Beta-N-acetyl hexosaminidase and application thereof in synthesis of lactose-N-trisaccharide II
By modifying the β-N-acetaminohexosidase Fmhex, mutants Fmhex(W291A) and Fmhex(I436S) were obtained, which improved the conversion rate of lactose-N-trisaccharide II, solved the problem of low conversion rate in the existing technology, and achieved more efficient LNT II synthesis.
Patent Information
- Application Number
- CN202511113498.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-11-07
AI Technical Summary
In existing technologies, β-N-acetaminohexosidase has a low conversion rate in the preparation of lactose-N-trisaccharide II (LNT II), which is difficult to meet the needs of industrial applications.
By discovering and modifying β-N-acetaminohexosidase Fmhex, mutants Fmhex(W291A) and Fmhex(I436S) were obtained, and their transglycosylation activity was improved. LNT II was synthesized under the action of the enzyme using chitobiose and lactose as substrates.
The conversion rates of mutants Fmhex(W291A) and Fmhex(I436S) were increased by 1.68 times and 1.88 times, respectively, which significantly improved the synthesis efficiency of LNT II and showed greater potential for industrial production.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to β - N Acetylhexosaminidase and its application in lacto- N - triose II synthesis, belongs to the technical field of functional enzymes. BACKGROUND
[0002] Human milk oligosaccharides (HMOs) are a class of complex and diverse oligosaccharides in human milk, which have multiple physiological activities such as promoting brain development and regulating intestinal flora, and are high-value substances that cannot be replaced by other mammalian milk. At present, more than 200 HMOs have been found in human milk, and the structures of more than 100 HMOs have been identified. Among them, lacto- N - triose II (LNT II) is an important core skeleton of HMOs, which has multiple physiological activities such as immunity, antibacterial, and prebiotic. Expanding the preparation route of LNT II has a significant promoting effect on the industrialization application of LNT II and social and economic development.
[0003] LNT II (GlcNAcβ1-3Galβ1-4Glc) is a non-reducing trisaccharide composed of β - N - acetylglucosamine (GlcNAc) and lactose, and its molecular formula is C 20 H 35 NO 16 . As a key component of LNT II, GlcNAc can usually be obtained by hydrolyzing chitin, and chitin is very rich in source and is the second most abundant natural polysaccharide in nature. Using chitinase to hydrolyze chitin to prepare (GlcNAc)2, and then using β - N - acetylhexosaminidase (EC 3.2.1.52) to hydrolyze (GlcNAc)2 can obtain GlcNAc. In recent years, it has been found that GH20 family β - N - acetylhexosaminidase not only has hydrolysis activity, but also has high transglycosylation activity, which can be used for the synthesis of LNT II. Specifically, it can use (GlcNAc)2 as a donor and lactose as an acceptor, and transfer the GlcNAc residue to the non-reducing end of lactose to generate LNT II through transglycosylation activity. SUMMARY
[0004] In view of the above prior art, the present application provides an β - NAcetylhexosaminidase Fmhex, and two mutants: Fmhex(W291A) and Fmhex(I436S), and their application in the synthesis of lacto- N - triose II.
[0005] The present application is realized by the following technical solutions: An β - N Acetylhexosaminidase Fmhex, the amino acid sequence of which is shown in SEQ ID NO. 1.
[0006] The β - N The coding gene of acetylhexosaminidase Fmhex, the nucleotide sequence of which is shown in SEQ ID NO. 2.
[0007] A mutant of Fmhex, β - N Acetylhexosaminidase Fmhex(W291A), the amino acid sequence of which is shown in SEQ ID NO. 3.
[0008] The β - N The coding gene of acetylhexosaminidase Fmhex(W291A), the nucleotide sequence of which is shown in SEQ ID NO. 4.
[0009] A mutant of Fmhex, β - N Acetylhexosaminidase Fmhex(I436S), the amino acid sequence of which is shown in SEQ ID NO. 5.
