Heat-resistant and acid-resistant enzyme for catalyzing fructose and inorganic ammonium to prepare glucosamine

Abstract

The invention discloses a heat-resistant and acid-resistant enzyme for catalyzing fructose and inorganic ammonium to prepare glucosamine, and belongs to the field of enzyme engineering and biological manufacturing. According to the amino sugar synthase, 6-phosphate glucosamine deaminase derived from thermoclostridium sterilis is modified, and the glucosamine synthase which can be used for converting fructose into glucosamine by using an amino donor is obtained. The glucosamine synthase can keep good activity under high-temperature and acidic conditions, has the advantages of cheap raw materials, low production cost, environmental friendliness, safety to human bodies and the like when being applied to preparation of glucosamine, and is suitable for production of glucosamine in various scenes.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme engineering and biomanufacturing, and relates to a heat-resistant and acid-resistant enzyme for catalyzing the preparation of glucosamine from fructose and inorganic ammonium, and specifically to an acid-resistant and heat-resistant enzyme mutant, and a method for preparing glucosamine using fructose and inorganic ammonium as raw materials through in vitro single enzyme catalysis. Background Art

[0002] Glucosamine (2-amino-2-deoxy-D-glucose, abbreviated as glucosamine) is a functional amino monosaccharide and a component of glycoproteins, proteoglycans and glycosaminoglycans. It is commonly found in higher plants, animals, bacteria and fungi. Glucosamine has multiple specific biological activities. It can stimulate and replenish synovial fluid in large quantities, making the joints flexible and free. It can also inhibit the inflammatory response of nonspecific factors in the joint cavity, greatly relieving joint pain. It has been identified as effective in the clinical treatment of cardiovascular diseases, neurological deficits, skin diseases and cancer. In addition, as a component of hyaluronic acid, glucosamine has good skin permeability and can be used to improve skin moisture, reduce facial pigmentation, and promote the proliferation of keratinocytes and fibroblasts.

[0003] Traditional methods for producing glucosamine involve acid hydrolysis or enzymatic hydrolysis of chitin and chitosan extracted from crab and shrimp shells. The large amount of acid used in the acid hydrolysis method creates significant environmental stress and places high demands on production equipment. Enzymatic hydrolysis requires the synergistic action of endochitinase, exochitinase, β-acetylglucosaminidase, and N-acetyl-D-glucosamine deacetylase, resulting in high enzyme costs and low solid-phase catalytic efficiency. Furthermore, glucosamine extracted from crab and shrimp shells may cause allergic reactions in some individuals. Currently, microbial fermentation is gaining increasing attention for producing glucosamine. However, because glucosamine inhibits bacterial growth, fermentation methods typically first produce acetyl glucosamine, which is non-toxic to microorganisms, followed by acidolysis to remove the acetyl group. Several studies have attempted to engineer platform strains capable of producing acetyl glucosamine using metabolic engineering approaches. However, the two-step fermentation followed by acidolysis inevitably results in low product yields, high capital investment, and environmental pollution.

[0004] The 2020 Chinese invention patent CN110714042A discloses an in vitro multi-enzyme system for preparing glucosamine using starch and inorganic ammonium as substrates through multi-enzyme cascade catalysis. The system does not contain shrimp and crab shell allergens, phosphate balance, and does not require coenzymes. It completes energy activation, material conversion and energy release processes through five enzymes. With 50g / L starch as a substrate, it can produce 23.7g / L glucosamine. It is safe, environmentally friendly and sustainable, but the conversion rate of the system does not reach the theoretical yield, mainly because the poor enzyme substrate specificity of the energy release step causes the intermediate products of the material conversion process to release energy prematurely and fail to complete efficient conversion. In addition, the 2021 Chinese invention patents CN115851647A and CN115896075A disclose systems that use single enzymes or whole cells to catalyze fructose and amino donors to prepare glucosamine at room temperature. However, in industrial production, using enzymes with high thermal stability to carry out reactions under high temperature conditions can reduce the risk of microbial contamination, lower enzyme preparation costs, and reduce the viscosity of the reaction solution, thereby improving mass transfer efficiency. Glucosamine easily undergoes self-reorganization and condensation to form compounds such as fructose, furfural, and pyrazine under alkaline conditions, but is more stable at acidic pH. Therefore, developing enzymes that can stably catalyze under high temperature and acidic conditions will be more conducive to the economical, green, and efficient production of glucosamine.

[0005] Therefore, it is urgent to develop a method for preparing glucosamine enzyme that is low-cost, low-pollution, simple and easy to operate and can ensure product stability. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] In existing research, there is still a lack of enzymes that can withstand acidic and high temperature conditions well and can be used to convert and produce glucosamine under high temperature and acidic conditions. There is also a lack of enzymes that can generate glucosamine simply and efficiently.

[0008] Solutions for solving problems

[0009] To address these current challenges, the present invention uses the wild-type glucosamine-6-phosphate deaminase (GlmD) with the amino acid sequence shown in SEQ ID NO: 2 as a starting protein. Through rational design, multiple rounds of site-directed mutagenesis were performed on it. The mutant has at least 90% identity to the wild-type GlmD amino acid sequence SEQ ID NO: 2, exhibits activity in catalyzing the conversion of fructose and inorganic ammonium to glucosamine, and is acid- and heat-resistant. The mutant is named glucosamine synthase (GNS). Furthermore, the present invention provides a method for preparing glucosamine using an enzyme-catalyzed reaction.

[0010] The specific technical solutions of the present invention are as follows:

[0011] [1] A glycosaminoglycan synthase that catalyzes the production of glucosamine from fructose and inorganic ammonium, wherein the glycosaminoglycan synthase is selected from any one of the following groups (I) to (V):

[0012] (I) the aminoglycosylation enzyme comprises a mutation at at least one of positions 40, 202, 169, 170, 168, 38, 167, 35, and 36 corresponding to the sequence of SEQ ID NO: 2, compared to the sequence of SEQ ID NO: 2;

[0013] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2;

[0014] (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0015] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I);

[0016] (b) the full-length complementary polynucleotide of (a);

[0017] (IV) a fragment of the mutant represented by any one of (I), (II) or (III), wherein the fragment still has aminoglycoside synthase activity;

[0018] (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

[0019] [2] The aminoglycosylate according to [1], wherein the aminoglycosylate corresponds to the sequence shown in SEQ ID NO: 2 and has the following (d1) to (d2) 36 ) any of the mutations shown:

[0020] (d1) mutation at position 40;

[0021] (d2) combined mutations at positions 40 and 35;

[0022] (d3) combined mutations at positions 40 and 36;

[0023] (d4) combined mutations at positions 40 and 38;

[0024] (d5) combined mutations at positions 40 and 167;

[0025] (d6) combined mutations at positions 40 and 168;

[0026] (d7) combined mutations at positions 40 and 169;

[0027] (d8) combined mutations at positions 40 and 170;

[0028] (d9) combined mutations at positions 40 and 202;

[0029] (d 10 ) combined mutations at positions 40, 202, and 35;

[0030] (d 11 ) combined mutations at positions 40, 202, and 36;

[0031] (d 12 ) combined mutations at positions 40, 202, and 38;

[0032] (d 13 ) combined mutations at positions 40, 202, and 167;

[0033] (d 14 ) combined mutations at positions 40, 202, and 168;

[0034] (d 15 ) combined mutations at positions 40, 202, and 169;

[0035] (d 16 ) combined mutations at positions 40, 202, and 170;

[0036] (d 17 ) combined mutations at positions 40 and 202 and 169 and 35;

[0037] (d 18 ) combined mutations at positions 40 and 202 and 169 and 36;

[0038] (d 19 ) combined mutations at positions 40 and 202 and 169 and 38;

[0039] (d 20 ) combined mutations at positions 40 and 202 and at positions 169 and 167;

[0040] (d 21 ) combined mutations at positions 40 and 202 and at positions 169 and 168;

[0041] (d 22) combined mutations at positions 40 and 202 and at positions 169 and 170;

[0042] (d 23 ) combined mutations at positions 40 and 202 and 169 and 170 and 35;

[0043] (d 24 ) combined mutations at positions 40, 202, 169, 170, and 36;

[0044] (d 25 ) combined mutations at positions 40 and 202 and 169 and 170 and 38;

[0045] (d 26 ) combined mutations at positions 40 and 202 and 169 and 170 and 167;

[0046] (d 27 ) combined mutations at positions 40 and 202 and 169 and 170 and 168;

[0047] (d 28 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 35;

[0048] (d 29 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 36;

[0049] (d 30 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38;

[0050] (d 31 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 167;

[0051] (d 32 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 35;

[0052] (d 33 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 36;

[0053] (d 34 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167;

[0054] (d 35) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167 and 35;

[0055] (d 36 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167 and 36;

[0056] Optionally, the aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions:

[0057] P40F, K202Q, K202H, K202G, K202E, F169K, F169Y, F169H, F169E, K170F, K170 Y, K170Q, K170V, K170L, F168T, F168E, F168S, F168N, F168D, S38K, S38H, S38 V, S38N, S38L, S38I, R167K, R167E, R167F, R167D, R167H, A35I, A35T, A35D, A 35Q, A35N, A35V, A35L, A35E, T36S, T36E, T36D, T36V, T36I, T36Q, T36H, T36Y.

