CN112852796A - Cellobiose epimerase mutant and application thereof in preparation of lactulose - Google Patents

Abstract

The invention discloses a cellobiose epimerase mutant and application thereof in preparing lactulose by a catalytic method. The mutant is obtained by carrying out single-point mutation or combined mutation on cellobiose epimerase CE with an amino acid sequence shown as SEQ ID NO. 1, and the cellobiose epimerase mutant with improved catalytic activity is obtained and can be used for producing lactulose by using lactose as a substrate through isomerization. Compared with the wild cellobiose epimerase, the single-site mutant and the combined mutant thereof have higher activity, particularly the combined mutant shows the additive effect of the single-site mutant activity, and the crude enzyme liquid enzyme activity of the mutant R56K/L187I/L254I is as high as 30U/ml. Under the reaction conditions of 80 ℃, pH7.0 and substrate concentration of 700g/L, the yield of lactulose generated by catalyzing lactose is 526g/L, the conversion rate reaches 75 percent, and the production cost is greatly reduced.

Description

Cellobiose epimerase mutant and application thereof in preparation of lactulose

Technical Field

The invention belongs to the technical field of biology, and relates to an application of a cellobiose epimerase mutant, in particular to an application of the cellobiose epimerase mutant in preparation of lactulose by taking lactose as a substrate for isomerization reaction.

Background

The chemical name of lactulose is 4-O-beta-D-galactopyranosyl-D-fructose, the lactulose is an isomer with lactose, the sweetness is equivalent to that of lactose less than that of sucrose, the lactulose has cool and mellow feeling, the viscosity is low, the heat value is low, the safety is high, the stability is good, and the Maillard reaction does not occur. Lactulose has unique physiological functions of regulating intestinal flora, improving immunity, improving metabolism, reducing blood ammonia and catharsis, is used for preventing and treating various hepatic diseases, can be used as a low-calorie sweetener and a functional food additive in food, and is a functional oligosaccharide used as both medicine and food.

At present, lactulose produced commercially is mainly obtained by chemical isomerization of lactose in an alkaline solution, a large amount of chemical catalysts are needed, the reaction conditions are generally severe, the obtained product has deep color and a large amount of byproducts, and the catalyst is difficult to separate, so that the further development of lactulose production by a chemical isomerization method is restricted. Therefore, enzymatic synthesis of lactulose is an economical and efficient alternative, and lactose can be efficiently produced by galactose acylation or isomerization. Among them, galactose acylation is usually the hydrolysis of lactose catalyzed by β -glucosidase and β -galactosidase (EC 3.2.1.23) to produce galactose and glucose, the galactose moiety is converted to fructose, and then fructose and galactose are reacted by transglycosidase to form lactulose; cellobiose-epimerase can catalyze the isomerization of lactose to produce lactulose.

β -glucosidase: fischer et al found in 1996 that the enzyme CelB produced by Pyrococcus furiosus had 100% beta-glucosidase and 60% beta-galactosidase activity. 2004, Mayer et al [1] found that the highest yield of lactulose catalyzed by recombinant CelB was 15.0g/L under the condition that fructose was a galactosyl receptor; in 2010, the research group developed a model for continuous production of lactulose in an enzyme membrane reactor or packed bed, the lactulose yields synthesized by using CelB immobilized on anion exchange resin Amberlite IRA-93 or Eupergit C as catalyst were all 14.6g/l, while the lactulose yield converted from free CelB was 13.9g/l [2 ].

Free beta-galactosidase: 2006, Doek et al [3], using genetically modified thermostable lactase, with lactose (400 g/L) and fructose (200 g/L) as substrates, reacting at 80 ℃ for 6h to produce 50g/L lactulose; 2008 Zhang Li Hui et al [4], using Kluyveromyces lactis, the screened strain after mutagenesis has beta-galactosidase enzyme activity of about 33.47U/mL, the optimum concentrations of lactose and fructose are 40% (w/v) and 20% (w/v), respectively, and the yield of lactulose is 57.83 g/under the optimum reaction conditions; 2009, Adamczak [5] finds that the capability of aspergillus oryzae beta-galactosidase to catalyze lactulose synthesis is strongest, and the yield reaches up to 65 g/L; 2011 Tan et al [6] discovered that Arthrobacter hypothermis (Arthrobacter sp.) beta-galactosidase can catalyze the production of lactulose under mild reaction conditions (20 ℃, p H6.0.0 ℃).