[0010] The β - N The coding gene of acetylhexosaminidase Fmhex(I436S), the nucleotide sequence of which is shown in SEQ ID NO. 6.
[0011] The β - N Acetylhexosaminidase Fmhex, β - N Acetylhexosaminidase Fmhex(W291A), β - N Acetylhexosaminidase Fmhex(I436S) in the synthesis of lacto- N - triose II. In specific application, chitobiose and lactose are used as substrates, and lacto- β - N - triose II is prepared under the action of acetylhexosaminidase. N - triose II.
[0012] Further, in specific applications, lactose and β - N - acetylhexosaminidase, the initial concentration of chitobiose is 25 mM, the initial concentration of lactose is 250 mM, β - N - the concentration of acetylhexosaminidase is 2 mM; water bath reaction at 40 DEG C for 2.5 h, to prepare lactose- N - triose II.
[0013] The present application has been obtained by β - N - acetylhexosaminidase Fmhex, research has found that it can prepare GlcNAc with p NP-GlcNAc, (GlcNAc) 2-6 and (GlcNAc)2 as substrate, and can also transfer glycosyl to prepare lactose- N - triose II with (GlcNAc)2 as donor, but the conversion rate is low. Then, the present application mutates and reforms β - N - acetylhexosaminidase Fmhex, and obtains two mutants with higher glycosyl transferase activity: β - N - acetylhexosaminidase Fmhex (W291A), β - N - acetylhexosaminidase Fmhex (I436S), the conversion rate of preparing lactose- N - triose II is β - N 1.68 times, 1.88 times of acetylhexosaminidase Fmhex, which has higher application potential. The research of the present application has important industrial production value and economic value.
[0014] The various terms and phrases used in the present application have the general meanings known to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 : SDS-PAGE electrophoresis detection results, wherein M1 and M2 are standard proteins; Fmhex represents β - N - acetylhexosaminidase Fmhex; W291A represents β - N - acetylhexosaminidase Fmhex (W291A); I436S represents β - N - acetylhexosaminidase Fmhex (I436S).
[0016] Figure 2 : β - N A schematic diagram of the active pocket and amino acid composition of acetaminohexosidase Fmhex.
[0017] Figure 3 : Schematic diagram of multiple sequence alignment.
[0018] Figure 4 : β - N A schematic diagram of the molecular docking of acetylaminohexosidase Fmhex with (GlcNAc)2.
[0019] Figure 5 : β - N - A schematic diagram of the molecular docking of acetylaminohexosidase Fmhex with lactose. Detailed Implementation
[0020] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.
[0021] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.
[0022] The invention adopts p NP-GlcNAc, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0023] The invention adopts α - Chitosan, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0024] The chitosan oligosaccharides used in this invention, namely chitobiose (GlcNAc)2, chitotriose (GlcNAc)3, chitotetraose (GlcNAc)4, chitopentose (GlcNAc)5, and chitohexaose (GlcNAc)6, were all purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd.
[0025] The lactose used in this invention was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.
[0026] Example 1 β - N -Discovery of acetaminohexosidase Fmhex In order to explore products with high hydrolytic activity β - N - Acetaminohexosidase, the inventors selected an enzyme from *Flavobacterium karyotes* from the NCBI website (…). Fulvivirga maritima The protein fragment (GenBank: WP_233776848.1) was predicted to have... β - N - Acetaminohexosidase activity, belonging to the GH20 family, its amino acid sequence after removing the signal peptide is shown in SEQ ID NO.1, and is named by this invention. β - N - Acetaminohexosidase Fmhex. The coding gene was codon optimized, and the optimized nucleotide sequence is shown in SEQ ID NO.2. To study its enzymatic properties (hydrolytic activity, transglycosylation activity, etc.), this invention performed heterologous expression, extraction, purification, and related experimental studies, as detailed in the following examples.