[0058] [3] The aminoglycosylate according to [1] or [2], wherein the aminoglycosylate corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m2) 57 ) is a mutation shown in any one of:

[0059] (m1)S38H;

[0060] (m2)K170Y;

[0061] (m3)K202G;

[0062] (m4)F168E;

[0063] (m5)A35T;

[0064] (m6)T36S;

[0065] (m7)R167E;

[0066] (m8)F169Y;

[0067] (m9)P40F;

[0068] (m 10 )P40F, F169H;

[0069] (m 11)P40F、T36S;

[0070] (m 12 )P40F、A35D;

[0071] (m 13 )P40F、K170Q;

[0072] (m 14 )P40F、K202H;

[0073] (m 15 )P40F、K202E;

[0074] (m 16 )P40F、R167F;

[0075] (m 17 )P40F、S38K;

[0076] (m 18 )P40F、F168S;

[0077] (m 19 )P40F、F169E;

[0078] (m 20 )P40F、K170V;

[0079] (m 21 )P40F、K202Q;

[0080] (m 22 )P40F、K202Q、S38V;

[0081] (m 23 )P40F、K202Q、A35Q;

[0082] (m 24 )P40F、K202Q、T36E;

[0083] (m 25 )P40F、K202Q、S38N;

[0084] (m 26 )P40F、K202Q、R167D;

[0085] (m 27 )P40F、K202Q、R170Q;

[0086] (m 28 )P40F、K202Q、F168T;

[0087] (m 29)P40F、K202Q、F169H;

[0088] (m 30 )P40F、K202Q、F169K;

[0089] (m 31 )P40F、K202Q、K170Q;

[0090] (m 32 )P40F、K202Q、S38L;

[0091] (m 33 )P40F、K202Q、F169K、R167E;

[0092] (m 34 )P40F、K202Q、F169K、T36D;

[0093] (m 35 )P40F、K202Q、F169K、A35N;

[0094] (m 36 )P40F、K202Q、F169K、K170L;

[0095] (m 37 )P40F、K202Q、F169K、K170F;

[0096] (m 38 )P40F、K202Q、F169K、K170F、R167K;

[0097] (m 39 )P40F、K202Q、F169K、K170F、F168D;

[0098] (m 40 )P40F、K202Q、F169K、K170F、S38I;

[0099] (m 41 )P40F、K202Q、F169K、K170F、T36V;

[0100] (m 42 )P40F、K202Q、F169K、K170F、A35Q;

[0101] (m 43 )P40F、K202Q、F169K、K170F、F168T;

[0102] (m 44)P40F、K202Q、F169K、K170F、F168T、R167H;

[0103] (m 45 )P40F、K202Q、F169K、K170F、F168T、T36I;

[0104] (m 46 )P40F、K202Q、F169K、K170F、F168T、S38V;

[0105] (m 47 )P40F、K202Q、F169K、K170F、F168T、A35E;

[0106] (m 48 )P40F、K202Q、F169K、K170F、F168T、S38K;

[0107] (m 49 )P40F、K202Q、F169K、K170F、F168T、S38K、R167H;

[0108] (m 50 )P40F、K202Q、F169K、K170F、F168T、S38K、A35V;

[0109] (m 51 )P40F、K202Q、F169K、K170F、F168T、S38K、T36E;

[0110] (m 52 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K;

[0111] (m 53 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35L;

[0112] (m 54 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、T36Q;

[0113] (m 55 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I;

[0114] (m 56)P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36H;

[0115] (m 57 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y; Preferably, the aminoglycoside synthase corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations:

[0116] (m1)S38H;

[0117] (m2)K170Y;

[0118] (m3)K202G;

[0119] (m4)F168E;

[0120] (m5)A35T;

[0121] (m6)T36S;

[0122] (m7)R167E;

[0123] (m8)F169Y;

[0124] (m9)P40F;

[0125] (m 10 )P40F, F169H;

[0126] (m 11 )P40F, T36S;

[0127] (m 14 )P40F, K202H;

[0128] (m 15 )P40F, K202E;

[0129] (m 16 )P40F, R167F;

[0130] (m 17 )P40F, S38K;

[0131] (m 20 )P40F, K170V;

[0132] (m 21 )P40F, K202Q;

[0133] (m 22 )P40F, K202Q, S38V;

[0134] (m 23 )P40F、K202Q、A35Q;

[0135] (m 24 )P40F、K202Q、T36E;

[0136] (m 26 )P40F、K202Q、R167D;

[0137] (m 27 )P40F、K202Q、R170Q;

[0138] (m 28 )P40F、K202Q、F168T;

[0139] (m 29 )P40F、K202Q、F169H;

[0140] (m 30 )P40F、K202Q、F169K;

[0141] (m 31 )P40F、K202Q、K170Q;

[0142] (m 33 )P40F、K202Q、F169K、R167E;

[0143] (m 34 )P40F、K202Q、F169K、T36D;

[0144] (m 35 )P40F、K202Q、F169K、A35N;

[0145] (m 36 )P40F、K202Q、F169K、K170L;

[0146] (m 37 )P40F、K202Q、F169K、K170F;

[0147] (m 39 )P40F、K202Q、F169K、K170F、F168D;

[0148] (m 40 )P40F、K202Q、F169K、K170F、S38I;

[0149] (m 41 )P40F、K202Q、F169K、K170F、T36V;

[0150] (m 42 )P40F、K202Q、F169K、K170F、A35Q;

[0151] (m 43 )P40F、K202Q、F169K、K170F、F168T;

[0152] (m 45 )P40F、K202Q、F169K、K170F、F168T、T36I;

[0153] (m 46 )P40F、K202Q、F169K、K170F、F168T、S38V;

[0154] (m 47 )P40F、K202Q、F169K、K170F、F168T、A35E;

[0155] (m 48 )P40F、K202Q、F169K、K170F、F168T、S38K;

[0156] (m 49 )P40F、K202Q、F169K、K170F、F168T、S38K、R167H;

[0157] (m 52 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K;

[0158] (m 53 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35L;

[0159] (m 55 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I;

[0160] (m 56 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I、T36H;

[0161] (m 57 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I、T36Y;

[0162] More preferably, the aminoglucose synthase corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations:

[0163] (m9)P40F;

[0164] (m 21 )P40F, K202Q;

[0165] (m 30 )P40F, K202Q, F169K;

[0166] (m 36 )P40F, K202Q, F169K, K170L;

[0167] (m 43 )P40F, K202Q, F169K, K170F, F168T;

[0168] (m 48 )P40F, K202Q, F169K, K170F, F168T, S38K;

[0169] (m 52 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K;

[0170] (m 55 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I;

[0171] (m 57 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y.

[0172] [4]. An isolated polynucleotide, wherein the polynucleotide encodes the aminoglucose synthase according to any one of [1] to [3].

[0173] [5]. A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide described in [4].

[0174] [6]. A recombinant host cell, wherein the recombinant host cell comprises the aminoglycosylation described in any one of [1] to [3], the isolated polynucleotide described in [4], or the recombinant expression vector described in [5].

[0175] [7]. A cell culture comprising the recombinant host cell as described in [6].

[0176] [8]. A product for preparing glucosamine or its derivatives, comprising the glucosamine synthase described in any one of [1] to [3], the polynucleotide described in [4], the recombinant expression vector described in [5], the recombinant host cell described in [6], or the cell culture described in [7].

[0177] [9]. A method for preparing glucosamine or its derivatives, wherein the method comprises providing the aminoglycosylation described in any one of [1-3], the polynucleotide described in [4], the recombinant expression vector described in [5], the recombinant host cell described in [6], the cell culture described in [7], or the product described in [8] to a reaction system to catalyze the conversion of the substrate into glucosamine or its derivatives;

[0178] Optionally, the substrate comprises fructose;

[0179] Optionally, the reaction system contains an amino donor; preferably, the amino donor is an inorganic ammonium salt;

[0180] Optionally, the concentration of the substrate is 1 to 2000 mM, preferably 2 to 500 mM;

[0181] Preferably, the pH of the reaction system is below 7, preferably 6.0±0.5;

[0182] Preferably, the reaction temperature of the reaction system is 40-80°C, preferably 50-70°C.

[0183]

[10] Use of the glucosamine synthase according to any one of [1] to [3], the polynucleotide according to [4], the recombinant expression vector according to [5], the recombinant host cell according to [6] or the cell culture according to [7] in the preparation of glucosamine or its derivatives;

[0184] Optionally, in the reaction system for preparing glucosamine or its derivatives, the substrate includes fructose; and / or,

[0185] In the reaction system for preparing glucosamine or its derivatives, the amino donor includes inorganic ammonium.

[0186] Effects of the Invention

[0187] Compared with the prior art, the present invention has the following positive effects:

[0188] 1. By constructing a series of heat-resistant and acid-resistant mutants, we screened out glucosamine synthase (GNS), which can catalyze the synthesis of glucosamine from fructose and inorganic ammonium in vitro under high temperature and acidic conditions. GNS can maintain good activity under high temperature and acidic conditions and is suitable for glucosamine production in various scenarios.

[0189] 2. The method provided by the present invention is a "one-pot" catalytic method. The reaction begins after the substrate and enzyme are added, without the need for any expensive cofactors. The final product, glucosamine, is obtained directly after the reaction. Compared with other existing production methods, the bioenzymatic preparation method of glucosamine provided by the present invention has the advantages of cheap raw materials, low production cost, environmental friendliness, and human safety, making it suitable for promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0190] Figure 1 Schematic diagram of the pathway for preparing glucosamine using a single enzyme-catalyzed reaction.

[0191] Figure 2 These are the results of enzyme screening for aminoglucose synthase activity.

[0192] Figure 3 The degradation of glucosamine under different pH conditions.

[0193] Figure 4 This is the time course curve of the production of glucosamine from fructose and inorganic ammonium catalyzed by glucosamine synthase. DETAILED DESCRIPTION

[0194] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0195] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0196] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0197] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0198] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0199] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0200] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0201] In this specification, "glucosamine synthase", "GNS", "glucosamine-6-phosphate deaminase" and "GlmD" are used interchangeably. GNS can replace the hydroxyl group at position 2 of fructose with the amino group in the ammonium ion to generate glucosamine.

[0202] In this specification, "glucosamine" and "glucosamine" are used interchangeably. The molecular formula is C6H 13 NO5, molecular weight 179.2, chemical structure formula:

[0203]

[0204] Throughout this specification, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0205] As used herein, the term "wild-type" refers to an object found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. As used herein, "naturally occurring" and "wild-type" are synonyms.

[0206] In this specification, the term "mutant" refers to a polynucleotide or polypeptide that contains an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to the removal of a nucleotide or amino acid occupying a position. An insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0207] As used herein, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion, or addition of one or more amino acids or nucleotides." In the present invention, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to a "deletion."