Immobilized β -galactosidase: 2010, Hua X and the like [7], the highest yield of lactulose can reach 151 g/L and the yield reaches 17% in a cyclohexane-buffer solution dual-phase system by using immobilized beta-galactosidase and glucose isomerase; 2015, Shenzuiyun et al [8], the enzyme activity is still maintained above 60% after 20 times of continuous use of beta-galactosidase embedded in alginate-gelatin-calcium phosphate (AGCaP) capsules, and the enzyme activity is still maintained at about 90% after 30 days of storage. Meanwhile, compared with the beta-galactosidase which is free and embedded in the calcium alginate capsule, the beta-galactosidase fixed on the AGCaP has the widest temperature and pH application range. At the temperature of 38 ℃, the pH value is 6.8, the concentrations of lactose and fructose are respectively 250 mg/mL, after reaction for 3 hours, the beta-galactosidase embedded in the AGCaP capsule catalyzes transglycosidation reaction to respectively generate 26.1 mg/mL lactulose and 74.2 mg/m L lactulose. 2018 Guerrero C and the like [9] adopt beta-galactosidase from immobilized aspergillus oryzae to continuously produce lactulose by taking fructose and lactose as raw materials, and the highest equivalent rate of the lactulose reaches 0.6 g/g. 2020, Maria de F-tima Matos de Fretias 1 et al [10], beta-galactosidase is produced by adding yeast extract to Kluyveromyces lactis NRRL Y1564 cheese whey at an optimum temperature of 30 ℃, and its enzymatic activity is 4418.37U/g. The conversion rates obtained by hydrolyzing lactose with both soluble and immobilized enzymes were 38.0% and 42.8%, respectively, and the yield of lactulose could also reach 17.32 g/L using biocatalysts from immobilized enzyme to chitosan carrier.

Although a plurality of researchers improve the traditional enzyme method for preparing lactulose in various ways, including that an organic two-aqueous-phase system reduces water activity, so that the transglycosidic activity of beta-galactosidase is improved, a two-enzyme co-immobilization system is adopted to catalyze the reaction of lactose serving as a single substrate, the use of permeable cells and the like, the traditional enzyme method for preparing lactulose still has the defects of low conversion rate (10% -43%), high concentration of fructose serving as a co-acting substrate (100-300 g/L), long reaction time, low production efficiency (less than or equal to 151 g/L) and the like, and the large-scale application of the traditional enzyme method in lactulose preparation is limited.

The cellobiose epimerase is a thermophilic enzyme, the substrate solubility and the reaction rate are improved at high temperature, and the application of the cellobiose epimerase has the advantages that substrate fructose does not need to be additionally added, the cost is low, and the prospect of commercially producing lactulose by using the cellobiose epimerase in the future is very wide. 2012, Yeong-Su et al [11] attempted to catalyze the lactose reaction with cellobiose epimerase (CsCE) derived from Caldicellulosriptor saccharolyticus DSM 890, which is capable of isomerizing aldose into ketose, and under the optimum conditions (pH 7.5, 80 ℃, 700g/l lactose), lactulose yield of 408g/l was achieved, the conversion of lactulose was 58%, and about 15% of the substrate lactose was converted into ipralactose, and both the concentration and conversion of lactulose were the highest reported in the enzymatic preparation of lactulose; 2013, the Yeong-Su research group [12] adds borate (molar ratio, borate: lactose =1:1) into the reaction system of CsCE catalyzing lactulose synthesis, the yield of lactulose is improved to 616g/l, the conversion rate is improved to 88%, although the method can improve the lactulose conversion rate, the addition of a large amount of borate improves the difficulty of separation and purification, and increases the cost of lactulose synthesis. The patent CN201910023301.9 discloses a recombinant Bacillus subtilis containing a cellobiose epimerase mutant, wherein the enzyme activity of the recombinant epimerase is 8U/mL, the yield of lactulose generated under the reaction conditions of 80 ℃, pH 7.5, enzyme addition amount of 20U/mL and substrate concentration of 400g/L is 200g/L, and the conversion rate can reach 51%.

In summary, the existing technologies for preparing lactulose by the conventional enzymatic method and cellobiose epimerase method have the problems of low substrate yield, high cost and the like, so that a new cellobiose epimerase with higher activity and better stability needs to be found.

Reference documents:

[1]Mayer J,Conrad J,Klaiber I,et al.Enzymatic production andcomplete nuclear magnetic resonance assignment of the sugarlactulose[J].Agric Food Chem,2004,52(23):6983-6990.

[2]Mayer J,Kranz B,Fischer L.Continuous production of lactulose by immobilized thermostableβ

-glycosidase from pyrococcus[J].Biotechnol,2010,145(4):387-393.

[3]Kim Y S,Park C S,Oh D K.Lactulose production from lactose and fructose by a thermostableβ

-galactosidase from sulfolobus solfataricus[J].Enzyme and Microbial Technology.2006,39(4):903-908.

[4] zhang Bao Li is suitable for screening beta-galactosidase high-yield strain prepared from lactulose and optimizing enzyme production conditions [ D ]. Wuxi, Shuoshi paper of Jiangnan university, 2008, 4-5.

[5]Adamczak M,Charubin D,Bednarski W.Influence of reactionmedium composition on enzymatic synthesis of galactooligosaccharides and lactulose from lactose concentrates prepared fromwhey permeate[J].Chem Pap,2009,63(2):111-116.

