Partial glyceride monoesterase mutant and its encoding gene and application

Abstract

The present invention discloses a partial glyceride monoesterase mutant, its encoding gene, and application. The amino acid sequence of the mutant is shown in SEQ ID NO. 3 or SEQ ID NO. 5. The present invention obtains partial glyceride monoesterase mutants EstGtA2-S147G and EstGtA2-S147A by introducing a suitable single-point mutation into the wild-type partial glyceride monoesterase EstGtA2. Under the same conditions, the esterification rate of the wild-type partial glyceride monoesterase is 48.86%, while the esterification rate of the partial glyceride monoesterase mutant EstGtA2-S147G is 65.93%, and the esterification rate of the partial glyceride monoesterase mutant EstGtA2-S147A is 63.80%. The esterification rate is significantly improved while the hydrolysis specific enzyme activity is not reduced, making it more suitable for industrial application.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and in particular relates to a partial glyceride monoesterase mutant and its encoding gene and application. Background Art

[0002] Lipase (EC 3.1.1.3) is a serine hydrolase that not only hydrolyzes oils and fats at the oil-water interface to produce fatty acids, diglycerides, monoglycerides, and glycerol, but also catalyzes reactions such as esterification, transesterification, alcoholysis, and acidolysis. Triglyceride lipase can hydrolyze or esterify to form triglycerides, diglycerides, or monoglycerides, while partial monoglyceride lipase can only decompose monoglycerides, not diglycerides or triglycerides, and the esterification reactions it participates in only produce monoglycerides.

[0003] Partial monoglyceride esterase plays an important role in the preparation of monoglycerides and the removal of monoglycerides from oils and fats, and therefore shows great market prospects in the fields of oil processing, medicine, feed additives, food and nutrition, and chemical industry.

[0004] Currently, there are relatively few partial glyceride monoesterases reported in the literature, and no commercial partial glyceride monoesterases are available. Therefore, the development of partial glyceride monoesterases is of great significance. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a partial glyceride monoesterase mutant and its encoding gene and application.

[0006] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] In a first aspect of the present invention, a partial glyceride monoesterase mutant is provided, wherein the amino acid sequence of the mutant is shown as SEQ ID NO.3, or the amino acid sequence of the mutant is shown as SEQ ID NO.5.

[0008] In a second aspect of the present invention, a gene encoding a partial glyceride monoesterase mutant is provided, wherein the nucleotide sequence of the gene encoding is shown in SEQ ID NO.4, or the nucleotide sequence of the gene encoding is shown in SEQ ID NO.6.

[0009] The third aspect of the present invention provides the use of the partial glyceride monoesterase mutant or the gene encoding the partial glyceride monoesterase mutant in catalyzing the preparation of monoglyceride from glycerol and oleic acid.

[0010] The fourth aspect of the present invention provides a recombinant expression vector into which the gene encoding the above-mentioned partial glyceride monoesterase mutant is inserted.

[0011] The fifth aspect of the present invention provides a recombinant engineered strain into which the above-mentioned recombinant expression vector is introduced.

[0012] The sixth aspect of the present invention provides the use of the above-mentioned recombinant expression vector or recombinant engineered strain in catalyzing the preparation of monoglyceride from glycerol and oleic acid.

[0013] In a seventh aspect, the present invention provides a method for preparing monoglyceride, using the above-mentioned partial glyceride monoesterase mutant to catalyze the reaction between glycerol and oleic acid.

[0014] The present invention introduces a suitable single-point mutation into the wild-type partial glycerol monoesterase EstGtA2 to obtain partial glycerol monoesterase mutants EstGtA2-S147G and partial glycerol monoesterase mutants EstGtA2-S147A. Under the same conditions, the esterification rate of the wild-type partial glycerol monoesterase is 48.86%, while the esterification rate of the partial glycerol monoesterase mutant EstGtA2-S147G is 65.93%, which is significantly improved (compared to the current The best partial glycerol monoesterase mutant, S26I, had an esterification rate increased by 8.27%, and its hydrolysis specific enzyme activity was not reduced compared with the wild type. The partial glycerol monoesterase mutant, EstGtA2-S147A, had an esterification rate of 63.80%, a significantly improved esterification rate, and a significantly improved hydrolysis specific enzyme activity compared with the wild type. Therefore, the partial glycerol monoesterase mutants EstGtA2-S147G and EstGtA2-S147A are more suitable for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a graph showing the results of SDS-PAGE detection of protein purity in Example 2 of the present invention; wherein lanes 1-8 respectively represent: wild-type partial glyceride monoesterase EstGtA2, partial glyceride monoesterase mutant EstGtA2-I145L, partial glyceride monoesterase mutant EstGtA2-V198I, partial glyceride monoesterase mutant EstGtA2-S147A, partial glyceride monoesterase mutant EstGtA2-A144S, partial glyceride monoesterase mutant EstGtA2-S158G, partial glyceride monoesterase mutant EstGtA2-S147G, and partial glyceride monoesterase mutant EstGtA2-S26I.