[0208] In some embodiments, the "mutation" of the present invention can be selected from "conservative mutations". In the present invention, the term "conservative mutation" refers to a mutation that can maintain the normal function of the protein. A representative example of a conservative mutation is a conservative substitution.

[0209] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0210] As used herein, the terms "sequence identity" or "percent identity" in the context of comparing two nucleic acids or polypeptides means that they are identical or have a specified percentage of identical sequences when compared and aligned for maximum correspondence using a nucleotide or amino acid residue sequence comparison algorithm or as measured by visual inspection. In other words, nucleotide or amino acid sequence identity can be defined as the ratio of the number of identical nucleotides or amino acids to the total number of nucleotides or amino acids in the aligned portions when two or more nucleotide or amino acid sequences are aligned to maximize the number of identical nucleotides or amino acids, adding gaps as needed.

[0211] As used herein, the term "recombinant polynucleotide" refers to a polynucleotide having sequences that are not linked together in nature. The recombinant polynucleotide can be contained in a suitable vector, and the vector can be used to transform into a suitable host cell. A host cell containing the recombinant polynucleotide is referred to as a "recombinant host cell." The polynucleotide is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide."

[0212] As used herein, the term "expression" includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0213] As used herein, the term "expression vector" refers to a DNA construct containing a DNA sequence operably linked to appropriate control sequences for expressing a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired exogenous polypeptide. A recombinant expression vector may include, for example, i) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) appropriate transcriptional and translational initiation and termination sequences. Recombinant expression vectors are constructed in any suitable manner. The nature of the vector is not critical, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from combinations of plasmids and phage DNA, and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. For example, the expression vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK or pQlink series, etc., which can be replicated and expressed in prokaryotic cells.

[0214] In this specification, the term "host cell" means any cell type that is easily transformed, transfected, transduced, etc. with a mutant polypeptide, a polynucleotide encoding a mutant polypeptide, or a recombinant expression vector comprising the present invention. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the polynucleotide or recombinant expression vector encoding the mutant polypeptide is introduced, and the recombinant host cell is specifically achieved by transformation. The host cell of the present invention can be a prokaryotic cell or a eukaryotic cell. In one embodiment, the host cell refers to a prokaryotic cell, and specifically, the host cell is derived from a microorganism of the genus Escherichia, the genus Bacillus, or the genus Corynebacterium. In some preferred embodiments, the host cell is derived from the genus Escherichia, more preferably Escherichia coli, including Escherichia coli DH5α, Escherichia coli Top10, Escherichia coli Trans T1, Escherichia coli MC1061, Escherichia coli BL21, etc.

[0215] In this specification, the term "cell culture" refers to a combination of cells and a cell culture medium, wherein the cells are cultured in the cell culture medium outside a living organism.

[0216] As used herein, the terms "transformation," "transfection," and "transduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0217] In this specification, "conversion" can also refer to the enzymatic conversion (or bioconversion) of substrate(s) to corresponding product(s). "Percent conversion rate" refers to the percentage of substrate converted to product under specified conditions within a period of time. Therefore, the "enzyme activity" or "activity" of an aminoglycosylation polypeptide can be expressed as the "percent conversion" of substrate to product within a specific period of time.

[0218] In this specification, "cultivation" refers to growing a microbial cell population under any suitable conditions (for example, using a liquid, gel or solid culture medium), including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

[0219] In this specification, the terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are removed from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0220] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0221] The technical solution of the present invention is described in detail below:

[0222] The inventors found in their previous studies that glucosamine is not very stable in solution and its degradation degree is mainly affected by pH. When 10g / L glucosamine was incubated at 37°C under different pH conditions, the glucosamine in the solution showed different degrees of degradation over time. When the pH was 8.0, about 50% of the glucosamine was degraded after 24h incubation (see Figure 3 ). In an acidic environment, glucosamine is relatively more stable. When the pH is 6.0, only about 5% of the glucosamine is degraded after 24 hours of incubation. When the temperature is raised to 50°C, only about 20% of the glucosamine remains after 24 hours of incubation at pH 8.0, while 90% of the glucosamine can still be detected without degradation after 24 hours of incubation at pH 6.0. This result shows that regardless of the temperature, glucosamine synthesis under relatively low pH conditions can effectively inhibit its degradation. In addition, the product glucosamine can be kept at a low pH after the reaction is completed, which can minimize the degradation of glucosamine caused by untimely product treatment.

[0223] <First Aspect>

[0224] Based on the above research, in a first aspect of the present invention, a series of acid-resistant and high-temperature-resistant glucosamine synthases are provided, which catalyze the production of glucosamine from fructose and inorganic ammonium, wherein the mutants are selected from any one of the following groups (I) to (V):

[0225] (I) the aminoglycosylation enzyme comprises a mutation at at least one of positions 40, 202, 169, 170, 168, 38, 167, 35, and 36 corresponding to the sequence of SEQ ID NO: 2, compared to the sequence of SEQ ID NO: 2;

[0226] (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2;

[0227] (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0228] (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I);

[0229] (b) the full-length complementary polynucleotide of (a);

[0230] (IV) a fragment of the mutant represented by any one of (I), (II) or (III), wherein the fragment still has aminoglycoside synthase activity;

[0231] (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

[0232] In some embodiments, the aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2, and has the following (d1) to (d 36 ) any of the mutations shown:

[0233] (d1) mutation at position 40;

[0234] (d2) combined mutations at positions 40 and 35;

[0235] (d3) combined mutations at positions 40 and 36;

[0236] (d4) combined mutations at positions 40 and 38;

[0237] (d5) combined mutations at positions 40 and 167;

[0238] (d6) combined mutations at positions 40 and 168;

[0239] (d7) combined mutations at positions 40 and 169;

[0240] (d8) combined mutations at positions 40 and 170;

[0241] (d9) combined mutations at positions 40 and 202;

[0242] (d 10 ) combined mutations at positions 40, 202, and 35;

[0243] (d 11 ) combined mutations at positions 40, 202, and 36;

[0244] (d 12 ) combined mutations at positions 40, 202, and 38;

[0245] (d 13 ) combined mutations at positions 40, 202, and 167;

[0246] (d 14 ) combined mutations at positions 40, 202, and 168;

[0247] (d 15 ) combined mutations at positions 40, 202, and 169;

[0248] (d 16 ) combined mutations at positions 40, 202, and 170;

[0249] (d 17 ) combined mutations at positions 40 and 202 and 169 and 35;

[0250] (d 18 ) combined mutations at positions 40 and 202 and 169 and 36;

[0251] (d 19 ) combined mutations at positions 40 and 202 and 169 and 38;

[0252] (d 20 ) combined mutations at positions 40 and 202 and at positions 169 and 167;

[0253] (d 21 ) combined mutations at positions 40 and 202 and at positions 169 and 168;

[0254] (d 22 ) combined mutations at positions 40 and 202 and at positions 169 and 170;

[0255] (d 23 ) combined mutations at positions 40 and 202 and 169 and 170 and 35;

[0256] (d 24 ) combined mutations at positions 40, 202, 169, 170, and 36;

[0257] (d 25 ) combined mutations at positions 40 and 202 and 169 and 170 and 38;

[0258] (d 26 ) combined mutations at positions 40 and 202 and 169 and 170 and 167;

[0259] (d 27 ) combined mutations at positions 40 and 202 and 169 and 170 and 168;

[0260] (d 28 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 35;

[0261] (d 29 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 36;

[0262] (d 30 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38;

[0263] (d 31 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 167;

[0264] (d 32 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 35;

[0265] (d 33 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 36;

[0266] (d 34 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167;

[0267] (d 35 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167 and 35;

[0268] (d 36 ) Combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167 and 36.

[0269] In some optional embodiments, the amino synthase corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions:

[0270] P40F, K202Q, K202H, K202G, K202E, F169K, F169Y, F169H, F169E, K170F, K170 Y, K170Q, K170V, K170L, F168T, F168E, F168S, F168N, F168D, S38K, S38H, S38 V, S38N, S38L, S38I, R167K, R167E, R167F, R167D, R167H, A35I, A35T, A35D, A 35Q, A35N, A35V, A35L, A35E, T36S, T36E, T36D, T36V, T36I, T36Q, T36H, T36Y.

[0271] In some specific embodiments, the aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2, and has the following (m1) to (m2) 57 ) is a mutation shown in any one of:

[0272] (m1) S38H (M0-1 in this specification);

[0273] (m2) K170Y (M0-2 in this specification);

[0274] (m3) K202G (M0-3 in this specification);

[0275] (m4) F168E (M0-4 in this specification);

[0276] (m5)A35T (M0-5 in this specification);

[0277] (m6) T36S (M0-6 in this specification);

[0278] (m7) R167E (M0-7 in this specification);

[0279] (m8) F169Y (M0-8 in this specification);

[0280] (m9) P40F (M1 in this specification);

[0281] (m 10 ) P40F, F169H (M1-1 in this manual);

[0282] (m 11 ) P40F, T36S (M1-2 in this manual);

[0283] (m 12 ) P40F, A35D (M1-3 in this manual);

[0284] (m 13) P40F, K170Q (M1-4 in this manual);

[0285] (m 14 ) P40F, K202H (M1-5 in this manual);

[0286] (m 15 )P40F, K202E (M1-6 in this manual);

[0287] (m 16 ) P40F, R167F (M1-7 in this specification);

[0288] (m 17 ) P40F, S38K (M1-8 in this manual);

[0289] (m 18 ) P40F, F168S (M1-9 in this manual);

[0290] (m 19 ) P40F, F169E (M1-10 in this manual);

[0291] (m 20 )P40F, K170V (M1-11 in this manual);

[0292] (m 21 )P40F, K202Q (M2 in this manual);

[0293] (m 22 )P40F, K202Q, S38V (M2-1 in this manual);

[0294] (m 23 ) P40F, K202Q, A35Q (M2-2 in this manual);

[0295] (m 24 ) P40F, K202Q, T36E (M2-3 in this manual);

[0296] (m 25 ) P40F, K202Q, S38N (M2-4 in this manual);

[0297] (m 26 ) P40F, K202Q, R167D (M2-5 in this manual);