[6]Tang L,Li Z A,Dong X X,et al.Lactulose biosynthesis by β-galactosidase from a newly isolated Arthrobacter sp. [J].Journal of Industrial Microbiology and Biotechnology.2011,38(3):471-476

[7]Hua X,Yang R,Zhang W,et al.Dual-enzymatic synthesis oflactulose in organic-aqueous two-phase media[J].Food Re-search International 2010,43(3):716-722.

[8] Shenzuyun, construction and application of enzyme for preparing lactulose study [ D ]. Wuxi, university of Jiangnan, 2015.

[9]Guerrero C, Valdivia F,Ubilla C, et al.Continuous enzymaticsynthesis of lactulose in packed-bedreactor with immobilized Aspergillusoryzae β- galactosidase [J].Bioresource Technology ,2018.

[10]Maria de Fátima Matos de Freitas1 , Lucas C. Hortêncio1 , Tiago Lima de Albuquerque1, et al.Simultaneous hydrolysis of cheese whey and lactulose productioncatalyzed by β-galactosidase from Kluyveromyces lactis NRRL Y1564 [J].Bioprocess and Biosystems Engineering. 2020, 43:711–722

[11]Kim YS, DK O. Lactulose production from lactose as a single substrate by a thermostable cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus[J]. Bioresour Technol. 2012, 104: 668-672.

[12] Kim YS, Kim JE, DK O. Borate enhances the production of lactulose from lactose by cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus[J]. Bioresource Technology. 2013, 128: 809-812。

Disclosure of Invention

The invention aims to solve the technical problem of providing a cellobiose epimerase mutant and application thereof in preparing lactulose by catalyzing lactose isomerization.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention provides a cellobiose epimerase mutant, which is obtained by mutating an amino acid sequence shown as SEQ ID NO. 1, wherein the mutation site is selected from one or more of R56K, L187I, L254I, L254M, W372Y and C374T.

Preferably, the cellobiose epimerase mutant has mutation sites of R56K, L254I, C374T, R56K/W372Y, R56K/L254M, L254I/C374T, R56K/L187I/L254I, R56K/L254I/C374T or R56K/L254I/W372Y.

Further preferably, the mutation site is R56K/L187I/L254I or R56K/L254I/W372Y.

According to the cellobiose epimerase mutant provided by the invention, the amino acid sequence of the single-point mutant corresponding to R56K is SEQ ID NO. 3; the amino acid sequence of the single-point mutant corresponding to the L254I is SEQ ID NO. 5; the amino acid sequence of the single-point mutant corresponding to C374T is SEQ ID NO. 7; the amino acid sequence of the combined mutant corresponding to R56K/W372Y is SEQ ID NO. 9; the amino acid sequence of the combined mutant corresponding to R56K/L254M is SEQ ID NO. 11; the amino acid sequence of the combined mutant corresponding to L254I/C374T is SEQ ID NO 13; the amino acid sequence of the combined mutant corresponding to R56K/L187I/L254I is SEQ ID NO. 15; the amino acid sequence of the combined mutant corresponding to R56K/L254I/C374T is SEQ ID NO 17; the amino acid sequence of the combined mutant corresponding to R56K/L254I/W372Y is SEQ ID NO 19.

The present invention also provides a gene encoding the cellobiose epimerase mutant as described above,

the gene sequence of the single-point mutant corresponding to R56K is SEQ ID NO. 4; the gene sequence of the single-point mutant corresponding to the L254I is SEQ ID NO. 6; the gene sequence of the single-point mutant corresponding to C374T is SEQ ID NO. 8; the gene sequence of the combined mutant corresponding to R56K/W372Y is SEQ ID NO 10; the gene sequence of the combined mutant corresponding to R56K/L254M is SEQ ID NO 12; the gene sequence of the combined mutant corresponding to L254I/C374T is SEQ ID NO. 14; the gene sequence of the combined mutant corresponding to R56K/L187I/L254I is SEQ ID NO 16; the gene sequence of the combined mutant corresponding to R56K/L254I/C374T is SEQ ID NO 18; the gene sequence of the combined mutant corresponding to R56K/L254I/W372Y is SEQ ID NO. 20.

The present invention also provides a recombinant expression vector comprising the gene as described above. The invention also provides a soluble protein, an immobilized enzyme or an engineering bacterium containing the cellobiose epimerase mutant.

The cellobiose epimerase mutant is applied to preparation of lactulose by catalyzing lactose isomerization.

The invention also provides a method for producing lactulose, which takes lactose as a substrate and takes the cellobiose epimerase mutant as a catalyst.

Preferably, the method for producing lactulose as described above is that the lactose concentration is 500-700g/L, the enzyme dosage is 20-30U/mL, the reaction temperature is 75-80 ℃, the pH is 7.0-7.5, and the reaction time is 1-4 h.