[0016] Figure 2 This is the HPLC analysis of the monoglyceride composition of the products of the reaction of glycerol and oleic acid catalyzed by each mutant in Example 3 of the present invention.

[0017] Figure 3The esterification rates of monoglyceride synthesized by the wild type and mutants of partial glyceride monoesterase in Example 3 of the present invention are shown; a, b, and c in the figure are significant difference analyses, and different letters indicate significant differences between them; a represents the wild type, b represents the best mutant S26I reported so far, and c represents mutants S147G and S147A. DETAILED DESCRIPTION

[0018] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0020] In the present invention, the inventors analyzed the crystal structures of existing monoesterases GMGL and MGLP (PDB ID: 5XKS and 4KEA), and based on the inventors' accumulated experience and using Modeller software, predicted the crystal structure of the partial glyceride monoesterase EstGtA2 from Geobacter thermodenitrificans (which can be used to synthesize high-purity monoglyceride and has the highest esterification rate compared to other partial glyceride monoesterases, but its esterification rate has not yet reached the level of industrial application, with an amino acid sequence as shown in SEQ ID NO.1 and a nucleotide sequence as shown in SEQ ID NO.2). After repeated comparisons, the inventors obtained six mutants of EstGtA2 by site-directed mutagenesis (using "original amino acid-position-substituted amino acid" to represent the mutated amino acid in the partial glyceride monoesterase mutant), namely, partial glyceride monoesterase mutant S147G (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4), partial glyceride monoesterase mutant S147A (amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.6). NO.6), partial glyceride monoesterase mutant V198I (amino acid sequence shown in SEQ ID NO.7, nucleotide sequence shown in SEQ ID NO.8), partial glyceride monoesterase mutant I145L (amino acid sequence shown in SEQ ID NO.9, nucleotide sequence shown in SEQ ID NO.10), partial glyceride monoesterase mutant A144S (amino acid sequence shown in SEQ ID NO.11, nucleotide sequence shown in SEQ ID NO.12), partial glyceride monoesterase mutant S158G (amino acid sequence shown in SEQ ID NO.13, nucleotide sequence shown in SEQ ID NO.14).

[0021] Amino acid sequence of partial glyceride monoesterase EstGtA2 (SEQ ID NO.1):

[0022] MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCL

[0023] PRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTL

[0024] YLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSIGSDLKNPDVKELS

[0025] YEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQ

[0026] SSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0027] Nucleotide sequence of the encoding gene of monoglyceride monohydrolase EstGtA2 (SEQ ID NO.2): ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGAAAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTATTCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTGCCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTACCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTGAAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCACCCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCCGATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTGGTGGTGAAGTTCCGCGTTACCTGGATAGCATCGGC AGC GATCTGAAAAACCCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCTGCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCACCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGGGTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATCGTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAGACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0028] Amino acid sequence of monoglyceride lipase mutant EstGtA2-S147G (SEQ ID NO.3): MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSIGGDLKNPDVKELSYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0029] Nucleotide sequence of the coding gene of monoglyceride lipase mutant EstGtA2-S147G (SEQ ID NO.4): ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGAAAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTATTCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTGCCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTACCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTGAAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCACCCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCCGATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTGGTGGTGAAGTTCCGCGTTACCTGGATAGCATCGGC GGCGATCTGAAAAACCCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCTGCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCACCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGGGTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATCGTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAGACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0030] Amino acid sequence of monoglyceride monohydrolase mutant EstGtA2 - S147A (SEQ ID NO.5): MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSIGADLKNPDVKELSYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0031] Nucleotide sequence of the coding gene of the monoglyceride lipase mutant EstGtA2-S147A (SEQ ID NO.6): ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGAAAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTATTCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTGCCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTACCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTGAAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCACCCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCCGATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTGGTGGTGAAGTTCCGCGTTACCTGGATAGCATCGGC GCC GATCTGAAAAACCCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCTGCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCACCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGGGTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATCGTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAGACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG C