[0298] (m 27 ) P40F, K202Q, R170Q (M2-6 in this manual);

[0299] (m 28) P40F, K202Q, F168T (M2-7 in this manual);

[0300] (m 29 ) P40F, K202Q, F169H (M2-8 in this manual);

[0301] (m 30 )P40F, K202Q, F169K (M3 in this manual);

[0302] (m 31 )P40F, K202Q, K170Q (M3-1 in this manual);

[0303] (m 32 ) P40F, K202Q, S38L (M3-2 in this manual);

[0304] (m 33 ) P40F, K202Q, F169K, R167E (M3-3 in this manual);

[0305] (m 34 ) P40F, K202Q, F169K, T36D (M3-4 in this manual);

[0306] (m 35 ) P40F, K202Q, F169K, A35N (M3-5 in this manual);

[0307] (m 36 ) P40F, K202Q, F169K, K170L (M3-6 in this manual);

[0308] (m 37 )P40F, K202Q, F169K, K170F (M4 in this manual);

[0309] (m 38 ) P40F, K202Q, F169K, K170F, R167K (M4-1 in this manual);

[0310] (m 39 ) P40F, K202Q, F169K, K170F, F168D (M4-2 in this manual);

[0311] (m 40 )P40F, K202Q, F169K, K170F, S38I (M4-3 in this manual);

[0312] (m 41)P40F, K202Q, F169K, K170F, T36V (M4-4 in this manual);

[0313] (m 42 )P40F, K202Q, F169K, K170F, A35Q (M4-5 in this manual);

[0314] (m 43 )P40F, K202Q, F169K, K170F, F168T (M5 in this manual);

[0315] (m 44 ) P40F, K202Q, F169K, K170F, F168T, R167H (M5-1 in this manual);

[0316] (m 45 ) P40F, K202Q, F169K, K170F, F168T, T36I (M5-2 in this manual);

[0317] (m 46 )P40F, K202Q, F169K, K170F, F168T, S38V (M5-3 in this manual);

[0318] (m 47 ) P40F, K202Q, F169K, K170F, F168T, A35E (M5-4 in this manual);

[0319] (m 48 )P40F, K202Q, F169K, K170F, F168T, S38K (M6 in this manual);

[0320] (m 49 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167H (M6-1 in this manual);

[0321] (m 50 )P40F, K202Q, F169K, K170F, F168T, S38K, A35V (M6-2 in this manual);

[0322] (m 51 ) P40F, K202Q, F169K, K170F, F168T, S38K, T36E (M6-3 in this manual);

[0323] (m 52)P40F, K202Q, F169K, K170F, F168T, S38K, R167K (M7 in this manual);

[0324] (m 53 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35L (M7-1 in this manual);

[0325] (m 54 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, T36Q (M7-2 in this manual);

[0326] (m 55 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I (M8 in this manual);

[0327] (m 56 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36H (M8-1 in this manual);

[0328] (m 57 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y (M9 in this manual);

[0329] In some preferred embodiments, the aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations:

[0330] (m1) S38H (M0-1 in this specification);

[0331] (m2) K170Y (M0-2 in this specification);

[0332] (m3) K202G (M0-3 in this specification);

[0333] (m4) F168E (M0-4 in this specification);

[0334] (m5)A35T (M0-5 in this specification);

[0335] (m6) T36S (M0-6 in this specification);

[0336] (m7) R167E (M0-7 in this specification);

[0337] (m8) F169Y (M0-8 in this specification);

[0338] (m9) P40F (M1 in this specification);

[0339] (m 10 ) P40F, F169H (M1-1 in this manual);

[0340] (m 11 ) P40F, T36S (M1-2 in this manual);

[0341] (m 14 ) P40F, K202H (M1-5 in this manual);

[0342] (m 15 )P40F, K202E (M1-6 in this manual);

[0343] (m 16 ) P40F, R167F (M1-7 in this specification);

[0344] (m 17 ) P40F, S38K (M1-8 in this manual);

[0345] (m 20 )P40F, K170V (M1-11 in this manual);

[0346] (m 21 )P40F, K202Q (M2 in this manual);

[0347] (m 22 )P40F, K202Q, S38V (M2-1 in this manual);

[0348] (m 23 ) P40F, K202Q, A35Q (M2-2 in this manual);

[0349] (m 24 ) P40F, K202Q, T36E (M2-3 in this manual);

[0350] (m 26 ) P40F, K202Q, R167D (M2-5 in this manual);

[0351] (m 27 ) P40F, K202Q, R170Q (M2-6 in this manual);

[0352] (m 28 ) P40F, K202Q, F168T (M2-7 in this manual);

[0353] (m29 ) P40F, K202Q, F169H (M2-8 in this manual);

[0354] (m 30 )P40F, K202Q, F169K (M3 in this manual);

[0355] (m 31 )P40F, K202Q, K170Q (M3-1 in this manual);

[0356] (m 33 ) P40F, K202Q, F169K, R167E (M3-3 in this manual);

[0357] (m 34 ) P40F, K202Q, F169K, T36D (M3-4 in this manual);

[0358] (m 35 ) P40F, K202Q, F169K, A35N (M3-5 in this manual);

[0359] (m 36 ) P40F, K202Q, F169K, K170L (M3-6 in this manual);

[0360] (m 37 )P40F, K202Q, F169K, K170F (M4 in this manual);

[0361] (m 39 ) P40F, K202Q, F169K, K170F, F168D (M4-2 in this manual);

[0362] (m 40 )P40F, K202Q, F169K, K170F, S38I (M4-3 in this manual);

[0363] (m 41 )P40F, K202Q, F169K, K170F, T36V (M4-4 in this manual);

[0364] (m 42 )P40F, K202Q, F169K, K170F, A35Q (M4-5 in this manual);

[0365] (m 43 )P40F, K202Q, F169K, K170F, F168T (M5 in this manual);

[0366] (m 45) P40F, K202Q, F169K, K170F, F168T, T36I (M5-2 in this manual);

[0367] (m 46 )P40F, K202Q, F169K, K170F, F168T, S38V (M5-3 in this manual);

[0368] (m 47 ) P40F, K202Q, F169K, K170F, F168T, A35E (M5-4 in this manual);

[0369] (m 48 )P40F, K202Q, F169K, K170F, F168T, S38K (M6 in this manual);

[0370] (m 49 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167H (M6-1 in this manual);

[0371] (m 52 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K (M7 in this manual);

[0372] (m 53 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35L (M7-1 in this manual);

[0373] (m 55 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I (M8 in this manual);

[0374] (m 56 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36H (M8-1 in this manual);

[0375] (m 57 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y (M9 in this manual).

[0376] In some more preferred embodiments, the aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations:

[0377] (m9)P40F;

[0378] (m 21 )P40F, K202Q;

[0379] (m 30 )P40F, K202Q, F169K;

[0380] (m 36 )P40F, K202Q, F169K, K170L;

[0381] (m 43 )P40F, K202Q, F169K, K170F, F168T;

[0382] (m 48 )P40F, K202Q, F169K, K170F, F168T, S38K;

[0383] (m 52 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K;

[0384] (m 55 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I;

[0385] (m 57 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y.

[0386] In some embodiments, the half-life of the above-mentioned glucosamine synthase at 70°C is more than 5 hours, and the synthase has good heat resistance and acid resistance, and can stably convert fructose into glucosamine for a long time under acidic conditions.

[0387] In some specific embodiments, aminosynthase is prepared and used in the form of cells expressing enzyme, as a crude extract or as a preparation separated or purified. In some exemplary embodiments, aminosynthase is prepared as a lyophilized powder, a powdered form (e.g., acetone powder) or as an enzyme solution. In some preferred embodiments, aminosynthase is in the form of a substantially pure preparation.

[0388] <Second Aspect>

[0389] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the polynucleotide encodes the aminoglycosylation enzyme as described in the first aspect of the present disclosure.

[0390] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0391] The polynucleotide encoding the mutant of the present invention includes: a coding sequence encoding only the mutant; a coding sequence of the mutant and various additional coding sequences; a coding sequence of the mutant (and optional additional coding sequences) and non-coding sequences.

[0392] <Third Aspect>

[0393] In the third aspect of the present disclosure, a recombinant expression vector is provided, wherein the recombinant expression vector comprises the polynucleotide described in the second aspect of the present disclosure.

[0394] In some embodiments, the polynucleotide of the second aspect is operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing a polypeptide.

[0395] <Fourth Aspect>

[0396] In the fourth aspect of the present invention, a recombinant host cell is provided, wherein the recombinant host cell comprises the aminoglycosylation described in the first aspect of the present invention, the isolated polynucleotide described in the second aspect of the present invention, or the recombinant expression vector described in the third aspect of the present invention.

[0397] In some embodiments, an expression vector containing a heterologous polynucleotide encoding an aminoglycosylate polypeptide is introduced into an appropriate host cell to express the corresponding aminoglycosylate polypeptide.

[0398] In some optional embodiments, the recombinant host cell is derived from a microorganism of the genus Escherichia, Erwinia, Serratia, Providencia, Enterobacteria, Salmonella, Streptomyces, Pseudomonas, Brevibacterium, Bacillus or Corynebacterium;

[0399] In some preferred embodiments, the recombinant host cell is derived from Escherichia coli, Corynebacterium glutamicum or Bacillus subtilis;

[0400] In some more preferred embodiments, the recombinant host cell is derived from Escherichia coli.

[0401] <Fifth Aspect>

[0402] In a fifth aspect of the present invention, a cell culture comprising the recombinant host cell according to the fourth aspect of the present invention is provided.

[0403] <Sixth Aspect>

[0404] In the sixth aspect of the present invention, a product is provided for preparing glucosamine or its derivatives, which comprises the glycosaminoglycans described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, and the cell culture described in the fifth aspect.

[0405] In some optional embodiments, the product may include enzyme preparations, bacterial preparations, kits, etc.

[0406] <Seventh Aspect>

[0407] In the seventh aspect of the present invention, a method for preparing glucosamine or its derivatives is provided, comprising providing the glycosaminoglycan synthase described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, or the product described in the sixth aspect into a reaction system to catalyze the conversion of the substrate into glucosamine or its derivatives.

