown as SEQ ID NO: 2.
2. An exo-type inulinase gene encoding an exo-type inulinase inunold according to claim 1.
3. The exo-type inulase gene according to claim 2, wherein the nucleotide sequence of the exo-type inulase gene is as set forth in SEQ ID NO: 1.
4. An expression cassette comprising an exoinulinase gene according to claim 2 or 3.
5. A recombinant expression vector comprising the exoinulinase gene of claim 2 or 3.
6. A recombinant strain comprising the exoinulase gene of claim 2 or 3.
7. The recombinant strain of claim 6, wherein the host cell of the recombinant strain comprises any one of escherichia coli, bacillus, aspergillus, and yeast.
8. The recombinant strain of claim 7, wherein the yeast comprises kluyveromyces lactis.
9. A method for preparing exo-type inumulase inumuld, characterized in that the recombinant strain according to any one of claims 5 to 7 is cultivated to obtain exo-type inumulase inumuld.
10. Use of exo-inulinase inunold according to claim 1 for the preparation of fructose.
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