[0027] β - N The amino acid sequence of acetaminohexosidase Fmhex is shown in SEQ ID NO.1, as follows: MSCSTS VPKEEEYNSPISIIPEPVSLEQGKGALIINENTKLLFAKDDEEAQKIAESFADQFSAASGISLAVEESAEGKVEGGILFSVNENLDVPEEGYELKVDDAGVSIVGKDHAGLFYGMQTLKQLLPPAIESTEKVEGEKWLVPLVSIQDYPRYKWRGLHLDVSRHFSTVDFVKKYIDNMAMHKLNTFHWHLTDDQGWRIEIKKYPKLTEIGAYRDETLVGHAGSTEFDGKRYGGFYTQEEIKEVVAYAQERHITVVPEIELPGHATAAVASYPELGVTGNRPKVVTEWGVFLDIYGVQDETFEFLQDVLTEVMELFPSKYIHIGGDEAWKDQWKASAEVQEKIKELGLKDEHELQSWFITRIEQFVNSKGRQIIGWDEILEGGLAPNAAVMSWRGEEGGIAAAKEKHNVVMTPAGYVYFDHYQGDPQFEPLQISGYTTLERVYSYDPTPEVLSPEEQKYILGAQANVWSEYLPTSEMVEYVVFPRLAALSEVLWTPLENKDWEAFKEKIPNQLKRYDYRGINYSKSIYYVTYDVEDKAGTEKLVVSLKNQFGLSKMYYTTDGSEPTKESSLYEGPLYLDEGTTLKAVAIQDGEPMSKVTEITVEKPKEEEK.
[0028] β - N The nucleotide sequence of the gene encoding acetylhexosaminidase Fmhex is shown as SEQ ID NO. 2, as follows (direction 5'-3'):
[0029] Example 2 β - N Preparation of acetylhexosaminidase Fmhex The steps are as follows: (1) Construction of recombinant expression vector: the gene fragment shown in SEQ ID NO. 2 was artificially synthesized and connected to the pET-28a(+) expression vector to construct a recombinant plasmid.
[0030] (2) Construction of recombinant engineering bacteria: the recombinant plasmid constructed above was transformed into E. coli BL21(DE3) competent cells, and positive transformant selection was performed using an LB plate containing kanamycin sulfate. The clones were verified by colony PCR using T7 universal primers.
[0031] (3) Culture of recombinant engineering bacteria: positive clones were inoculated in 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C, 220 rpm for 12 h. The culture was inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate at a 1% inoculation amount, and cultured at 37°C, 220 rpm until the OD value was 0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added at a concentration of 0.1 mM, and expression was induced at 18°C for 16 h.
[0032] (3) Extraction: the culture solution was centrifuged at 8000 rpm for 5 min at 4°C, the bacterial cells were collected, resuspended in Tirs-HCl buffer (10 mM, pH 7.5), and sonicated for 30 min. The supernatant was centrifuged at 12000 rpm for 30 min, and the supernatant was the crude enzyme solution.
[0033] (4) Purification: the crude enzyme solution was purified by affinity chromatography using a Ni-NTA nickel column. The column was equilibrated with 10 mM imidazole solution (10 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl), then the weakly bound impurities were eluted with 20 mM imidazole solution (20 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl), and the target protein was eluted with 200 mM imidazole solution (200 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl), and the eluate was collected; the eluate was concentrated using an ultrafiltration tube with a molecular weight cutoff of 30 kDa to obtain a concentrated protein solution containing β - N Purified enzyme solution of acetylhexosaminidase Fmhex. SDS-PAGE detection was performed, and the results are shown in Figure 1 The purified protein showed a single band with a molecular weight of about 75 kDa, which was consistent with the prediction.