[0032] It should be noted that there may be some inaccuracies in the translation due to the complexity of the biological sequence content. It is recommended to double-check with relevant professionals for accurate understanding.Amino acid sequence of monoglyceride lipase mutant EstGtA2-V198I (SEQ ID NO.7): MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSIGSDLKNPDVKELSYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHIVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0033] Nucleotide sequence of the encoding gene of monoglyceride lipase mutant EstGtA2-V198I (SEQ ID NO.8): ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGAAAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTATTCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTGCCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTACCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTGAAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCACCCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCCGATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTGGTGGTGAAGTTCCGCGTTACCTGGATAGCATCGGCAGCGATCTGAAAAACCCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCTGCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCACCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCAC ATAGTTCCGCCGGGTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATCGTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAGACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0034] Amino acid sequence of monoglyceride lipase mutant EstGtA2-I145L (SEQ ID NO.9): MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSLGSDLKNPDVKELSYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0035] Nucleotide sequence of the encoding gene of monoglyceride lipase mutant EstGtA2-I145L (SEQ ID NO.10): ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGAAAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTATTCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTGCCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTACCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTGAAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCACCCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCCGATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTGGTGGTGAAGTTCCGCGTTACCTGGATAGC CTCGGCAGCGATCTGAAAAACCCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCTGCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCACCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGGGTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATCGTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAGACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0036] Amino acid sequence of monoglyceride lipase mutant EstGtA2 - S144A (SEQ ID NO.11): MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDAIGSDLKNPDVKELSYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0037] Nucleotide sequence of the coding gene of monoglyceride lipase mutant EstGtA2 - S144A (SEQ ID NO:12):

[0038] ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGA

[0039] AAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTAT

[0040] TCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTG

[0041] CCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTA

[0042] CCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTG

[0043] AAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCAC

[0044] CCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCC

[0045] GATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTG

[0046] GTGGTGAAGTTCCGCGTTACCTGGAT GCC ATCGGCAGCGATCTGAAAAAC

[0047] CCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCT

[0048] GCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCA

[0049] CCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGG

[0050] GTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATC

[0051] GTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAG

[0052] ACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0053] The amino acid sequence of the partial glyceride monoesterase mutant EstGtA2-S158G (SEQ ID NO. 13): MKERYPVLPGAEPFYAENGPVGVLLSHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSIGSDLKNPDVKELGYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0054] The nucleotide sequence of the gene encoding the partial glyceride lipase mutant EstGtA2-S158G (SEQ ID NO.14):

[0055] ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGA

[0056] AAACGGTCCGGTTGGTGTTCTGCTGTCTCATGGTTTTACTGGTACTCCGTAT

[0057] TCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTG

[0058] CCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTA

[0059] CCACCTTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTG

[0060] AAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCAC

[0061] CCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCC

[0062] GATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTG

[0063] GTGGTGAAGTTCCGCGTTACCTGGATAGCATCGGCAGCGATCTGAAAAAC

[0064] CCGGATGTTAAAGAACTG GGC TACGAAAAAACCCCGACCGCGTCCCTGCT

[0065] GCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCA

[0066] CCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGG

[0067] GTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATC

[0068] GTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAG

[0069] ACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0070] In some embodiments of the present invention, a partial glyceride monoesterase mutant is disclosed, the amino acid sequence of the mutant is shown as SEQ ID NO.3, or the amino acid sequence of the mutant is shown as SEQ ID NO.5.

[0071] In other embodiments of the present invention, the use of the above-mentioned partial glyceride monoesterase mutant in the preparation of monoglyceride is disclosed.

[0072] In other embodiments of the present invention, a gene encoding a partial glyceride monoesterase mutant is disclosed, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.4, or the nucleotide sequence of the encoding gene is shown as SEQ ID NO.6.