[0408] In some alternative embodiments, the substrate comprises fructose.

[0409] In some optional embodiments, the system for preparing glucosamine or its derivatives comprises an amino donor. Preferably, the amino donor comprises an inorganic ammonium salt, and further, the inorganic ammonium salt includes ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium dimolybdate, ammonium chloroplatinite, ammonium chloroplatinate, ammonium chromate, ammonium fluoroborate, ammonium metavanadate, ammonium tetrachloroaurate, ammonium formate, water-soluble ammonium polyphosphate, ammonium phosphomolybdate, and ammonium oxalate.

[0410] In some preferred embodiments, the amino donor comprises ammonium sulfate, ammonium chloride, and aqueous ammonia, or any mixture of two or more thereof.

[0411] In some optional embodiments, the concentration of the substrate fructose in the system for preparing glucosamine or its derivatives is 1 to 2000 mM, preferably 500 mM. The fructose can be obtained or prepared by any method known in the art, for example, by converting glucose in glucose or fructose syrup into fructose, including converting glucose into fructose by glucose isomerase (GI). A solution containing at least one of glucose, fructose syrup, and molasses, or a solution in which fructose is mixed with at least one of glucose, fructose syrup, and molasses, can be treated with glucose isomerase to convert the glucose in the solution into fructose, and then glucosamine can be generated under the action of the aminoglycoside synthase.

[0412] In some optional embodiments, the concentration of glycosaminoglycan in the system for preparing glucosamine or its derivatives is 0.1-10 g / L, preferably 5 g / L. In other optional embodiments, the concentration of glycosaminoglycan in the system for preparing glucosamine or its derivatives is not less than 1 U / L, preferably not less than 1 U / L, 12 U / L, 35 U / L, 78 U / L, 140 U / L, 180 U / L, 248 U / L, 320 U / L, 360 U / L, and the enzyme concentration can be 5 U / L, 10 U / L, 12 U / L, 13 U / L, 30 U / L, 35 U / L, 78 U / L, 140 U / L, 180 U / L, 248 U / L, 320 U / L, 360 U / L. / L, 40U / L, 75U / L, 80U / L, 100U / L, 110U / L, 120U / L, 130U / L, 140U / L, 150U / L, 160U / L, 170U / L, 180U / L, 190U / L, 200U / L, 240U / L, 250U / L, 300U / L, 350U / L, 400U / L, 1000U / L, 1500U / L, etc.

[0413] In some optional embodiments, the concentration of inorganic ammonium in the system for preparing glucosamine or its derivatives is 10-1000 mM, preferably 500 mM.

[0414] In some optional embodiments, a buffer is further included in the system for preparing glucosamine or its derivatives to maintain the pH of the reaction system at 6.0±0.5. It will be appreciated by those skilled in the art that various buffers can be used in the present invention, such as HEPES buffer, Tris-HCl buffer, MOPS buffer, citrate buffer, etc. The concentration of the buffer in the reaction system is 20 to 300 mM, preferably 80 to 150 mM.

[0415] In some optional embodiments, the system for preparing glucosamine or its derivatives does not contain cofactors conventionally used in the art, such as NAD(H), ATP, and pyridoxal phosphate (PLP).

[0416] In some embodiments, the reaction temperature is 40-80°C, preferably 50-70°C.

[0417] In some embodiments, the reaction time is not less than 2 h, preferably, not less than 4 h, more preferably 12 h.

[0418] <Eighth Aspect>

[0419] In the eighth aspect of the present invention, provided is the use of the glycosamine synthase described in the first aspect, the polynucleotide described in the second aspect, the recombinant expression vector described in the third aspect, the recombinant host cell described in the fourth aspect, the cell culture described in the fifth aspect, or the product described in the sixth aspect in the preparation of glucosamine or its derivatives.

[0420] In some optional embodiments, in the reaction system for preparing glucosamine or its derivatives, the substrate includes fructose.

[0421] In some optional embodiments, an amino donor is included in the reaction system for preparing glucosamine or its derivatives. Preferably, the amino donor includes an inorganic ammonium salt, and further, the inorganic ammonium salt includes ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium dimolybdate, ammonium chloroplatinite, ammonium chloroplatinate, ammonium chromate, ammonium fluoroborate, ammonium metavanadate, ammonium tetrachloroaurate, ammonium formate, water-soluble ammonium polyphosphate, ammonium phosphomolybdate, and ammonium oxalate.

[0422] Example

[0423] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0424] The following materials are used in the embodiments of the present invention:

[0425] Fructose, product of Sigma, product number: 57-48-7;

[0426] pET20b vector, Novagen, Madison, WI;

[0427] Escherichia coli expression strain BL21 (DE3), Invitrogen, Carlsbad, CA;

[0428] All enzymes of the present invention can be purchased from Sigma, and all enzymes can also be obtained by prokaryotic expression according to genetic engineering methods.

[0429] Vector construction method for GNS mutants: Using the Phusion site-directed saturation mutagenesis method, degenerate primer NNK was designed. PCR amplification was performed using the pET20b-gns vector containing wild-type GNS or the vector containing the optimal mutant from each round as a template. The PCR reaction system consisted of 25 μL of 2× PrimerStar mixture, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 1 μL of template DNA, and 22 μL of double-distilled water. PCR amplification conditions were: initial denaturation at 98°C for 45 seconds, followed by 30 cycles of 98°C for 15 seconds, 55°C for 15 seconds, and 72°C for 45 seconds, followed by a final incubation at 72°C for 10 minutes. The PCR product was digested with Dpn I for 30 minutes and then purified using a PCR product purification kit. Mix 3 μL of the purified PCR product with 1 μL of T4 PNK, 1 μL of 10× T4 PNK buffer, 1 μL of 100 mM ATP, and 4 μL of double-distilled water. Phosphorylate the product at 37°C for 2 hours. Mix 10 μL of the phosphorylated PCR product with 1 μL of Rapid Ligase and 10 μL of Rapid Ligase buffer. Incubate at 25°C for 10 minutes for blunt-end ligation. Ligation products were transformed into competent E. coli TOP10 cells, plated onto antibiotic-containing LB solid medium plates, and cultured overnight. Single colonies were cultured overnight in LB liquid medium, after which plasmids were extracted and verified by sequencing.

[0430] Expression of GNS mutants: BL21(DE3) strains containing the sequenced mutant gene vector were inoculated into LB liquid medium and cultured at 37°C, 200 rpm until the OD600nm reached 0.6-0.8. IPTG was added for induction at a final concentration of 0.1 mM and cultured at 25°C for 12 hours. The cells were harvested and ultrasonically disrupted to obtain a crude mutant protein solution. Purification by nickel column, ultrafiltration, and buffer exchange were performed to obtain electrophoretically pure mutant protein.

[0431] Glucosamine synthase is an enzyme that catalyzes the addition of amino groups to fructose and inorganic ammonium as substrates to produce glucosamine.

[0432] Example 1: Screening of enzymes with aminoglycosylation activity

[0433] Based on the nucleotide sequences of the glucosamine-6-phosphate deaminase (GlmD) genes from the extremely thermophilic archaeon Thermococcus kodakarensis, Thermoclostridium stercorarium, and Thermotoga maritima (respectively, as shown in Genebank: TK0809, Genebank: Cst_c19900, and Genebank: TM0813), the complete gene was synthesized and linked to the pET20b plasmid via simple cloning. The gene was then transformed into competent Escherichia coli TOP10 cells, plated on LB plates containing 100 μg / ml ampicillin, and cultured at 37°C for 12 hours. Positive transformants were picked, identified, and sequenced. The confirmed positive single clone was inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C. The recombinant plasmid was then extracted and transformed into the expression host E. coli BL21(DE3). Pure enzyme was obtained by protein expression and purification. A reaction solution containing 100 mM potassium phosphate buffer (pH 7.0), 100 mM fructose, 100 mM NH4Cl, and 1 g / L wild-type pure enzyme was prepared. The reaction was incubated at 50°C for 12 h, and perchloric acid was added to terminate the reaction. The pH was neutralized to 7.0 with potassium hydroxide and the mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was collected and the glucosamine concentration was detected by two methods: one was a colorimetric method, in which 50 μL of sample was mixed with 100 μL of acetylacetone solution (3 mL of acetylacetone solution dissolved in 100 mL of 1.25 M Na2CO3 solution, prepared freshly), and boiled for 15 min; cooled to room temperature, 1 mL of ethanol was added, and 100 μL of DMAB solution (1.6 g of DMAB dissolved in 30 mL of concentrated hydrochloric acid and 30 mL of ethanol) was added. The mixture was allowed to stand at room temperature for 30 min for color development, and the OD530 nm was measured; the other was high-performance liquid chromatography to determine the glucosamine concentration. The chromatographic column used was an Agilent ZORBAX SB-C18 (300 mm), the derivatization reagent was o-phthalaldehyde (OPA), the mobile phase was 40 mM disodium hydrogen phosphate (phase A, pH 7.8) and acetonitrile:methanol:water = 45:45:10 (phase B), the flow rate was 1 mL / min, the column temperature was 40°C, and the detector used was an ultraviolet detector with a detection wavelength of 338 nm. GlmD derived from Thermoclostridium stercorarium catalyzed the highest yield of glucosamine ( Figure 2), confirming it as the wild-type glucosamine synthase, with a calculated enzymatic activity of approximately 1.80 mU / mg and a half-life of 5 hours at 70°C. After a 12-hour reaction, GlmD from the extremely thermophilic archaeon Thermococcus kodakarensis co-produced 4.2 mM glucosamine; GlmD from the thermophilic bacterium Thermoclostridium stercorarium co-produced 10.1 mM glucosamine; and GlmD from the extremely thermophilic archaeon Thermotoga maritima co-produced 5.9 mM glucosamine.