[0034] Example 3 β - N - Enzymatic activity determination of acetylhexosaminidase Fmhex Using p NP chromogenic method to determine the enzyme activity, the reaction system consists of: 90 μL of 1 mM p-nitrophenyl-β-acetylglucosamine (p-NP-GlcNAc) solution, 10 μL of pure enzyme solution (prepared in Example 2), 100 μL of 50 mM phosphate buffer. Reaction at 40°C for 10 min. After the reaction, 200 μL of 200 mM sodium carbonate solution was added to terminate the reaction, and the absorbance value at 410 nm was detected. Detection three times, take the average value. β - N - Enzymatic activity determination of acetylglucosaminidase (p-NP-GlcNAc) p NP-GlcNAc) solution, 10 μL of pure enzyme solution (prepared in Example 2), 100 μL of 50 mM phosphate buffer. Reaction at 40°C for 10 min. After the reaction, 200 μL of 200 mM sodium carbonate solution was added to terminate the reaction, and the absorbance value at 410 nm was detected. Detection three times, take the average value.
[0035] One unit (U) of β - N - Acetylhexosaminidase activity is defined as: the amount of enzyme required to release 1 μmol of NP per minute. p NP per minute.
[0036] Determined, β - N - The activity of acetylhexosaminidase Fmhex is 1291 ± 43.69 U / mg.
[0037] Example 4 β - N - Determination of the substrate hydrolysis specificity of acetylhexosaminidase Fmhex The enzyme activity was detected under standard enzyme activity determination conditions using chitin and chitooligosaccharide as substrates, respectively, to determine the substrate specificity of the enzyme. The results are shown in Table 1.
[0038]
[0039] Note: The released reducing sugar in chitin was determined by DNS method. The enzyme activity determination method for chitooligosaccharide is: take 3 μM enzyme solution, add 2 mg / mL chitooligosaccharide, and 40 μL of pH 6.0 phosphate buffer, react at 40°C for 10 min. Then determine the amount of acetylglucosamine released by high performance liquid chromatography (HPLC). HPLC determination conditions: HPLC-RID detection system, the temperature of the chromatographic column (Sugar Pak I) is 75°C, the mobile phase is calcium disodium ethylenediaminetetraacetate (50 mg / L) solution, the flow rate is 0.5 mL / min, and the time is 20 min. β - N - Enzymatic activity determination of acetylglucosaminidase (p-NP-GlcNAc) β - N- The activity unit of acetylhexosaminidase is defined as: 1 μmol of (GlcNAc)2 generated per minute under the above reaction conditions. β - N - The amount of enzyme required for acetylglucosamine is one enzyme activity unit (1 U).
[0040] Results: β - N - Acetylhexosaminidase Fmhex has no activity on chitin. It has an activity of close to 30 U / mg on (GlcNAc)4 and (GlcNAc)6. The activity on (GlcNAc)5 is about 20 U / mg, and the activity on (GlcNAc)2 is 15.68±1.28 U / mg. It can be seen that, β - N - Acetylhexosaminidase Fmhex has activity in breaking the (GlcNAc)2 glycosidic bond, but the activity is low and needs to be improved.
[0041] The chitin is prepared from chitin by acid treatment α - The chitin is prepared from chitin by acid treatment α - 10 mg of chitin powder is added to 100 ml of concentrated hydrochloric acid, quickly stirred to be uniform, and stored at 4℃ for 24 h; then 300 mL of 50% ethanol solution (volume percentage) is added, quickly stirred, and the precipitated chitin is washed with distilled water repeatedly until neutral, and then freeze-dried to obtain chitin powder.
[0042] Example 5 β - N - Activity determination of acetylhexosaminidase Fmhex in transglycosylation synthesis of LNT II (1) The (GlcNAc)2 required for the preparation of LNT II is of high concentration, and the cost of directly purchasing (GlcNAc)2 is too high, so the present application uses self-made (GlcNAc)2, and the preparation method is: chitin is hydrolyzed by chitinase.