[0073] In other embodiments of the present invention, the use of the gene encoding the partial glyceride monoesterase mutant in catalyzing the preparation of monoglyceride from glycerol and oleic acid is disclosed.

[0074] In other embodiments of the present invention, a recombinant expression vector into which the above-mentioned encoding gene is inserted is disclosed.

[0075] In other embodiments of the present invention, a recombinant engineered strain into which the above-mentioned recombinant expression vector is introduced is disclosed.

[0076] In other embodiments of the present invention, disclosed is the use of the above-mentioned recombinant expression vector or the above-mentioned recombinant engineered strain in catalyzing the preparation of monoglyceride from glycerol and oleic acid.

[0077] In other embodiments of the present invention, a method for constructing the above-mentioned recombinant engineering strain is disclosed, comprising the following steps: cloning the coding gene of the above-mentioned partial glyceride monoesterase mutant into an expression vector, and transforming Escherichia coli competent cells to obtain the obtained strain.

[0078] In other embodiments of the present invention, a method for preparing monoglyceride is disclosed, using the above-mentioned partial glyceride monoesterase mutant to catalyze the reaction of glycerol and oleic acid.

[0079] In the following examples, a plasmid extraction kit was purchased from Omega Trading Co., Ltd., a Primer STAR kit was purchased from TAKARA, TOP10 Escherichia coli competent cells were purchased from Tiangen Biotechnology Co., Ltd., and mutant primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.; a PCR product purification and recovery kit was purchased from Dalian Bao Biotechnology Co., Ltd.; the expression vector pET30a(+)-EstGtA2 for partial glyceride monoesterase EstGtA2 was constructed by the applicant in the early stage of the experiment, that is, the EstGtA2 gene (Genbank ID: JN031579.1) was inserted into the vector pET30a(+) according to the existing conventional method, and the restriction enzyme cutting sites were Nde I and Xho I; LB and ZYM-5052 culture media were prepared according to the Invitrogen Escherichia coli expression kit operating manual, and the remaining reagents were all analytical grade purchased domestically and abroad.

[0080] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0081] Example 1 Construction of partial glyceride monoesterase mutant expression vector

[0082] 1. Design primers

[0083] The primer sequences are shown in Table 1.

[0084] Table 1

[0085]

[0086]

[0087] 2. PCR amplification

[0088] Using pET30a(+)-EstGtA2 as a template and the primers listed in Table 1, PCR reactions were performed to construct expression vectors for the partial glyceride monoesterase mutant EstGtA2-I145L, partial glyceride monoesterase mutant EstGtA2-V198I, partial glyceride monoesterase mutant EstGtA2-S147A, partial glyceride lipase mutant EstGtA2-A144S, partial glyceride monoesterase mutant EstGtA2-S158G, and partial glyceride monoesterase mutant EstGtA2-S147G.

[0089] Reaction system: 1 μL of each primer, 12.5 μL of Primer Star (2X), 1 μL of template, and 9.5 μL of ddH2O. PCR amplification conditions: 98°C for 3 min; 30 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 5 min; extension at 72°C for 5 min.

[0090] 3. The amplified product was digested with DnpⅠ enzyme template. After the size of the mutant band was detected by agarose gel electrophoresis, the mutant plasmid was transformed into E. coli TOP10 competent cells using the heat shock method and spread on LB (Kana concentration was 50 μg / mL) solid plates. The cells were cultured at 37°C overnight and positive transformants were selected for plasmid sequencing.

[0091] In addition, for comparative screening, the gene of the mutant with the highest esterification efficiency, EstGtA2-S26I (JP2020-92615A, Osamura, T, et al. Efficient monoacylglycerol synthesis bycarboxylesterase EstGtA2 from Geobacillus thermodenitrificans in asolvent-free two-phase system, J.Biosci.Bioeng.2022), was inserted into the vector pET30a(+), with the restriction enzyme sites of Nde I and Xho I, and the above operation was performed.

