CN114262701A - Exo-type inugulase INUGold, preparation method and application thereof - Google Patents
Exo-type inugulase INUGold, preparation method and application thereof Download PDFInfo
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- CN114262701A CN114262701A CN202111457065.5A CN202111457065A CN114262701A CN 114262701 A CN114262701 A CN 114262701A CN 202111457065 A CN202111457065 A CN 202111457065A CN 114262701 A CN114262701 A CN 114262701A
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Abstract
The invention discloses an exo-type inugulase INUGold, a preparation method and application thereof, and relates to the technical field of bioengineering. The amino acid sequence of the exo-type inugulase INUGold is shown as SEQ ID NO: 2, respectively. The exo-type inugulase INUGold provided by the invention takes an amino acid sequence (X57202.1) of Kluyveromyces marxianus exo-type INU1 as an original material, and the exo-type INU1 is subjected to site-directed mutation transformation through artificial design transformation to obtain the exo-type inugulase INUGold (SEQ ID NO: 2) with high activity, so that the exo-type inugulase INUGold can be used for preparing fructose, the production efficiency of the fructose is obviously improved, and the exo-type inugulase INUGold can be used for industrial production of the fructose.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to exo-type inugulase INUGold, a preparation method and application thereof.
Background
Fructose is an excellent monosaccharide, has good mouthfeel, high sweetness and a low glycemic index, and is called as "healthy sugar". Fructose is the major edible sugar. Especially, with the change of consumption concept, the demand of fructose in food and beverage industries is increasing, and the consumption of fructose and high fructose syrup is already a main product of edible sugar.
Currently, there are two main methods for industrially producing fructose: firstly, the hydrolysis of glucose into glucose by amylase and the subsequent conversion of glucose into fructose by glucose isomerase are two-enzyme processes, but usually only a mixture of fructose and glucose (commonly called high fructose syrup) can be obtained, and if pure or high fructose content is required, a complicated purification process is required; secondly, sucrose is hydrolyzed into glucose and fructose by an acid method and an enzyme method, and the fructose is separated from the glucose by ion exclusion chromatography, while the sucrose is hydrolyzed by the acid method with potential pollution, and the fructose syrup mixture prepared by converting the glucose by isomerase usually has the fructose content of only 42 percent. If high purity fructose is required, a subsequent complicated separation and purification system is required.
Inulin (also called inulin) is an edible and storable polysaccharide next to starch, is widely present in compositae plants, and is a renewable resource with abundant sources and low price. It is mainly found in Jerusalem artichoke and chicory, so it is called inulin. Inulin, a natural macromolecular polymer formed by the polymerization of fructose monomers, was first extracted from plants in 1804 by german scientists. Inulin is a chain polysaccharide formed by connecting a plurality of fructosyl groups by beta-D-fructosyl bonds. The natural inulin is a mixture of fructose polymers with different lengths and different polymerization degrees, and the polymerization Degree (DP) of the natural inulin is approximately between 2 and 100. Therefore, inulin is an ideal material for producing fructose. Hydrolysis of inulin by exo-inulase to produce fructose is an efficient, green and advanced technique.
Exo-type inulinases are distributed in both plants and microorganisms, but the activity of exo-type inulinases produced from microorganisms obtained from nature is low, thereby limiting the industrial application of exo-type inulinases.
Disclosure of Invention
The invention mainly aims to provide an exo-type inugulase INUGold, a preparation method and application thereof, and aims to provide a high-activity exo-type inugulase.
In order to achieve the purpose, the invention provides an exo-type inugulase INUGold, wherein the amino acid sequence of the exo-type inugulase INUGold is shown as SEQ ID NO: 2, respectively.
In addition, the invention also provides an excision-type inulase gene which is used for coding the excision-type inulase INUGold.
Alternatively, the nucleotide sequence of the excised inulase gene is as defined in ID NO:1 is shown.
Furthermore, the invention also provides an expression cassette, which comprises the exo-type inulase gene.
In addition, the invention also provides a recombinant expression vector which comprises the exo-type inulase gene.
Furthermore, the present invention also proposes a recombinant strain comprising the exo-type inulase gene as described above.
Optionally, the host cell of the recombinant strain comprises any one of escherichia coli, bacillus, aspergillus and yeast.
Optionally, the yeast comprises kluyveromyces lactis.
In addition, the invention also provides a preparation method of the exo-type inugulase INUGold, which cultures the recombinant strain to obtain the exo-type inugulase.