The invention also provides application of the cellobiose epimerase mutant in the fields of food, feed or medicine.

In the field of food, lactulose can stimulate the growth of probiotics in the intestinal tracts of adults and juveniles, and can be used as an ideal nutrient addition source in functional food based on the characteristic of benefiting health; some special functional foods prepared for patients with specific diseases (such as diabetes) may also contain lactulose; lactulose as an additive to dairy products, e.g. lactulose yoghurt, can be used to treat constipation in children, and has a better therapeutic effect than yoghurt containing other dietary fibres (such as galacto-oligosaccharides, resistant starch, etc.). In the field of feed, lactulose can be used as animal feed to increase the immunity of livestock. In the field of medicine, lactulose has the effects of inhibiting salmonella, reducing blood ammonia value, enhancing mineral absorption, preventing bile acid formation and the like, can be used for treating constipation, hepatic encephalopathy, enteritis and osmotic pressure regulation type cathartics.

Has the advantages that: the invention carries out single-point mutation or combined mutation on cellobiose epimerase with an amino acid sequence shown as SEQ ID NO. 1 for the first time and is applied to catalytic preparation of lactulose, the catalytic activity is obviously improved, wherein the enzyme activity of crude enzyme liquid of a combined variant R56K/L187I/L254I is up to 30U/ml, the yield of lactulose generated by catalyzing lactose is 526g/L under the reaction conditions of 80 ℃, pH7.0 and substrate concentration of 700g/L, the conversion rate is up to 75 percent, and the production cost is greatly reduced.

Drawings

FIG. 1: shaking flask fermentation SDS-PAGE electrophoresis of recombinant bacteria, 1: coli BL21 (DE3)/pET-28a empty cell wall-broken supernatant; 2: coli BL21 (DE3)/pET-28a empty cell wall-broken sediment: 3: coli BL21 (DE3)/pET-28a-CE cell-wall-broken supernatant; 4: coli BL21 (DE3)/pET-28a-CE wall-breaking precipitate; m: molecular weight Marker in standard protein.

FIG. 2: the graph shows the change of the content of lactulose in the course of the reaction of example 5.

FIG. 3: the liquid chromatogram of lactose and lactulose in the assay system after the end of the reaction of mutant R56K/L187I/L254I in example 5 was used.

Detailed Description

Measuring lactulose by HPLC method with Daian U3000, BP-100 calcium ion hydrocarbon column and RI detector at 80 deg.C and flow rate of 0.5 mL/min as mobile phase. The yield of lactulose was determined by the retention time of the peak and the area of the peak using an external standard method.

The enzyme activity of cellobiose epimerase is defined as the amount of enzyme required to catalyze lactose to produce 1. mu. mol lactulose in 50mM buffer at 80 ℃ and pH7.0, and the reaction time is 20min, with one U being the concentration of lactose as a reaction substrate of 150 g/L. Example 1: transformation of recombinant plasmid pET-28a-CE

A recombinant plasmid pET-28a-CE containing a cellobiose epimerase CE (GenBank: HGB 31345.1) gene having an amino acid sequence shown in SEQ ID NO:1 was synthesized by general biosystems (Anhui) Co., Ltd, and the recombinant plasmid was transformed into E.coli BL21 competence purchased in Novonopathy to obtain recombinant E.coli BL21 (DE3)/pET-28a-CE, which was spread on an LB plate containing kanamycin resistance (50. mu.g/mL) and cultured overnight at 37 ℃. Selecting single colony to 5mL liquid LB culture medium containing kanamycin (50 mug/mL) resistance, culturing at 37 ℃ for 8h, preserving the strain liquid in a glycerin tube, storing in a refrigerator at-20 ℃, and performing shake flask fermentation to produce enzyme after verification of correctness by common biological sequencing.

Example 2: obtaining of mutants

The site-directed mutagenesis technology is adopted to obtain single-point mutation or combined mutant, the Primer of the site-directed mutagenesis is designed by Primer premier 5.0, and the principle of Primer design is as follows: the 5' end of the forward and reverse amplification primers comprises a 15-21bp reverse complementary region, the length of each primer non-complementary region is at least 15 bp, and the mutation to be introduced is contained in the complementary region. The mutant primers are shown in Table 1.