[0434] Example 2: First round of single-site saturation mutagenesis

[0435] The crystal structure of wild-type GNS was predicted using AlphaFold2, and molecular docking was performed using AutoDock Vina to identify the amino acid sites within the 21 substrate pockets. Multiple sequence alignment analysis was then performed to identify conserved amino acids, and site-directed saturation mutagenesis was performed at the nine non-conserved sites. Expression vectors for GNS mutants were constructed by PCR amplification using degenerate primers using the pET20b-gns vector containing the codon-optimized wild-type GNS gene (shown in SEQ ID NO:1) as a template. The constructed mutant library was transformed, and the cells were cultured in 96-well plates to obtain the mutant protein expression supernatants. The amino acid synthase activity of the mutants was roughly determined using a chromogenic assay. The enzyme activity of wild-type GNS (shown in SEQ ID NO:2) at 50°C was only 1.80 mU / mg. The screening conditions were: 100 mM potassium phosphate buffer (pH 6.5), 100 mM fructose, and 100 mM NH₄Cl. The supernatant was aspirated in a deep-well plate, and the amino acid concentration was measured after incubation at 50°C for 12 hours. This round of mutagenesis yielded nine mutants with enhanced enzyme activity after point mutations: GNS-S38H, GNS-K170Y, GNS-K202G, GNS-F168E, GNS-A35T, GNS-T36S, GNS-R167E, GNS-F169Y, and GNS-P40F. Their enzyme activities at 50°C were 2.1, 3.6, 2.3, 2.5, 4.0, 3.0, 2.5, 2.2, and 5.2 mU / mg, respectively. The GNS-P40F mutant exhibited the highest enzyme activity at 50°C and comparable stability to the wild type. The amino acid sequence of mutant GNS-S38H is shown in SEQ ID NO: 3; the amino acid sequence of mutant GNS-K170Y is shown in SEQ ID NO: 4; the amino acid sequence of mutant GNS-K202G is shown in SEQ ID NO: 5; the amino acid sequence of mutant GNS-F168EF is shown in SEQ ID NO: 6; the amino acid sequence of mutant GNS-A35T is shown in SEQ ID NO: 7; the amino acid sequence of mutant GNS-T36S is shown in SEQ ID NO: 8; the amino acid sequence of mutant GNS-R167E is shown in SEQ ID NO: 9; the amino acid sequence of mutant GNS-F169Y is shown in SEQ ID NO: 10; the amino acid sequence of the optimal mutant GNS-P40F obtained in this round is shown in SEQ ID NO: 11, and is named M1.

[0436] Example 3: Iterative saturation mutation

[0437] Starting with M1, single-site saturation mutagenesis was performed at the other eight sites. A mutant library was constructed using screening conditions: 100 mM potassium phosphate buffer (pH 6.0), 100 mM fructose, and 100 mM NH₄Cl. The supernatant was crushed in a deep-well plate and incubated at 50°C for 12 hours before measuring the glucosamine concentration. M2 (GNS-P40F-K202Q) exhibited the highest enzyme activity, at 12.8 mU / mg, with a half-life of 5.4 hours at 70°C. Subsequently, starting with M2, single-site saturation mutagenesis was performed at the other seven sites. A mutant library was constructed, and the supernatants of these mutants were crudely assayed for glucosamine synthase activity using a chromogenic assay. M3 (GNS-P40F-K202Q-F169K) exhibited the highest enzyme activity, at 35.5 mU / mg, with a half-life of 5.2 hours at 70°C. Starting with M3, single-point saturation mutagenesis was performed at the other six sites. After constructing a mutant library, the mutant supernatants were obtained, and the amino synthase activity of the mutants was roughly determined using a chromogenic method. Among them, M4 (GNS-P40F-K202Q-F169K-K170F) had the highest enzyme activity, 78.5 mU / mg, and a half-life of 5.1 hours at 70°C. Starting with M4, single-point saturation mutagenesis was performed at the other five sites. After constructing a mutant library, the mutant supernatants were obtained, and the amino synthase activity of the mutants was roughly determined using a chromogenic method. Among them, M5 (GNS-P40F-K202Q-F169K-K170F-F168T) had the highest enzyme activity, 140.0 mU / mg, and a half-life of 5.2 hours at 70°C. Starting with M5, single-site saturation mutagenesis was performed at the other four sites. A mutant library was constructed, and the supernatants of the mutants were obtained. The amino synthase activity of these mutants was roughly determined using a chromogenic assay. Among them, M6 (GNS-P40F-K202Q-F169K-K170F-F168T-S38K) exhibited the highest enzyme activity, at 180.7 mU / mg, with a half-life of 5.3 hours at 70°C. Starting with M6, single-site saturation mutagenesis was performed at the other three sites. A mutant library was constructed, and the supernatants of the mutants were obtained. The amino synthase activity of these mutants was roughly determined using a chromogenic assay. Among them, M7 (GNS-P40F-K202Q-F169K-K170F-F168T-S38K-R167K) exhibited the highest enzyme activity, at 248.8 mU / mg, with a half-life of 5.1 hours at 70°C. Starting with M7, single-site saturation mutagenesis was performed at the other two sites. A mutant library was constructed, and the supernatants of the mutants were obtained. The aminoglycosylation activity of the mutants was roughly determined using a chromogenic assay. M8 (GNS-P40F-K202Q-F169K-K170F-F168T-S38K-R167K-A35I) exhibited the highest activity, at 320.8 mU / mg, with a half-life of 5.2 hours at 70°C.Starting with M8, single-site saturation mutagenesis was performed at one other site. A mutant library was constructed, and the supernatants of these mutants were obtained. The aminoglycosylation activity of these mutants was roughly determined using a chromogenic assay. M9 (GNS-P40F-K202Q-F169K-K170F-F168T-S38K-R167K-A35I-T36Y) exhibited the highest activity, at 367.0 mU / mg, with a half-life of 5.1 h at 70°C. The amino acid sequence of mutant M2 is shown in SEQ ID NO:23; the amino acid sequence of mutant M3 is shown in SEQ ID NO:32; the amino acid sequence of mutant M4 is shown in SEQ ID NO:39; the amino acid sequence of mutant M5 is shown in SEQ ID NO:45; the amino acid sequence of mutant M6 is shown in SEQ ID NO:50; the amino acid sequence of mutant M7 is shown in SEQ ID NO:54; the amino acid sequence of mutant M8 is shown in SEQ ID NO:57; and the amino acid sequence of the optimal mutant M9 obtained in this round is shown in SEQ ID NO:59. The information and relative enzyme activities of other multi-site combination mutations screened by the iterative saturation mutagenesis process are listed in Table 1.

[0438] Table 1: Mutants generated during the mutation process and enzyme activity data

[0439]

[0440] Example 4: High-temperature and acid-resistant enzyme mutants catalyze the synthesis of glucosamine from low-concentration fructose

[0441] The preparation method comprises: using the glucosamine synthase M9 prepared in Example 4 to convert fructose and inorganic ammonium into glucosamine.

[0442] The reaction system contained 50 mM fructose as the substrate and 50 mM ammonium chloride as the amino donor. The reaction temperature was 50°C, the pH was 6.0, and the reaction time was 12 hours. The concentration of glucosamine synthase in the reaction system was 1 g / L (specific enzyme activity 367.0 mU / mg). A 0.5 mL reaction mixture was incubated at 50°C for 12 hours. Samples were collected at various times, the reaction terminated by adding perchloric acid, the pH was neutralized to 6.0 with potassium hydroxide, and the supernatant was centrifuged at 12,000 rpm for 10 minutes. The supernatant was then analyzed by high-performance liquid chromatography to determine the glucosamine concentration. The chromatographic column used was an Agilent ZORBAX SB-C18 (300 mm), the derivatization reagent was o-phthalaldehyde (OPA), the mobile phase was 40 mM disodium hydrogen phosphate (phase A, pH 7.8) and acetonitrile:methanol:water = 45:45:10 (phase B), the flow rate was 1 mL / min, the column temperature was 40°C, and the detector used was an ultraviolet detector with a detection wavelength of 338 nm. After 12 hours of reaction with the multienzyme system containing mutant M9, the concentration of glucosamine was 18 mM and the conversion rate was 36% ( Figure 4).

[0443] Example 5: High-temperature and acid-resistant enzyme mutants catalyze the synthesis of glucosamine from higher fructose concentrations

[0444] The preparation method comprises: using the glucosamine synthase M9 prepared in Example 3 to convert fructose and inorganic ammonium into glucosamine. The concentration of the substrate fructose in the reaction system is 500 mM, and the concentration of the amino donor ammonium chloride is 500 mM; the temperature of the catalytic reaction is 50°C; the pH of the catalytic reaction is 6.0, and the catalytic reaction time is 12 h. The concentration of glucosamine synthase in the reaction system is 5 g / L, and 0.5 mL of the reaction mixture is incubated at 50°C for 12 h. Samples are taken at different times, the reaction is terminated by adding perchloric acid, the pH is neutralized to 6.0 with potassium hydroxide, centrifuged at 12,000 rpm for 10 min, and the supernatant is taken to determine the concentration of glucosamine by high performance liquid chromatography. When the multi-enzyme system containing mutant M9 reacts for 12 h, the concentration of glucosamine is 152 mM, and the conversion rate is 30.4% ( Figure 4 ).

[0445] Example 6: High-temperature and acid-resistant enzyme mutants catalyze the synthesis of glucosamine from high-concentration fructose

[0446] The preparation method comprises: using the glucosamine synthase M9 prepared in Example 4 to convert fructose and inorganic ammonium into glucosamine.

[0447] The concentration of substrate fructose in the reaction system is 2M, and the concentration of amino donor ammonium chloride is 2M; the temperature of the catalytic reaction is 50°C; the pH of the catalytic reaction is 6.0, and the catalytic reaction time is 12h. The concentration of glucosamine synthase in the reaction system is 12g / L (specific enzyme activity is 367.0mU / mg), and 0.5mL of the reaction mixture is incubated at 50°C for 12h. Samples were taken at different times, the reaction was terminated by adding perchloric acid, the pH was neutralized to 6.0 with potassium hydroxide, and the supernatant was centrifuged at 12000 rpm for 10min. The concentration of glucosamine was determined by high performance liquid chromatography. When the multi-enzyme system containing mutant M9 was reacted for 12h, the concentration of glucosamine was 410mM, and the conversion rate was 20.5%( Figure 4 ).