In addition, the invention also provides the application of the exo-type inugulase INUGold in the preparation of fructose.
The exo-type inugulase INUGold provided by the invention takes an amino acid sequence (X57202.1) of Kluyveromyces marxianus exo-type INU1 as an original material, and the exo-type INU1 is subjected to site-directed mutation transformation through artificial design transformation to obtain the exo-type inugulase INUGold with high activity, wherein the amino acid sequence of the exo-type inugulase INUGold is shown as SEQ ID NO: 2, when the compound is used for preparing fructose, the production efficiency of the fructose is obviously improved, and the compound 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 used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 shows the three-dimensional structure of exo-type INU1 of the starting material in example 1 of the present invention;
FIG. 2 is a three-dimensional structure of an exo-type inugulase INUGold active center in example 1 of the present invention;
FIG. 3 is a map of recombinant expression vector pGKLAC-inuguld in example 3 of the present invention;
FIG. 4 is a gel electrophoresis diagram of the recombinant expression vector pGKLAC-inuguld of example 3 of the present invention after double digestion;
FIG. 5 shows the results of SDS-PEGE of the supernatants of the respective fermentations 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 implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides an exo-type inumold, the amino acid sequence of which is shown as SEQ ID NO: 2, respectively.
In the embodiment, an amino acid sequence (X57202.1) of Kluyveromyces marxianus Exo-type INU1 is used as a starting material, and is artificially designed, transformed and optimized to obtain the novel exo-type inumol, so that the activity of the original inumol is improved, and when the exo-type inumol with high activity is used for preparing fructose, the production efficiency of the fructose is remarkably improved, and the exo-type inumol can be used for industrial production of the fructose.
Specifically, on the basis of the protein sequence of the candidate inulase INU1, the invention researches the relationship between the sequence-structure-function of the protein on the basis of the statistical analysis of the sequence characteristics, and designs and obtains the novel excised inulase INUGold with improved sequence substrate/product catalytic efficiency on the basis of the information of the protein structure and large-scale data statistics and deep learning. The amino acid sequence of the novel excision inugulase INUGold is SEQ ID NO: 2, respectively. The sequence difference from the original inum 1 mainly lies in that aspartic acid at position 65 is mutated into leucine (D65L), glycine at position 241 is mutated into aspartic acid (G241D), methionine at position 306 is mutated into phenylalanine (M306F), tyrosine at position 326 is mutated into leucine (Y326L), and leucine at position 476 is mutated into proline (L476P). It should be noted that various mutation methods conventional in the art can be used for point mutation, and are not described herein.
The invention also provides an excision-type inulase gene, and the invention does not limit the specific nucleotide sequence of the excision-type inulase gene as long as the excision-type inulase gene can be coded by the excision-type inulase gene (SEQ ID NO: 2).
Completely designing and optimizing exogenetic inulase codons based on evolution information of an expression host, preference of the expression host to the codons, evolution information of enzyme molecules and the like; optimizing the adapted host cell. In this example, the nucleotide sequence of the artificially synthesized excised inulase gene is shown in SEQ ID NO:1, the excised inusold gene was named "excised inusold". After the recombinant strain containing the exo-type inulase gene inuguld is induced and expressed, the exo-type inuguld with high yield can be obtained, and the obtained exo-type inuguld has high activity.
The invention also provides an expression cassette which comprises the exo-type inugol gene (SEQ ID NO: 1) as described above. Specifically, an expression cassette induced by lactose is formed by combining a lactose-inducible promoter PLAC2, an exo-type inusold gene and a lactose operon termination sequence TT.
It will be appreciated that the expression cassette includes a lactose promoter for driving expression of the exo-type inusold enzyme gene described above. In the culture process of the recombinant strain, when lactose is added into a culture medium, the lactose can be carbon nutrition produced by the recombinant strain and can induce and start strong expression of the exo-type inusold.
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 exo-type inusold gene for directing the trans-membrane transfer of exo-type inulinase out of the cell.
The invention also provides a recombinant expression vector which comprises the exo-type inugol gene inugold (SEQ ID NO: 1). Further, the recombinant expression vector comprises the expression cassette. In case the nucleotide sequence of the excision-type inusold inusulase gene and the amino acid sequence of the excision-type inusold inusulase are determined, the skilled person will be able to select suitable expression vectors as well as other functional units. The expression vector may be a vector suitable for expression in a host such as bacteria, yeast, or fungi. In a preferred embodiment, the expression vector is any one of a pichia pastoris expression vector and a kluyveromyces lactis expression vector. Preferably, the expression vector is a kluyveromyces lactis expression vector.