TABLE 1 mutant primers

Mutations	Nucleotide sequence of mutant primer	Corresponding serial number
			R56K-upstream primer	AGTTTTAACTACAAAAATATTATGGTTTTTTTCTG	SEQ ID NO :21
R56K-downstream primer	AAAACCATAATATTTTTGTAGTTAAAACTAAGCT	SEQ ID NO :22
			L187I-upstream primer	GTATGAATTCTCTAATTCATATATTGGAAGCTTAT	SEQ ID NO :23
L187I-downstream primer	TTCCAATATATGAATTAGAGAATTCATACTTTTTT	SEQ ID NO :24
			L254I-upstream primer	TTGAGGCAACTTGGATATTAGATGAATCCTTAAAA	SEQ ID NO :25
L254I-downstream primer	GGATTCATCTAATATCCAAGTTGCCTCAATATCAT	SEQ ID NO :26
			L254M-upstream primer	GAGGCAACTTGGATGTTAGATGAATCCTTAAAATA	SEQ ID NO :27
L254M-downstream primer	GGATTCATCTAACATCCAAGTTGCCTCAATATCATGT	SEQ ID NO :28
			W372Y-upstream primer	ATAGTAGAACCCTACAAATGCCCTTATCATAATGG	SEQ ID NO :29
W372Y-downstream primer	GATAAGGGCATTTGTAGGGTTCTACTATGGGCATA	SEQ ID NO :30
			C374T-upstream primer	AGAACCCTGGAAAACCCCTTATCATAATGGAAGAA	SEQ ID NO :31
C374T-downstream primer	CCATTATGATAAGGGGTTTTCCAGGGTTCTACTATGG	SEQ ID NO :32

Site-directed mutagenesis takes pET-28a-CE recombinant plasmid as a template, PrimerStar Mix is used for full plasmid amplification, a reaction system is set according to table 2, the template in a PCR reaction system is removed by digesting an amplification product through Dpn I enzyme, and then homologous recombination is carried out on a 5 'end and a 3' end under the catalysis of recombinase, so that the cyclization of the plasmid is completed. Finally, the circularized amplification product was transferred to E.coli BL21(DE3) host bacteria, spread on kanamycin-containing plates, and cultured overnight in a 37 ℃ incubator.

TABLE 2 site-directed mutagenesis System

Name (R)	Volume (μ l)
		Recombinant plasmid pET-28a-CE	1
Upstream primer	1
		Downstream primer	1
PrimerStar Mix	25
		Supplement of ddH2O to	50

The PCR procedure was: pre-denaturation at 95 ℃ for 300s, denaturation at 98 ℃ for 10s, annealing at 65 ℃ for 15s, extension at 72 ℃ for 300s, reaction for 30 cycles, extension at 72 ℃ for 5min, and final heat preservation at 4 ℃. After the PCR reaction, the PCR product was detected by 0.8% agarose gel electrophoresis. Then, 1 μ l of Dpn I is added into each PCR tube, gently mixed and placed in a metal bath at 37 ℃ for 2 h, and then the digested amplification product is subjected to recombination reaction according to the formula in the table 3.

TABLE 3 recombination reaction System

Components	Volume (μ l)
		200 ng of linearized plasmid	6
5 x Ligation Free Cloning	4
		Supplement of ddH2O to	20

The next day, three recombinant E.coli BL21 strains containing mutant plasmids were selected from the plates, and the above recombinant bacteria were inoculated from the plates into 50mL shake tubes containing 5mL of liquid LB medium (LB (g/L): peptone 10, sodium chloride 10, yeast extract 5) containing the corresponding resistance, respectively, and the corresponding resistance was added, followed by incubation at 37 ℃ on a shaker for 12 h at a rotation speed of 200 rpm. After the culture was completed, the plasmid was extracted and sequenced by Kinshire. Finally, the sequencing result is compared with the wild type enzyme protein nucleic acid sequence to determine whether the mutation is successful.

According to the method, single site mutation is firstly carried out to obtain recombinant expression bacteria E.coli BL21 (DE3)/pET-28a-R56K, E.coli BL21 (DE3)/pET-28aL187I, E.coli BL21 (DE3)/pET-28a-L254I, E.coli BL21(DE 21)/pET-28 a-L254 21, E.coli BL21(DE 21)/pET-28 a-W372 21, E.coli BL21(DE 21)/pET-28 a-C21, and the recombinant plasmid subjected to the primary mutation is taken as a template for secondary mutation or tertiary mutation, and finally the double-site comprising the CE mutant with correct sequencing and the three-site mutation recombinant expression bacteria E.coli BL21(DE 21)/pET-28 a-3656/W21 a/21/E.coli BL 21/21 a/21 E.coli BL21 (DE3)/pET-28a-R56K/L187I/L254I, E.coli BL21 (DE3)/pET-28a-R56K/L254I/C374T, E.coli BL21 (DE3)/pET-28 a-R56K/L254I/W372Y.