[0448] Example 7: High-temperature and acid-resistant enzyme mutants catalyze in vitro multi-enzyme synthesis of glucosamine

[0449] The preparation method includes: using the aminoglycosylation enzyme M9 prepared in Example 4 to convert glucose and inorganic ammonium into glucosamine. The concentration of the substrate glucose in the reaction system is 2M, and the concentration of the amino donor ammonium chloride is 2M; the temperature of the catalytic reaction is 50°C; the pH of the catalytic reaction is 6.0, and the catalytic reaction time is 12h. The concentration of glucose isomerase (GlucoseIsomerase, purchased from Meryer, catalog number: M83989) in the reaction system is 15g / L, the concentration of aminoglycosylation enzyme is 12g / L (specific enzyme activity is 367.0mU / mg), and 0.5mL of the reaction mixture is incubated at 50°C for 12h. Samples are taken at different times, the reaction is terminated by adding perchloric acid, the pH is neutralized to 6.0 with potassium hydroxide, centrifuged at 12000 rpm for 10min, and the supernatant is taken to determine the concentration of glucosamine by high performance liquid chromatography. When the multi-enzyme system containing mutant M9 and glucose isomerase was reacted for 12 hours, the concentration of glucosamine was 390 mM and the conversion rate was 19.5%.

[0450] SEQ ID NO: 1

[0451] ATGCGTATCATTAACGTGAAAGACTACGAGGAAATGAGCCGTAAGGCGGCGGATCTGATTGCGGCGCAGATCATTCTGAACCCGAAAAGCGTGCTGGGTCTGGCGACCGGCAGCAGCCCGATTGGTACCTATGAGCGTCTGGTTGAACTGAACCGTAACGGCGTGATCGACTTCAGCCACGTTACCACCATTAACCTGGATGAGTACTATGGTCTGGACCCGACCCACGATCAGAGCTACCGTTATTTCATGAACAAGCACCTGTTTAGCCGTGTGAACATCAACATGGCGAACACCCACCTGCCGGATGGCAAGGCGAAAGACATTGATGCGGAGTGCCGTCGTTACGACGATCTGATCGAAAGCGTTGGTGGCATTGACCTGCAACTGCTGGGTATCGGCCACAACGGTCACATTGGCTTCAACGAGCCGAGCGATGAATTTATCCCGGGTACCCACTGCGTTAGCCTGAGCGAGAGCACCATTAACGCGAACAGCCGTTTCTTTAAAAGCCGTGACGAAGTGCCGCGTAAGGCGATCACCATGGGCATCAAAGCGATTATGCAAGCGCGTAAGGTTCTGCTGATCGCGAGCGGCGAGGATAAGAAAGAAATTCTGAAGAAAGCGCTGTTTGGTCCGATCACCCCGCAGGTGCCGGCGAGCATTCTGCAACTGCACAAGGACCTGACCGTTATCACCCCGCTGGATATT

[0452] SEQ ID NO:2

[0453] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0454] SEQ ID NO:3

[0455] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGHSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0456] SEQ ID NO:4

[0457] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFYSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0458] SEQ ID NO:5

[0459] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDGKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0460] SEQ ID NO:6

[0461] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSREFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0462] SEQ ID NO:7

[0463] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLTTGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0464] SEQ ID NO:8

[0465] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLASGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0466] SEQ ID NO:9

[0467] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSEFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0468] SEQ ID NO:10

[0469] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSPIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFYKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0470] SEQ ID NO:11

[0471] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0472] SEQ ID NO:12

[0473] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFHKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0474] SEQ ID NO:13

[0475] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLASGSSFIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0476] SEQ ID NO:14

[0477] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLDTGSSFIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0478] SEQ ID NO:15

[0479] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKQSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0480] SEQ ID NO:16

[0481] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEYYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGHNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDHKEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0482] SEQ ID NO:17

[0483] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0484] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRYDDLIESVGGIDLQLLGIGH

[0485] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDEKEI

[0486] LKKALFGPITPQVPASILQLHKDLTVITPLDI

[0487] SEQ ID NO:18

[0488] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0489] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRYDDLIESVGGIDLQLLGIGH

[0490] NGHIGFNEPSDEFIPGTHCVSLSESTINANSFFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKEI

[0491] LKKALFGPITPQVPASILQLHKDLTVITPLDI

[0492] SEQ ID NO:19

[0493] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0494] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0495] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKK

[0496] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0497] SEQ ID NO:20

[0498] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0499] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0500] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRSFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKE

[0501] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0502] SEQ ID NO:21

[0503] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0504] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0505] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFEKSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKE

[0506] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0507] SEQ ID NO:22

[0508] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0509] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0510] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFVSRDEVPRKAITMGIKAIMQARKVLLIASGEDKKE

[0511] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0512] SEQ ID NO:23

[0513] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0514] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0515] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0516] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0517] SEQ ID NO:23

[0518] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0519] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0520] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0521] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0522] SEQ ID NO:24

[0523] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGVSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0524] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0525] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0526] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0527] SEQ ID NO:25

[0528] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLQTGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0529] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0530] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0531] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0532] SEQ ID NO:26

[0533] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLAEGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0534] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0535] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0536] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0537] SEQ ID NO:27

[0538] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGNSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0539] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0540] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0541] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0542] SEQ ID NO:28

[0543] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0544] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0545] NGHIGFNEPSDEFIPGTHCVSLSESTINANSDFFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0546] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0547] SEQ ID NO:29

[0548] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0549] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0550] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFFQSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0551] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0552] SEQ ID NO:30

[0553] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0554] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0555] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRTFKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0556] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0557] SEQ ID NO:31

[0558] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0559] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0560] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFHKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0561] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0562] SEQ ID NO:32

[0563] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0564] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0565] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0566] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0567] SEQ ID NO:33

[0568] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0569] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0570] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRNKKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0571] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0572] SEQ ID NO:34

[0573] MRIINVKDYEEMSRKAADLIAAQIILNPKSVGLATGLSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0574] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0575] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQ

[0576] KEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0577] SEQ ID NO:35

[0578] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0579] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRYDDLIESVGGIDLQLLGIGH

[0580] NGHIGFNEPSDEFIPGTHCVSLSESTINANSEFKKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0581] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0582] SEQ ID NO:36

[0583] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGADGSSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0584] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0585] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQ

[0586] KEILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0587] SEQ ID NO:37

[0588] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLNTGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0589] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0590] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKKSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0591] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0592] SEQ ID NO:38

[0593] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0594] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0595] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKLSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0596] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0597] SEQ ID NO:39

[0598] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0599] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0600] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0601] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0602] SEQ ID NO:40

[0603] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0604] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0605] NGHIGFNEPSDEFIPGTHCVSLSESTINANSKFKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0606] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0607] SEQ ID NO:41

[0608] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0609] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0610] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRDKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0611] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0612] SEQ ID NO:42

[0613] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGISFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0614] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0615] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0616] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0617] SEQ ID NO:43

[0618] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLAVGSSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0619] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0620] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0621] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0622] SEQ ID NO:44

[0623] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLQTGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0624] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0625] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRFKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0626] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0627] SEQ ID NO:45

[0628] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0629] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0630] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0631] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0632] SEQ ID NO:46

[0633] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0634] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0635] NGHIGFNEPSDEFIPGTHCVSLSESTINANSHTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0636] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0637] SEQ ID NO:47

[0638] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLAIGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0639] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0640] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0641] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0642] SEQ ID NO:48

[0643] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGVSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0644] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0645] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0646] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0647] SEQ ID NO:49

[0648] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLETGSSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0649] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRYDDLIESVGGIDLQLLGIGH

[0650] NGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0651] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0652] SEQ ID NO:50

[0653] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0654] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0655] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0656] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0657] SEQ ID NO:51

[0658] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0659] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0660] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSHTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0661] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0662] SEQ ID NO:52

[0663] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLVTGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0664] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0665] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0666] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0667] SEQ ID NO:53

[0668] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLAEGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0669] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0670] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSRTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0671] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0672] SEQ ID NO:54

[0673] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLATGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0674] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0675] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSKTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0676] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0677] SEQ ID NO:55

[0678] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLLTGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0679] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0680] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSKTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0681] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0682] SEQ ID NO:56

[0683] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLAQGKSFIGTYERLVELNRNGVIDFSHVTTINLDE

[0684] YYGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIG

[0685] HNGHIGFNEPSDEFIPGTHCVSLSESTINANSKTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQK

[0686] EILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0687] SEQ ID NO:57

[0688] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLITGKSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0689] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0690] NGHIGFNEPSDEFIPGTHCVSLSESTINANSKTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0691] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0692] SEQ ID NO:58

[0693] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLIHGKSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0694] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0695] NGHIGFNEPSDEFIPGTHCVSLSESTINANSKTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0696] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0697] SEQ ID NO:59

[0698] MRIINVKDYEEMSRKAADLIAAQIILNPKSVLGLIYGKSFIGTYERLVELNRNGVIDFSHVTTINLDEY

[0699] YGLDPTHDQSYRYFMNKHLFSRVNINMANTHLPDGKAKDIDAECRRYDDLIESVGGIDLQLLGIGH

[0700] NGHIGFNEPSDEFIPGTHCVSLSESTINANSKTKFSRDEVPRKAITMGIKAIMQARKVLLIASGEDQKE

[0701] ILKKALFGPITPQVPASILQLHKDLTVITPLDI

[0702] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0703] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Ammonia synthase that catalyzes the production of glucosamine from fructose and inorganic ammonium, wherein: The aminoglycosylation enzyme is selected from any one of the following groups (I) to (V): (I) the aminoglycosylation enzyme comprises a mutation at at least one of positions 40, 202, 169, 170, 168, 38, 167, 35, and 36 corresponding to the sequence of SEQ ID NO: 2, compared to the sequence of SEQ ID NO: 2; (II) has at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (I), and does not include mutants of the sequence shown in SEQ ID NO: 2; (III) A mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions: (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (I); (b) the full-length complementary polynucleotide of (a); (IV) a fragment of the mutant represented by any one of (I), (II) or (III), wherein the fragment still has aminoglycoside synthase activity; (V) A polypeptide having an amino acid sequence as shown in (I), (II), (III) or (IV) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus.