In one embodiment, the recombinant expression vector is prepared by the steps of:
step S10, introducing Nde I restriction sites and EcoR I restriction sites at two ends of the excision type inumold gene fragment, using Nde I and EcoR I to double-restriction the excision type inumold gene fragment, under the action of T4 ligase, staying overnight at 16 ℃, connecting the restriction enzyme-cleaved inumold gene fragment to the lactic acid Kluyveromyces expression vector pGKLAC which is subjected to the same restriction enzyme and is recovered by glue, and obtaining a recombinant expression vector which is named as pGKLAC-inumold.
The invention also provides a recombinant strain, which comprises the exo-type inugol gene inugold (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 inugulase INUGold (SEQ ID NO: 2). Wherein the host cell of the recombinant strain comprises any one of escherichia coli, bacillus, aspergillus and yeast. Preferably, the host cell of the recombinant strain is Kluyveromyces lactis in yeast.
In one embodiment, the recombinant strain is prepared by the following steps:
step S20, linearizing the recombinant expression vector pGKLAC-inugold prepared in the step S10 by using restriction enzyme;
step S21, transforming the linearized recombinant expression vector into Kluyveromyces lactis by using an electrotransfer;
s22, screening single colonies growing in a flat plate containing acetamide;
and S23, after streaking and purifying, verifying the positive transformant by PCR to obtain the yeast recombinant strain containing the exo-enzyme inugulase inuguld gene.
The invention also provides a preparation method of the exo-type inugulase INUGold, which comprises the following steps: the recombinant strain is cultured to obtain the exo-type inugulase INUGold. Specifically, the recombinant strain containing the exo-type inulinase gene inunolid is cultured in YPGal culture solution containing lactose, and the exo-type inulinase is gradually induced, 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 application of the exo-type inugulase INUGold in preparation of fructose. Specifically, exo-type inugulase INUGold is contacted with inulin, and fructose is obtained through the hydrolysis of the exo-type inugulase INUGold on the inulin.
Further, the excision-type inumold is encoded by the excision-type inumold gene inumold. Because the artificially designed and synthesized excision-type inusold gene has the characteristics of high yield and high activity, compared with the traditional fructose preparation method, the method for obtaining the fructose provided by the invention is more efficient and can be suitable for industrial production of the fructose.
Preferably, the hydrolysis parameters of the exo-type inugulase inuguld on inulin are as follows: the pH value is 5.0, the temperature is 50 ℃, the inulin concentration is 15%, the ratio of the exo-type inulinase/inulin is 5000U/g (namely the mass ratio of the enzyme activity unit to the substrate is 5000U/g), the hydrolysis time is 4 hours, and under the hydrolysis parameters, the high-purity fructose syrup can be efficiently and conveniently obtained.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
Example 1 obtaining of Exo-type inugulase INUGold
Taking an amino acid sequence (X57202.1) of Kluyveromyces marxianus Exo-type INU1 as a starting material, wherein the nucleotide sequence of the starting Ex-type INU1 gene (named as excision-type inulase gene INU1) is represented by SEQ ID NO: 3, the amino acid sequence of the exo-type INU1 consists of SEQ ID NO: 4, respectively. It is understood that the last TGA in the nucleotide sequence (SEQ ID NO: 3) is a stop codon.
The invention is based on a protein sequence database of candidate exo-type inulase INU1, 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 sequence-structure-function relationship of the protein; based on the information of protein structure and large-scale data statistics and deep learning, with the assistance of TrRosetta and the like, a novel excised type inugulase INUGold with improved sequence substrate/product catalytic efficiency is designed and obtained, wherein the amino acid sequence of the novel excised type inugulase INUGold is SEQ ID NO: 2, the sequence difference from the original inum 1 mainly lies in that aspartic acid at position 65 is mutated into leucine (D65L), glycine at position 241 is mutated into aspartic acid (G241D), methionine at position 306 is mutated into phenylalanine (M306F), tyrosine at position 326 is mutated into leucine (Y326L), and leucine at position 476 is mutated into proline (L476P).