Example 3: recombinant bacterium escherichia coli fermentation culture

10 strains (not containing E.coli BL21 (DE3)/pET-28a-L187I, E.coli BL21 (DE3)/pET-28a-L254M, E.coli BL21 (DE3)/pET-28 a-W372Y) of the recombinant bacteria E.coli strains obtained in examples 1 and 2 were inoculated into 5mL of LB medium containing kanamycin resistance (50. mu.g/mL), cultured at 37 ℃ for 8 hours, transferred to 50mL of TB fermentation medium in 2% inoculum size, and incubated at 37 ℃ and 200rpm until the OD of the bacterial cells reached₆₀₀When the temperature reaches 0.6 ℃, the mixture is transferred to 30 ℃ and is subjected to shake flask induction fermentation for 16h at 200 rpm. And after fermentation is finished, the fermentation liquor is subjected to sound crushing and then is centrifuged, and the supernatant is the recombinase CE and the mutant enzyme. The activity of the recombinase CE can reach 16U/mL, the protein electrophoresis result shows that a band with the same theoretical molecular weight exists at the position of 43kDa (figure 1), and the enzyme activity of the mutant is shown in Table 4.

TABLE 4 enzymatic Activity of recombinase CE and mutants

Mutant enzymes	Enzyme activity relative recombination CE (%)
		R56K	136.8
L254I	128.7
		C374T	111.2
R56K/ W372Y	143.4
		R56K/ L254M	135.3
L254I/C374T	109.2
		R56K/L187I/L254I	187.6
R56K/L254I/C374T	125.6
		R56K/L254I/W372Y	152.1

Example 4: recombinase CE and mutant enzyme catalysis

The reaction system is as follows: lactose concentration 500g/L, enzyme amount 20U/mL (recombinant enzyme CE or mutant enzyme), using pH7.0 sodium phosphate buffer, at 75 degrees C, 150r/min water bath shaking table reaction, sampling every 1h boiling termination reaction, HPLC detection product content, 4h after the termination of the whole reaction.

The enzyme catalysis result is shown in Table 5, and the detection shows that when the reaction is carried out for 3 hours, the recombinant enzyme CE achieves the equilibrium conversion rate of 56 percent, and the conversion rate of the mutant enzyme R56K/L187I/L254I is as high as 75 percent.

TABLE 5 catalytic results

Catalytic enzymes	Lactulose conversion
		Recombinant CE	56%
R56K	66%
		L254I	61%
C374T	57%
		R56K/ W372Y	69%
R56K/ L254M	65%
		L254I/C374T	58%
R56K/L187I/L254I	75%
		R56K/L254I/C374T	60%
R56K/L254I/W372Y	72%

Example 5: method for producing lactulose

Using lactose as a substrate, and using cellobiose epimerase mutant R56K/W372Y, R56K/L187I/L254I or R56K/L254I/W372Y as a catalyst, wherein the reaction conditions are as follows: the concentration of lactose is 700g/L, the enzyme dosage is 30U/mL, the reaction temperature is 80 ℃, the pH value is 7.5, the reaction is carried out in a water bath shaking table at 150r/min, samples are taken every 1h, the boiling is carried out, the reaction is stopped, and the content of the product is detected by HPLC until the reaction reaches the balance.

The enzyme conversion process is shown in FIG. 2, when the reaction is carried out for 3h, the mutant enzyme reactions reach equilibrium, the conversion rate of R56K/L187I/L254I reaches up to 75%, the yield is 526g/L, and the liquid chromatogram peak is shown in FIG. 3.

Sequence listing

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tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttggt tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 3

<211> 392

<212> PRT

<213> Artificial sequence

<400> 3

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Lys Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Leu Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Cys Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 4

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 4

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaaaaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttggt tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 5

<211> 392

<212> PRT

<213> Artificial sequence

<400> 5

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Arg Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Ile Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Cys Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 6

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 6

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaagaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttgga tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 7

<211> 390

<212> PRT

<213> Artificial sequence

<400> 7

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Arg Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Leu Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Thr Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn

385 390

<210> 8

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 8

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaagaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttggt tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaaa ccccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 9

<211> 392

<212> PRT

<213> Artificial sequence

<400> 9

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Lys Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Leu Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Tyr Lys Cys Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 10

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 10

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaaaaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttggt tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctacaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 11

<211> 392

<212> PRT

<213> Artificial sequence

<400> 11

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Arg Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Ile Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Thr Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 12

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 12

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaaaaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttgga tgttagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 13

<211> 392

<212> PRT

<213> Artificial sequence

<400> 13

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Arg Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Ile Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Thr Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 14

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 14

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaagaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttgga tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaaa ccccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 15

<211> 392

<212> PRT

<213> Artificial sequence

<400> 15

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Lys Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Ile His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Ile Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Cys Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 16

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 16

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaaaaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctaat tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttgga tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 17

<211> 392

<212> PRT

<213> Artificial sequence

<400> 17

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Lys Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Ile Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Trp Lys Thr Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 18

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 18

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaaaaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttgga tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctggaaaa ccccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 19