2. The aminoglucose synthase according to claim 1, wherein The aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2 and has the following (d1) to (d 36 ) any of the mutations shown: (d1) mutation at position 40; (d2) combined mutations at positions 40 and 35; (d3) combined mutations at positions 40 and 36; (d4) combined mutations at positions 40 and 38; (d5) combined mutations at positions 40 and 167; (d6) combined mutations at positions 40 and 168; (d7) combined mutations at positions 40 and 169; (d8) combined mutations at positions 40 and 170; (d9) combined mutations at positions 40 and 202; (d 10 ) combined mutations at positions 40, 202, and 35; (d 11 ) combined mutations at positions 40, 202, and 36; (d 12 ) combined mutations at positions 40, 202, and 38; (d 13 ) combined mutations at positions 40, 202, and 167; (d 14 ) combined mutations at positions 40, 202, and 168; (d 15 ) combined mutations at positions 40, 202, and 169; (d 16 ) combined mutations at positions 40, 202, and 170; (d 17 ) combined mutations at positions 40 and 202 and 169 and 35; (d 18 ) combined mutations at positions 40 and 202 and 169 and 36; (d 19 ) combined mutations at positions 40 and 202 and 169 and 38; (d 20 ) combined mutations at positions 40 and 202 and at positions 169 and 167; (d 21 ) combined mutations at positions 40 and 202 and at positions 169 and 168; (d 22 ) combined mutations at positions 40 and 202 and at positions 169 and 170; (d 23 ) combined mutations at positions 40 and 202 and 169 and 170 and 35; (d 24 ) combined mutations at positions 40, 202, 169, 170, and 36; (d 25 ) combined mutations at positions 40 and 202 and 169 and 170 and 38; (d 26 ) combined mutations at positions 40 and 202 and 169 and 170 and 167; (d 27 ) combined mutations at positions 40 and 202 and 169 and 170 and 168; (d 28 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 35; (d 29 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 36; (d 30 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38; (d 31 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 167; (d 32 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 35; (d 33 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 36; (d 34 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167; (d 35 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167 and 35; (d 36 ) combined mutations at positions 40 and 202 and 169 and 170 and 168 and 38 and 167 and 36; Optionally, the aminoglycosylation enzyme corresponds to the sequence shown in SEQ ID NO: 2, and has a mutated amino acid at at least one of the following positions: P40F, K202Q, K202H, K202G, K202E, F169K, F169Y, F169H, F169E, K170F, K170 Y, K170Q, K170V, K170L, F168T, F168E, F168S, F168N, F168D, S38K, S38H, S38 V, S38N, S38L, S38I, R167K, R167E, R167F, R167D, R167H, A35I, A35T, A35D, A 35Q, A35N, A35V, A35L, A35E, T36S, T36E, T36D, T36V, T36I, T36Q, T36H, T36Y.

3. The aminoglycosylate according to claim 1 or 2, wherein the aminoglycosylate corresponds to the sequence shown in SEQ ID NO: 2 and has the following (m1) to (m2) 57 ) is a mutation shown in any one of: (m1)S38H; (m2)K170Y; (m3)K202G; (m4)F168E; (m5)A35T; (m6)T36S; (m7)R167E; (m8)F169Y; (m9)P40F; (m 10 )P40F、F169H; (m 11 )P40F、T36S; (m 12 )P40F、A35D; (m 13 )P40F、K170Q; (m 14 )P40F、K202H; (m 15 )P40F、K202E; (m 16 )P40F、R167F; (m 17 )P40F、S38K; (m 18 )P40F、F168S; (m 19 )P40F、F169E; (m 20 )P40F、K170V; (m 21 )P40F、K202Q; (m 22 )P40F、K202Q、S38V; (m 23 )P40F、K202Q、A35Q; (m 24 )P40F、K202Q、T36E; (m 25 )P40F、K202Q、S38N; (m 26 )P40F、K202Q、R167D; (m 27 )P40F、K202Q、R170Q; (m 28 )P40F、K202Q、F168T; (m 29 )P40F、K202Q、F169H; (m 30 )P40F、K202Q、F169K; (m 31 )P40F、K202Q、K170Q; (m 32 )P40F、K202Q、S38L; (m 33 )P40F、K202Q、F169K、R167E; (m 34 )P40F、K202Q、F169K、T36D; (m 35 )P40F、K202Q、F169K、A35N;(m 36 )P40F、K202Q、F169K、K170L; (m 37 )P40F、K202Q、F169K、K170F; (m 38 )P40F、K202Q、F169K、K170F、R167K; (m 39 )P40F、K202Q、F169K、K170F、F168D; (m 40 )P40F、K202Q、F169K、K170F、S38I; (m 41 )P40F、K202Q、F169K、K170F、T36V; (m 42 )P40F、K202Q、F169K、K170F、A35Q; (m 43 )P40F、K202Q、F169K、K170F、F168T; (m 44 )P40F、K202Q、F169K、K170F、F168T、R167H; (m 45 )P40F、K202Q、F169K、K170F、F168T、T36I; (m 46 )P40F、K202Q、F169K、K170F、F168T、S38V; (m 47 )P40F、K202Q、F169K、K170F、F168T、A35E; (m 48 )P40F、K202Q、F169K、K170F、F168T、S38K; (m 49 )P40F、K202Q、F169K、K170F、F168T、S38K、R167H; (m 50 )P40F、K202Q、F169K、K170F、F168T、S38K、A35V; (m 51 )P40F、K202Q、F169K、K170F、F168T、S38K、T36E; (m 52 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K; (m 53 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35L; (m 54 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、T36Q; (m 55 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I; (m 56 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36H; (m 57 ) P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y; Preferably, the aminoglycoside synthase corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations: (m1) S38H; (m2)K170Y; (m3)K202G; (m4)F168E; (m5)A35T; (m6)T36S; (m7)R167E; (m8)F169Y; (m9)P40F; (m 10 )P40F、F169H; (m 11 )P40F、T36S; (m 14 )P40F、K202H; (m 15 )P40F、K202E; (m 16 )P40F、R167F; (m 17 )P40F、S38K; (m 20 )P40F、K170V; (m 21 )P40F、K202Q; (m 22 )P40F、K202Q、S38V; (m 23 )P40F、K202Q、A35Q; (m 24 )P40F、K202Q、T36E; (m 26 )P40F、K202Q、R167D; (m 27 )P40F、K202Q、R170Q; (m 28 )P40F、K202Q、F168T; (m 29 )P40F、K202Q、F169H; (m 30 )P40F、K202Q、F169K; (m 31 )P40F、K202Q、K170Q; (m 33 )P40F、K202Q、F169K、R167E; (m 34 )P40F、K202Q、F169K、T36D; (m 35 )P40F、K202Q、F169K、A35N; (m 36 )P40F、K202Q、F169K、K170L; (m 37 )P40F、K202Q、F169K、K170F; (m 39 )P40F、K202Q、F169K、K170F、F168D; (m 40 )P40F、K202Q、F169K、K170F、S38I; (m 41 )P40F、K202Q、F169K、K170F、T36V; (m 42 )P40F、K202Q、F169K、K170F、A35Q; (m 43 )P40F、K202Q、F169K、K170F、F168T; (m 45 )P40F、K202Q、F169K、K170F、F168T、T36I; (m 46 )P40F、K202Q、F169K、K170F、F168T、S38V; (m 47 )P40F、K202Q、F169K、K170F、F168T、A35E; (m 48 )P40F、K202Q、F169K、K170F、F168T、S38K; (m 49 )P40F、K202Q、F169K、K170F、F168T、S38K、R167H; (m 52 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K; (m 53 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35L; (m 55 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I; (m 56 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36H; (m 57 )P40F, K202Q, F169K, K170F, F168T, S38K, R167K, A35I, T36Y; More preferably, the aminoglycoside synthase corresponds to the sequence shown in SEQ ID NO: 2, and has any of the following mutations: (m9) P40F; (m 21 )P40F、K202Q; (m 30 )P40F、K202Q、F169K; (m 36 )P40F、K202Q、F169K、K170L; (m 43 )P40F、K202Q、F169K、K170F、F168T; (m 48 )P40F、K202Q、F169K、K170F、F168T、S38K; (m 52 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K; (m 55 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I; (m 57 )P40F、K202Q、F169K、K170F、F168T、S38K、R167K、A35I、T36Y。 4. An isolated polynucleotide, wherein The polynucleotide encodes the aminoglucose synthase according to any one of claims 1 to 3.

5. A recombinant expression vector, wherein: The recombinant expression vector comprises the polynucleotide according to claim 4.

6. A recombinant host cell, wherein The recombinant host cell comprises the aminoglycosylation according to any one of claims 1 to 3, the isolated polynucleotide according to claim 4, or the recombinant expression vector according to claim 5.

7. A cell culture comprising the recombinant host cell of claim 6.

8. A product for preparing glucosamine or its derivatives, comprising the glycosamine synthase according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, or the cell culture according to claim 7.

9. A method for preparing glucosamine or its derivatives, wherein: The method comprises providing the glucosamine synthase according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6, the cell culture according to claim 7, or the product according to claim 8 into a reaction system to catalyze the conversion of the substrate into glucosamine or its derivatives; Optionally, the substrate comprises fructose; Optionally, the reaction system contains an amino donor; preferably, the amino donor is an inorganic ammonium salt; Optionally, the concentration of the substrate is 1 to 2000 mM, preferably 2 to 500 mM; Preferably, the pH of the reaction system is below 7, preferably 6.0±0.5; Preferably, the reaction temperature of the reaction system is 40-80°C, preferably 50-70°C.

10. Use of the glucosamine synthase according to any one of claims 1 to 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the recombinant host cell according to claim 6 or the cell culture according to claim 7 in the preparation of glucosamine or its derivatives; Optionally, in the reaction system for preparing glucosamine or its derivatives, the substrate includes fructose; and / or, In the reaction system for preparing glucosamine or its derivatives, the amino donor includes inorganic ammonium.

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