FIG. 1 shows the three-dimensional structure of excision-type INU1, and it can be seen from FIG. 1 that excision-type INU1 surrounds 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-inundase INUGold, and it can be seen from FIG. 2 that the redesigned exo-inundase INUGold surrounds the 5 key amino acids Leu65, Asp241, Phe306, Leu326 and Phe476 of the active center.
EXAMPLE 2 obtaining of exo-type inusold Gene inugold
Based on evolution information of an expression host, preference of the expression host to codons, evolution information of enzyme molecules, sequence information of enzyme genes and the like, completely new design and optimization of an excised inusold codon of the inusulase Gene are completed based on Gene Designer and the like; optimizing the adapted host cell. The exo-type inusold gene with high activity and high yield is obtained by artificial optimization, design and synthesis, and the nucleotide sequence of the exo-type inusold gene is shown as SEQ ID NO. 1. It is understood that the last TAA of the nucleotide sequence SEQ ID NO. 1 is a stop codon.
EXAMPLE 3 construction of recombinant expression vectors
(1) Nde I and EcoR I enzyme cutting sites are respectively introduced into two ends of the fragment of the exo-type inumold (SEQ ID NO: 1) synthesized in the embodiment 1, after the exo-type inumold is subjected to double enzyme cutting by the Nde I and the EcoR I, under the action of T4 ligase, the temperature is kept overnight at 16 ℃, the enzyme-cut inumold gene fragment is connected to a lactate Kluyveromyces expression vector pGKLAC subjected to the same enzyme cutting and recovered by glue, a recombinant expression vector is obtained and named pGKLAC-inumold, and the map of the pGKLAC-inumold recombinant expression vector is shown in figure 3. The exo-type inusold gene is expressed by driving of a lactose inducer and is terminated by a lactose TT terminator.
(2) The recombinant expression vector of the starting material exo-type inulase gene inu1 is constructed in the same manner as the step (1) and named as pGKLAC-inu 1.
The plasmid pGKLAC-inuguld vector was extracted and verified by double digestion with Nde I and EcoR I, and the results are shown in FIG. 4 (in FIG. 4: M is DNA Marker, 1 represents the undigested pGKLAC-inuguld vector, and 2 represents the double digested pAO-INU vector). As can be seen from FIG. 4, there is a band at 1600bp position, which indicates that the exo-type inusold gene 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-inuguld prepared in example 3 above was linearized with restriction enzymes under the following conditions: every 200L volume contains 4 ug of plasmid, 10U of Sac II enzyme, and after linearization at 37 ℃ for 2h, the pellet is dried for use.
(2) And transforming the linearized recombinant expression vector into kluyveromyces lactis by using an electrotransformation machine, wherein the condition parameters of electrotransformation are that V is 1500V, PC is 200 omega, and C is 25 mu F.
(3) And (3) placing the plate containing acetamide in an incubator at 28 ℃ for 2-3 d to screen single colonies.
(4) After streak purification, the positive transformant is verified by PCR, and the yeast recombinant strain containing the exo-enzyme inuguld gene is obtained.
(5) The recombinant strain of the starting material exo-type inulase gene inu1 is constructed in the same manner as the steps (1), (2) and (3) to obtain the recombinant strain containing the exo-type inulase gene inu 1.
Through identification, the obtained lactate Kluyveromyces lactis recombinant strain cell containing the exonuclease inugold gene is white and spherical, the temperature is 28-30 ℃ in the growth process, and lactose is a main carbon source.
EXAMPLE 5 preparation of Exo-type inugulase INUGold
The recombinant strains prepared in step (4) and step (5) of example 4 were inoculated into 250mL volumetric flasks containing 50mL YPD medium, and cultured with shaking at 28 ℃ and 180r/min at constant temperature. When OD is reached600When the concentration is about 3.0-6.0, the cells are transferred into YPLac culture medium containing 100mL of culture medium (the components of the YPLac culture medium are 10g of yeast powder, 20g of peptone and 20g of lactose in each 1000mL of liquid), fermentation liquor is taken at different time points (12/24/36/48 h, 0h as the fermentation starting point during inoculation), the fermentation liquor is centrifuged for 5min at 10000rpm, and supernate (respectively exo-inulase INUGold and exo-inulase INU1) is taken.
The results of SDS-PAGE detection of the above-mentioned fermentation supernatants are shown in FIG. 5. As can be seen from FIG. 5, the protein content of the exo-type inusold recombinant strain obtained by artificial design and optimization is obviously higher than that of the recombinant strain containing the original inulinase gene inu 1.