<211> 392

<212> PRT

<213> Artificial sequence

<400> 19

Met Asp Leu Lys Thr Leu Lys Asn Glu Val Lys Asn His Leu Thr Glu

1 5 10 15

Lys Ile Ile Pro Phe Trp Ala Lys Leu Met Asp Lys Glu Asn Gly Gly

20 25 30

Tyr Ile Gly Tyr Val Ser Phe Asp Leu Lys Lys Asp Pro Tyr Ala His

35 40 45

Lys Ser Leu Val Leu Thr Thr Lys Ile Leu Trp Phe Phe Ser Ala Val

50 55 60

Tyr Asn Leu Thr Lys Glu Glu Asn Leu Ile Pro Tyr Met Asn His Ala

65 70 75 80

Tyr Ser Phe Leu Val Gln Lys Leu Trp Asp His Lys Asn Lys Gly Phe

85 90 95

Tyr Trp Met Val Asp Tyr Lys Gly Glu Pro Ile Asp Lys Arg Lys His

100 105 110

Ile Tyr Gly His Ala Phe Ser Ile Tyr Ala Leu Ser Glu Phe Tyr Lys

115 120 125

Ala Thr Lys Lys Asp Glu Ala Leu Asn Ile Ala Leu Glu Thr Tyr Asn

130 135 140

Leu Leu Glu Glu Lys Cys Lys Asp Glu Tyr Ala Tyr Leu Glu Glu Phe

145 150 155 160

Asp Glu Tyr Trp Asn Pro Lys Glu Asn Lys Ala Ile Ser Glu Tyr Gly

165 170 175

Ile Ile Thr Glu Lys Ser Met Asn Ser Leu Leu His Ile Leu Glu Ala

180 185 190

Tyr Thr Asn Leu Tyr Thr Thr Trp Pro His Glu Asn Leu Lys Lys Asn

195 200 205

Ile Glu Asn Leu Val Lys Ile Phe Lys Asp Lys Ile Phe Asn Pro Glu

210 215 220

Thr Lys His Leu Gly Val Phe Phe Asp Arg Lys Leu Asn Pro Ile Ile

225 230 235 240

Asp Ala Ile Ser Tyr Gly His Asp Ile Glu Ala Thr Trp Ile Leu Asp

245 250 255

Glu Ser Leu Lys Tyr Ile Asn Asp Ala Asn Leu Lys Glu Glu Val Asn

260 265 270

Arg Ile Thr Leu Glu Ile Ala Asp Gln Val Leu Glu Glu Ala Phe Glu

275 280 285

Asn Gly Ser Leu Ile Asn Glu Lys Val Arg Asn Ile Leu Asp Lys Ser

290 295 300

Arg Ile Trp Trp Val Glu Ala Glu Ala Leu Val Gly Phe Leu Asn Ala

305 310 315 320

Tyr Gln Lys Ser Arg Glu Glu Lys Phe Leu Asn Ala Val Ile Glu Leu

325 330 335

Trp Lys Phe Ile Lys Asn Tyr Met Val Asp Gln Arg Pro Asp Ser Glu

340 345 350

Trp Phe Trp Lys Leu Asp Glu Asn Tyr Ile Pro Ala Pro Met Pro Ile

355 360 365

Val Glu Pro Tyr Lys Cys Pro Tyr His Asn Gly Arg Met Cys Ile Glu

370 375 380

Ala Ile Lys Arg Ile Asn Glu Asp

385 390

<210> 20

<211> 1179

<212> DNA

<213> Artificial sequence

<400> 20

atggatttaa aaacattaaa aaatgaggta aagaatcacc ttacagaaaa aattatacct 60

ttttgggcaa aactcatgga taaagaaaat ggtggatata ttggatatgt atcttttgat 120

ttaaaaaaag atccttatgc tcacaaaagc ttagttttaa ctacaaaaat attatggttt 180

ttttctgctg tatataatct tacaaaggaa gaaaacctta ttccttatat gaatcatgca 240

tacagcttct tagtacaaaa actttgggac cataaaaaca aaggttttta ttggatggta 300

gactacaaag gagagcctat tgataaaaga aaacatatat atgggcatgc tttcagtatc 360

tatgccttat cagaatttta taaagccaca aaaaaagatg aggcacttaa tattgcactg 420

gaaacttata atcttttaga ggaaaagtgt aaagatgaat atgcttactt agaagagttt 480

gatgaatact ggaatccaaa agaaaataaa gctattagtg aatatggaat aataactgaa 540

aaaagtatga attctctact tcatatattg gaagcttata ctaatcttta tacaacatgg 600

cctcatgaga accttaagaa aaatatagaa aatctcgtaa aaatctttaa agacaagatt 660

tttaatccag aaacaaaaca tttaggtgta tttttcgata gaaaattgaa tcctataatt 720

gatgccattt catacggaca tgatattgag gcaacttgga tattagatga atccttaaaa 780

tatattaatg atgctaatct taaagaagaa gttaatagaa taaccttaga gattgctgat 840

caagtattag aagaagcctt tgaaaatggt agtttaataa atgaaaaagt aagaaatata 900

ttagataaaa gtagaatttg gtgggttgaa gcagaagctt tagttggctt tttaaatgca 960

tatcaaaagt caagagaaga aaaatttcta aatgcagtga tagaactttg gaaatttatt 1020

aaaaattaca tggtagatca aagaccagat agtgaatggt tctggaaact cgatgaaaat 1080

tatattccag ctcctatgcc catagtagaa ccctacaaat gcccttatca taatggaaga 1140

atgtgcatag aagcaataaa aagaataaat gaagactaa 1179

<210> 21

<211> 35

<212> DNA

<213> Artificial sequence

<400> 21

agttttaact acaaaaatat tatggttttt ttctg 35