[0043] Specifically, 20 mg / mL of chitin solution was added with chitinase ChiB (chitinase ChiB is a chitinase reported in the prior art, which is expressed in E. coli by a conventional method and extracted to obtain a crude enzyme solution. Chitinase ChiB is described in the following literature: Brurberg et al. Comparative Studies of Chitinases A and B from Serratia marcescens. Microbiology 1996, 142, 1581-1589), and the amount of enzyme added was 3 μM of chitinase ChiB per milliliter of substrate solution, and the reaction was carried out at 45°C for 12 h; boiling, filtering, freeze-drying concentration, and water re-dissolution into a high-concentration (GlcNAc) 2 solution. The concentration of (GlcNAc) 2 was measured by HPLC method. The HPLC determination conditions are the same as those in Example 4.
[0044] (2) using β - N - acetylhexosaminidase Fmhex to transfer the GlcNAc residue in (GlcNAc) 2 to the non-reducing end of lactose to prepare LNT II.
[0045] Specifically, lactose and β - N - acetylhexosaminidase Fmhex were added to the (GlcNAc) 2 solution. After addition, the initial concentration of (GlcNAc) 2 was 25 mM, the initial concentration of lactose was 250 mM, β - N - acetylhexosaminidase Fmhex was 2 μM. The reaction was carried out in a water bath at 40°C for 2.5 h, and the yield of LNT II was determined by HPLC method.
[0046] The HPLC method is: HPLC-RID detection system, the temperature of the chromatographic column (XBridge BEH Amide 5 μm) is 55°C, the mobile phase is 72% acetonitrile solution, the flow rate is 0.4 mL / min, and the time is 55 min.
[0047] Conversion rate = LNT II concentration (mM) / initial (GlcNAc) 2 concentration (mM) x 100%.
[0048] Results: using β - N - acetylhexosaminidase Fmhex to prepare LNT II has a conversion rate of 9.23%, which is relatively low and has a large room for improvement.
[0049] Example 6 β - NModification and initial activity screening of acetaminohexosidase Fmhex. The above experimental results show that β - N - Acetaminohexosidase Fmhex possesses the ability to synthesize LNT II via transglycosylation, but the conversion rate is only 9.23%, which is relatively low. Therefore, this invention aims to mutate it to obtain a transglycosylation enzyme with higher activity. β - N - Acetaminohexosidase.
[0050] First, build using the SWISS MODEL protein modeling server. β - N Structural model of acetylaminohexosidase Fmhex, predicted using PrankWeb 3 server. β - N The active pocket and corresponding amino acids of acetaminohexosidase Fmhex were determined, and the results are as follows: Figure 2 As shown.
[0051] Then, β - N Multiple sequence alignment (MSA) was performed between acetaminohexosidase Fmhex and the GH20 family to predict amino acid conservation. Results are as follows: Figure 3 As shown, only W291, G398, G417, H424, and I436 of the active pocket amino acids were found to be non-conserved, while the others were semi-conserved or highly conserved.
[0052] Then, β - N -Acetaminohexosidase Fmhex was molecularly docked with (GlcNAc)2 and lactose, respectively. The results of docking with (GlcNAc)2 are as follows: Figure 4 As shown, the results of lactose docking are as follows: Figure 5 As shown. By Figure 4 As can be seen, E330, H424, and W471 form hydrogen bonds with (GlcNAc)2, while W291, W396, and I436 form hydrophobic interactions with (GlcNAc)2. As shown in the figure, G398, G417, and H424 form hydrogen bonds with lactose, while I436 forms a hydrophobic interaction with lactose.
[0053] Since transglycosylation activity is based on the breaking of the (GlcNAc)2 glycosidic bond, this invention ultimately selects the non-conservative sites W291, H424, and I436 for site-directed saturation mutagenesis. Specifically, the method involves: β - N The amino acid codons at the three sites of -acetaminohexosidase Fmhex were changed to the codons of 19 other commonly used amino acids.
[0054] For each mutation site, 19 mutant plasmids were constructed by the conventional method, and the 19 mutant plasmids were expressed heterologously by the method of Example 2 to obtain 19 kinds of mutant crude enzyme solutions. The transglycosylation activity of each mutant crude enzyme solution was determined according to the method shown in Example 5 (with the activity of the acetylhexosaminidase Fmhex as the reference). β - N - acetylhexosaminidase Fmhex as the reference).