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 activity in U/mL, and the abscissa represents different time points). As can be seen from FIG. 6, the activity of the exo-type inumold after the optimized design and modification is obviously higher than that of the original exo-type inumold gene INU1, specifically, at 48h, the enzyme activity reaches 21500U/mL, while the original gene is 6150U/mL, and the activity is improved by about 3.5 times.
In conclusion, the exo-type inugulase INUGold with high yield and high activity can be obtained after the recombinant strain constructed by the exo-type inugulase gene inuguold through optimization is fermented and cultured.
Example 6 preparation of fructose
The exo-type inugulase INUGold obtained by culturing the recombinant strain constructed by the exonuclease-containing inugulase gene inuguld in the embodiment 5 is contacted with inulin, and fructose is obtained by hydrolysis of the inulin.
Optimizing a reaction system:
1. the effect of inulin concentration on hydrolysis; at the pH of 5.0 and the temperature of 50 ℃, the volume ratio of the dosage of the enzyme solution mentioned in example 5 to the inulin substrate is 1:1, the hydrolysis time is 0.5h, and under the conditions that a series of inulin concentrations are respectively 8%, 10%, 15%, 30% and 50%, the hydrolysis product is detected, the substrate concentration has a remarkable influence on the hydrolysis rate of the inulin, when the substrate concentration is 15%, the hydrolysis rate is close to 100%, the substrate concentration is higher than 15%, and the hydrolysis rate is obviously reduced. 15% inulin was used for the following experiments.
2. Influence of enzyme activity unit and inulin quality ratio on inulin hydrolysis rate; under the conditions of pH5.0, temperature 50 ℃, reaction inulin substrate concentration 15% and hydrolysis time 0.5h, a series of different weight ratios (U/g) of the enzyme activity unit of the excision-type inulase to the inulin substrate are set, and respectively: 25000. 12000, 10500, 7500, 5000 and 4000, and the hydrolysis rate of inulin at each ratio was measured. The results show that the hydrolysis rate of inulin is increased with the increase of exo-type inulinase. Meanwhile, when the amount of enzyme was changed from 5000 to 12000, the hydrolysis rates thereof were very similar. Considering that the hydrolysis time was set to 0.5 hours, a ratio of 5000U/g was selected for subsequent experiments.
3. The influence of the hydrolysis time on the rate of hydrolysis of inulin. The effect of hydrolysis time on the rate of inulin hydrolysis was investigated at pH5.0, a temperature of 50 ℃, a substrate concentration of 15% and an enzyme/inulin ratio of 10000U/g. When the reaction time is 2 hours, about 95% of the inulin is hydrolyzed, and after the time is extended to more than 4 hours, the inulin is hydrolyzed to almost 100% into monosaccharide.
4. According to the above optimization test, the optimal hydrolysis parameters of inulin are pH5.0, temperature 50 deg.C, inulin concentration 15%, enzyme/inulin ratio 5000U/g, and hydrolysis time 4 hr. After 4 hours 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 the other substance glucose of at most 5%.
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Kingkosey science and technology Limited
<120> exo-type inugulase INUGold, preparation method and application thereof
<130> 20211124
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1668
<212> DNA
<213> Artificial Synthesis
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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> Artificial 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 inumold, wherein the amino acid sequence of the exo-type inumold is as shown in SEQ ID NO: 2, respectively.
2. An exo-inulinase gene encoding the exo-inulinase of claim 1.
3. The exo-type inulase gene as claimed in claim 2 wherein the nucleotide sequence of the exo-type inulase gene is as set forth in SEQ ID NO:1 is shown.
4. An expression cassette comprising the exo-type inulase gene as claimed in claim 2 or 3.
5. A recombinant expression vector comprising the exo-type inulase gene as claimed in claim 2 or 3.
6. A recombinant strain comprising the exo-type inulase gene as defined in claim 2 or 3.
7. The recombinant strain of claim 6, wherein the host cell of the recombinant strain comprises any one of E.coli, Bacillus, Aspergillus, and yeast.
8. The recombinant strain of claim 7, wherein the yeast comprises kluyveromyces lactis.
9. A process for the preparation of exo-inulinase inuguld, characterized in that recombinant strains according to any one of claims 5 to 7 are cultivated to obtain exo-inulinase inuguld.
10. Use of an exo-inulinase inuguld as defined in claim 1 for the preparation of fructose.
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