[0092] The amino acid sequence of the partial glyceride monoesterase mutant EstGtA2-S26I is shown in SEQ ID NO.27: MKERYPVLPGAEPFYAENGPVGVLL IHGFTGTPYSMRPLAEAYAQAGYTVCLPRLKGHGTHYEDMERTTFHDWIASVEEGYEWLKQRCQTIFVTGLSMGGTLTLYLAEQHPEICGIVPINAAVDIPAIAAGMTGGGEVPRYLDSIGSDLKNPDVKELSYEKTPTASLLQLAQLMERVKEELGRITCPALIFVSDEDHVVPPGNADIIFQGVQSSEKEIVRLHNSYHVATLDYDQQTIIERSLQFFAKHA

[0093] The nucleotide sequence of the coding gene of the monoglyceride lipase mutant EstGtA2-S26I is shown in SEQ ID NO.28:

[0094] ATGAAAGAACGTTACCCGGTTCTGCCGGGTGCTGAACCGTTCTACGCTGA

[0095] AAACGGTCCGGTTGGTGTTCTGCTG ATT CATGGTTTTACTGGTACTCCGTAT

[0096] TCTATGCGTCCGCTGGCGGAAGCGTACGCGCAGGCGGGCTACACCGTTTG

[0097] CCTGCCGCGTCTGAAAGGCCACGGCACCCACTACGAAGATATGGAACGTA

[0098] CCACCTTCCACGATTGGATCGCGTCCGTTGAAGAAGGCTACGAATGGCTG

[0099] AAACAGCGTTGCCAGACCATCTTCGTTACCGGTCTGAGCATGGGTGGCAC

[0100] CCTGACCCTGTACCTGGCGGAACAGCACCCGGAAATCTGCGGTATCGTTCC

[0101] GATCAACGCGGCGGTTGACATCCCGGCGATCGCGGCGGGCATGACCGGTG

[0102] GTGGTGAAGTTCCGCGTTACCTGGATAGCATCGGCAGCGATCTGAAAAAC

[0103] CCGGATGTTAAAGAACTGAGCTACGAAAAAACCCCGACCGCGTCCCTGCT

[0104] GCAGCTGGCGCAGCTGATGGAACGTGTTAAAGAAGAACTGGGTCGTATCA

[0105] CCTGCCCGGCGCTGATCTTCGTTAGCGATGAAGATCACGTGGTTCCGCCGG

[0106] GTAACGCGGACATCATCTTCCAGGGTGTTCAGAGCAGCGAAAAAGAAATC

[0107] GTTCGTCTGCACAACTCTTACCACGTTGCGACCCTGGATTACGATCAGCAG

[0108] ACCATCATCGAACGTAGCCTGCAGTTCTTCGCGAAACACGCG

[0109] Example 2 Construction, expression and purification of a partial glyceride monoesterase mutant expression strain

[0110] After sequencing correctly positive transformants were amplified and cultured overnight in LB liquid medium, the plasmids were extracted and then transformed into E. coli BL21 competent cells using the heat shock method. The cells were spread on LB solid plates (Kana concentration was 50 μg / mL) and cultured overnight at 37°C to select positive transformants.

[0111] A single colony of the engineered strain was inoculated into 50 mL of LB medium and cultured at 37°C and 220 rpm to an OD of 0.8-1.0. The inoculation volume was then inoculated into 500 mL of ZYM-5052 medium at 10% and fermented at 30°C and 220 rpm for 20 h.

[0112] The fermentation broth was centrifuged at 7000 rpm for 40 minutes at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter membrane and purified using a nickel affinity chromatography column. The column was first equilibrated with phosphate buffer (pH 8.0) containing 20 mM imidazole at a flow rate of 4 mL / min. After equilibration, the supernatant was combined with the column, and then impurities were eluted with phosphate buffer (pH 7.4) containing 20 mM imidazole. The target protein was then eluted with phosphate buffer (pH 8.0) containing 500 mM imidazole. The eluted target protein was further desalted with a G-25 desalting column to remove imidazole, and the enzyme purity was finally detected by reducing SDS-PAGE vertical electrophoresis.

[0113] The results show Figure 1 As shown, the results showed that the purity of all proteins was above 95%.

[0114] Example 3 Synthesis of Monoglyceride Catalyzed by Partial Monoglyceride Monoesterase Mutant

[0115] Weigh 2 g of a mixture of oleic acid and glycerol (oleic acid to glycerol molar ratio of 1:5) into a conical flask and preheat it at 60°C for 10 min on a constant temperature magnetic stirrer at a speed of 450 rpm. After preheating, add 100 μl of 100 mg / mL wild-type partial glyceride monoesterase and partial glyceride lipase mutant, respectively. Then, place it in a constant temperature oscillator at 60°C with a magnetic stirrer for reaction at a speed of 450 rpm. Extract oil samples at different reaction times and analyze the monoglyceride composition of the reaction products catalyzed by each mutant using HPLC. The results are as follows: Figure 2 shown.