[0055] Results: Among the 3x19 mutants, the transglycosylation activity increased, decreased, or remained unchanged, and the increase or decrease in the magnitude and significance was different, and there was no obvious rule. Among them, the transglycosylation activity of mutant W291A and mutant I436S increased the most, which had great application potential. The present application further studied these two mutants. The pure enzyme solution of these two mutants was purified based on the crude enzyme solution (the method was the same as that of Example 2). The pure enzyme solution of these two mutants was detected by SDS-PAGE, and the SDS-PAGE electrophoresis detection result is shown in Figure 1 . The purified protein showed a single band with a molecular weight of about 75 kDa.
[0056] Example 7 Activity determination of mutant Fmhex (W291A) and mutant Fmhex (I436S) in synthesizing LNT II The pure enzyme solution of these two mutants was subjected to activity determination in synthesizing LNT II (the method was the same as that of Example 5), and the results were as follows: the conversion rate of mutant Fmhex (W291A) in synthesizing LNT II from (GlcNAc)2 was 15.53%, and the conversion rate of mutant Fmhex (I436S) in synthesizing LNT II from (GlcNAc)2 was 17.32%, which were 1.68 times and 1.88 times of that of acetylhexosaminidase Fmhex, respectively, and the conversion rate was greatly improved, indicating that mutant Fmhex (W291A) and mutant Fmhex (I436S) had higher application potential. β - N - acetylhexosaminidase Fmhex as the reference).
[0057] The mutant Fmhex (W291A) is named as β - N - acetylhexosaminidase Fmhex (W291A), the amino acid sequence of which is shown in SEQ ID NO. 3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 4. The mutant Fmhex (I436S) is named as β - N - acetylhexosaminidase Fmhex (I436S), the amino acid sequence of which is shown in SEQ ID NO. 5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 6.
[0058] β - N The amino acid sequence of acetylhexosaminidase Fmhex (W291A) is shown in SEQ ID NO. 3, as follows: MSCSTSVPKEEEYNSPISIIPEPVSLEQGKGALIINENTKLLFAKDDEEAQKIAESFADQFSAASGISLAVEESAEGKVEGGILFSVNENLDVPEEGYELKVDDAGVSIVGKDHAGLFYGMQTLKQLLPPAIESTEKVEGEKWLVPLVSIQDYPRYKWRGLHLDVSRHFSTVDFVKKYIDNMAMHKLNTFHWHLTDDQGWRIEIKKYPKLTEIGAYRDETLVGHAGSTEFDGKRYGGFYTQEEIKEVVAYAQERHITVVPEIELPGHATAAVASYPELGVTGNRPKVVTEAGVFLDIYGVQDETFEFLQDVLTEVMELFPSKYIHIGGDEAWKDQWKASAEVQEKIKELGLKDEHELQSWFITRIEQFVNSKGRQIIGWDEILEGGLAPNAAVMSWRGEEGGIAAAKEKHNVVMTPAGYVYFDHYQGDPQFEPLQISGYTTLERVYSYDPTPEVLSPEEQKYILGAQANVWSEYLPTSEMVEYVVFPRLAALSEVLWTPLENKDWEAFKEKIPNQLKRYDYRGINYSKSIYYVTYDVEDKAGTEKLVVSLKNQFGLSKMYYTTDGSEPTKESSLYEGPLYLDEGTTLKAVAIQDGEPMSKVTEITVEKPKEEEK.