<210> 22

<211> 34

<212> DNA

<213> Artificial sequence

<400> 22

aaaaccataa tatttttgta gttaaaacta agct 34

<210> 23

<211> 35

<212> DNA

<213> Artificial sequence

<400> 23

gtatgaattc tctaattcat atattggaag cttat 35

<210> 24

<211> 35

<212> DNA

<213> Artificial sequence

<400> 24

ttccaatata tgaattagag aattcatact ttttt 35

<210> 25

<211> 35

<212> DNA

<213> Artificial sequence

<400> 25

ttgaggcaac ttggatatta gatgaatcct taaaa 35

<210> 26

<211> 35

<212> DNA

<213> Artificial sequence

<400> 26

ggattcatct aatatccaag ttgcctcaat atcat 35

<210> 27

<211> 35

<212> DNA

<213> Artificial sequence

<400> 27

gaggcaactt ggatgttaga tgaatcctta aaata 35

<210> 28

<211> 37

<212> DNA

<213> Artificial sequence

<400> 28

ggattcatct aacatccaag ttgcctcaat atcatgt 37

<210> 29

<211> 35

<212> DNA

<213> Artificial sequence

<400> 29

atagtagaac cctacaaatg cccttatcat aatgg 35

<210> 30

<211> 35

<212> DNA

<213> Artificial sequence

<400> 30

gataagggca tttgtagggt tctactatgg gcata 35

<210> 31

<211> 35

<212> DNA

<213> Artificial sequence

<400> 31

agaaccctgg aaaacccctt atcataatgg aagaa 35

<210> 32

<211> 37

<212> DNA

<213> Artificial sequence

<400> 32

ccattatgat aaggggtttt ccagggttct actatgg 37

Claims (10)

1. A cellobiose epimerase mutant, which is characterized in that the cellobiose epimerase mutant is mutated at an amino acid sequence shown in SEQ ID NO. 1, and the mutation sites are selected from one or more of R56K, L187I, L254I, L254M, W372Y and C374T.

2. The cellobiose epimerase mutant according to claim 1, wherein the mutation site is R56K, L254I, C374T, R56K/W372Y, R56K/L254M, L254I/C374T, R56K/L187I/L254I, R56K/L254I/C374T or R56K/L254I/W372Y.

3. The cellobiose epimerase mutant according to claim 1 or 2,

the amino acid sequence of the single-point mutant corresponding to R56K is SEQ ID NO. 3;

the amino acid sequence of the single-point mutant corresponding to the L254I is SEQ ID NO. 4;

the amino acid sequence of the single-point mutant corresponding to C374T is SEQ ID NO. 5;

the amino acid sequence of the combined mutant corresponding to R56K/W372Y is SEQ ID NO 6;

the amino acid sequence of the combined mutant corresponding to R56K/L254M is SEQ ID NO. 7;

the amino acid sequence of the combined mutant corresponding to L254I/C374T is SEQ ID NO 8;

the amino acid sequence of the combined mutant corresponding to R56K/L187I/L254I is SEQ ID NO 9;

the amino acid sequence of the combined mutant corresponding to R56K/L254I/C374T is SEQ ID NO 10;

the amino acid sequence of the combined mutant corresponding to R56K/L254I/W372Y is SEQ ID NO. 11.

4. A gene encoding the cellobiose epimerase mutant of any one of claims 1 to 3.

5. A recombinant expression vector comprising the gene of claim 4.

6. A soluble protein, immobilized enzyme or engineered bacterium comprising the cellobiose epimerase mutant of any one of claims 1 to 3.

7. Use of the cellobiose epimerase mutant of any one of claims 1 to 3 for the preparation of lactulose.

8. A process for producing lactulose, characterized in that lactose is used as a substrate and a cellobiose epimerase mutant as defined in any one of claims 1 to 3 is used as a catalyst.

9. The method as claimed in claim 8, wherein the lactose concentration is 500-700g/L, the enzyme dosage is 10-30U/mL, the reaction temperature is 75-80 ℃, the pH is 7.0-7.5, and the reaction time is 1-4 h.

10. Use of the cellobiose epimerase mutant of any one of claims 1 to 3 in the fields of food, feed or medicine.

Images