[0116] The results are shown in Table 2 and Figure 3 shown.

[0117] Table 2 Esterification rate determination results

[0118]

[0119]

[0120] From Table 2 and Figure 3 The results showed that after 27 hours of reaction, the MAG (monoglyceride) content of the esterification product of the wild-type partial glyceride monoesterase was 48.86%, the MAG content of the partial glyceride monoesterase mutant S26I was 57.66%, the MAG content of the partial glyceride monoesterase mutant S147A was 63.80%, and the MAG content of the partial glyceride monoesterase mutant S147G was 65.93%. This shows that the partial glyceride mutants S147A and S147G are more effective in synthesizing monoglycerides and have better application prospects.

[0121] Example 4 Determination of specific activity of wild-type and mutant partial glyceride monoesterase

[0122] Enzyme activity was measured using an emulsion of glycerol-stearic acid-rich oil as a substrate: 150 g of 4% PVA solution and 50 g of monoglyceride were weighed and homogenized in a high-speed homogenizer for 6 minutes to prepare an emulsion. In a 100 mL conical flask, 4 g of the emulsion and 5 mL of 50 mM phosphate buffer (pH 8.0) were added and incubated at 60°C for 5 minutes. The purified enzyme was diluted, and 1 mL of the enzyme solution was added to the conical flask. The reaction was allowed to proceed for 10 minutes, and then terminated with 15 mL of 95% ethanol. Phenolphthalein was added and titrated with 0.05 M sodium hydroxide solution to determine the amount of free fatty acids released during the reaction. For the control group, the enzyme solution was replaced with an equal volume of buffer. Enzyme activity (U) was defined as the amount of enzyme consumed to produce 1 μmol of fatty acid per minute.

[0123] The specific activity assay results are shown in Table 3.

[0124] Table 3 Specific activity determination results

[0125] Specific activity (U / mg) wild type 82.5 I145L 84.2 V198I 95.13 S147A 100.0 A144S 35.7 S158G 86.3 S147G 87.5 S26I 89.0

[0126] Combining the results of Example 2 and Table 3, we can see that compared with the wild-type lipase EstGtA2, mutant S147A significantly improved both the esterification rate and the specific enzymatic activity. Mutants S26I and S147G increased the esterification rate while maintaining a slight improvement in the specific enzymatic activity. Mutants V198I, while showing a significant improvement in specific enzymatic activity, did not improve the esterification rate. Therefore, the partial glyceride mutants S147A and S147G have promising application prospects.

[0127] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A partial glyceride monoesterase mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.3, or the amino acid sequence of the mutant is shown in SEQ ID NO.

5.

2. Use of the partial glyceride monoesterase mutant according to claim 1 in catalyzing the preparation of monoglyceride from glycerol and oleic acid.

3. A gene encoding the partial glyceride monoesterase mutant according to claim 1.

4. The gene encoding the partial glyceride monoesterase mutant according to claim 3, characterized in that The nucleotide sequence of the encoding gene is shown as SEQ ID NO.4, or the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

6.

5. Use of the gene encoding the partial glyceride monoesterase mutant according to claim 3 or 4 in catalyzing the preparation of monoglyceride from glycerol and oleic acid.

6. A recombinant expression vector into which is inserted the gene encoding the partial glyceride monoesterase mutant according to claim 3 or 4.

7. A recombinant engineered strain transformed with the recombinant expression vector according to claim 6.

8. A method for constructing the recombinant engineered strain according to claim 7, characterized in that: The method comprises the following steps: cloning the coding gene of the partial glyceride monoesterase mutant according to claim 3 or 4 into an expression vector, and transforming competent Escherichia coli cells to obtain the product.

9. Use of the recombinant expression vector according to claim 6 or the recombinant engineered strain according to claim 7 in catalyzing the preparation of monoglyceride from glycerol and oleic acid.

10. A method for preparing monoglyceride, characterized in that: The partial glyceride monoesterase mutant according to claim 1 is used to catalyze the reaction of glycerol and oleic acid.

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