[0059] β - N The nucleotide sequence of the coding gene for acetylhexosaminidase Fmhex (W291A) is shown in SEQ ID NO. 4, as follows (direction 5'-3'):
[0060] β - N The amino acid sequence of acetylhexosaminidase Fmhex (I436S) is shown in SEQ ID NO. 5, as follows: MSCSTSVPKEEEYNSPISIIPEPVSLEQGKGALIINENTKLLFAKDDEEAQKIAESFADQFSAASGISLAVEESAEGKVEGGILFSVNENLDVPEEGYELKVDDAGVSIVGKDHAGLFYGMQTLKQLLPPAIESTEKVEGEKWLVPLVSIQDYPRYKWRGLHLDVSRHFSTVDFVKKYIDNMAMHKLNTFHWHLTDDQGWRIEIKKYPKLTEIGAYRDETLVGHAGSTEFDGKRYGGFYTQEEIKEVVAYAQERHITVVPEIELPGHATAAVASYPELGVTGNRPKVVTEWGVFLDIYGVQDETFEFLQDVLTEVMELFPSKYIHIGGDEAWKDQWKASAEVQEKIKELGLKDEHELQSWFITRIEQFVNSKGRQIIGWDEILEGGLAPNAAVMSWRGEEGGIAAAKEKHNVVMTPAGYVYFDHYQGDPQFEPLQSSGYTTLERVYSYDPTPEVLSPEEQKYILGAQANVWSEYLPTSEMVEYVVFPRLAALSEVLWTPLENKDWEAFKEKIPNQLKRYDYRGINYSKSIYYVTYDVEDKAGTEKLVVSLKNQFGLSKMYYTTDGSEPTKESSLYEGPLYLDEGTTLKAVAIQDGEPMSKVTEITVEKPKEEEK.
[0061] β - N The nucleotide sequence of the encoding gene of acetylhexosaminidase Fmhex (I436S) is shown in SEQ ID NO. 6, as follows (direction 5'-3'):
[0062] The foregoing examples are provided to give the skilled person in the art complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications apparent to the skilled person are to be within the scope of the claims appended hereto.
Claims
1. A kind β - N - Acetaminohexosidase Fmhex(I436S), characterized by: The amino acid sequence is shown as SEQ ID NO.
5.
2. The use according to claim 1 β - N - the coding gene of acetylhexosaminidase Fmhex (I436S), characterized in that: The nucleotide sequence is shown as SEQ ID NO.
6.
3. The method of claim 1 β - N - Use of acetylhexosaminidase Fmhex (I436S) in the synthesis of lacto- N - triose II.
4. Use according to claim 3, characterized in that: with chitobiose and lactose as substrates, under the action of β - N - acetylhexosaminidase Fmhex (I436S) to produce lactose- N - trisaccharide II.
5. Use according to claim 4, characterized in that: In a specific application, lactose and β - N - Acetylhexosaminidase Fmhex (I436S), initial concentration of chitobiose 25 mM, initial concentration of lactose 250 mM, β - N - Acetylhexosaminidase Fmhex (I436S) concentration 2 μM; water bath reaction at 40°C for 2.5 h, to prepare lactose- N - trisaccharide II.
6. A kind β - N - Acetaminohexosidase Fmhex(W291A), characterized by: The amino acid sequence is shown as SEQ ID NO.
3.
7. The method of claim 6 β - N - the gene encoding acetylhexosaminidase Fmhex (W291A) characterized in that: The nucleotide sequence is shown as SEQ ID NO.
4.
8. The method of claim 6 β - N - Use of acetylhexosaminidase Fmhex (W291A) in the synthesis of lacto- N - triose II.
9. Use according to claim 8, characterized in that: with chitobiose and lactose as substrates, under the action of β - N - acetylhexosaminidase Fmhex (W291A) to produce lactose- N - trisaccharide II.
10. Use according to claim 9, characterized in that: In a specific application, lactose and β - N - Acetylhexosaminidase Fmhex (W291A) at a concentration of 2 μM; incubation at 40°C in a water bath for 2.5 h, to produce lactose- β - N - Acetylhexosaminidase Fmhex (W291A) at a concentration of 2 μM; incubation at 40°C in a water bath for 2.5 h, to produce lactose- N - Trisaccharide II.