CN116334050B - Artificially designed lysyl endonuclease, coding sequence and fermentation method - Google Patents
Artificially designed lysyl endonuclease, coding sequence and fermentation method Download PDFInfo
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- CN116334050B CN116334050B CN202310376133.8A CN202310376133A CN116334050B CN 116334050 B CN116334050 B CN 116334050B CN 202310376133 A CN202310376133 A CN 202310376133A CN 116334050 B CN116334050 B CN 116334050B
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Abstract
The invention discloses an artificially designed lysyl endonuclease, a coding sequence and a fermentation method. The artificially designed lysyl endonuclease is obtained by mutating at least one amino acid of 167 th, 193 rd and 207 th positions in wild lysyl endonuclease as follows: valine at position 167 is mutated to leucine or isoleucine; serine at position 193 is mutated to alanine or glycine; glycine at position 207 is mutated to valine or leucine. The lysyl endonuclease provided by the invention has high enzyme activity which can reach more than 8000U/L, and has the advantage of high stability.
Description
Technical Field
The invention belongs to the technical field of biology, and in particular relates to an artificially designed lysyl endonuclease, a coding sequence and a fermentation method.
Background
Lysyl-specific endonucleases are polypeptides consisting of 268 amino acid residues, in which the peptide chain contains three disulfide bonds (Cys 6-Cys216, cys12-Cys80, cys36-Cys 58) inside, the triplet consisting of His57, asp113 and Ser194 determines the catalytic activity of the enzyme, while Asp225 determines its specific selectivity towards lysine.
Lysyl-specific endonucleases have only 20% homology with bovine trypsin, but the amino acid sequence and spatial structure that determine the catalytic activity of the enzyme and the lysine specificity are completely identical, and thus lysyl-specific endonucleases are classified as trypsin family. Compared with bovine trypsin, lysyl-specific endonucleases have higher selectivity for lysine carbon-terminal, and have 10 times of activity than trypsin, a wider optimal pH range (pH 8.5-10.5), and still normal stability in 4M urea or 0.1% SDS. These properties make lysyl-specific endonucleases a very useful tool enzyme in biomedical production.
In 1978, masaki et al, university of arrowhead, separated from the supernatant of the fermentation broth of Achromobacter Achromobacter lyticus M497-1 an alkaline protease-Achromobacter lysyl endonuclease having a molecular weight of 30kDa, which enzyme specifically hydrolyzes lysyl groups including lysyl-proline groups [ Masaki, T., et al, A new proteolytic enzyme from Achromobacter lyticus M497-1.Agricultural and Biological Chemistry,1978.42 ]; tsunasawa et al obtained the gene sequence and protein sequence of Achromobacter lysylase by sequencing, found by analysis that lysylase belongs to serine protease, and lysylase precursor comprises a mature peptide fragment containing 268 amino acid residues, a propeptide of 205 amino acid residues at the N-terminus and a large peptide fragment containing 180 amino acid residues at the C-terminus, the substrate binding site of which consists of His210-Trp211-Gly212, and found by sequence alignment that lysylase protein sequence has only 20% homology with bovine trypsin [ Tsunasawa, S., et al The primary structure and structural characteristics of Achromobacter lyticus protease I, analysis-specific serine protease J Biol Chem 1989.264 (7): p.3832-9 ].
Because the achromobacter lysyl endonuclease has the characteristics of high specificity, wide and stable tolerance pH range, not only can be used for cutting leader peptide and C peptide of biological medicine such as insulin and other proteins, but also can be used for peptide pattern mass spectrum research of monoclonal antibody and other macromolecular proteins, and because the achromobacter lysyl endonuclease has wide application and high commercial value, the expression quantity of the lysyl endonuclease produced by using natural achromobacter Achromobacter lyticus M497-1 is low, the period is long, the cost is obviously higher, and the price of the lysyl endonuclease is high.
Ohara et al cloned the achromobacter lysyl endonuclease zymogen gene into recombinant plasmid by genetic engineering means, expressed in recombinant E.coli, and finally harvested active lysyl endonuclease in E.coli periplasmic space, but no protease expression was detected in the fermentation broth supernatant [ Ohara, T., et al, cloning, nucleotide sequence, and expression of Achromobacter protease I gene.J Biol Chem,1989.264 (34): p.20625-31 ]; the gene amplification technology is adopted by Noand Nord company to clone the achromobacter lysyl endonuclease zymogen gene which does not contain C-terminal leader peptide, and then the gene is transformed into escherichia coli, and finally the lysyl endonuclease enzyme activity is detected in the supernatant of the recombinant escherichia coli fermentation broth, but the enzyme activity is not clear [ process for producing extracellular proteins in bacteria.US6171823B 1].
Disclosure of Invention
The invention aims at overcoming the defects and shortcomings of the prior art and providing an artificially designed lysyl endonuclease.
It is another object of the present invention to provide a nucleotide sequence encoding the above-mentioned artificially designed lysyl endonuclease.
It is still another object of the present invention to provide a fermentation method of the artificially designed lysyl endonuclease.
The aim of the invention is achieved by the following technical scheme: an artificially designed lysyl endonuclease is obtained by mutating at least one amino acid of 167 th, 193 rd and 207 th positions in a wild-type lysyl endonuclease as follows: valine at position 167 is mutated to leucine or isoleucine; serine at position 193 is mutated to alanine or glycine; glycine at position 207 is mutated to valine or leucine. Thus, the artificially designed lysyl endonuclease has higher enzyme activity than the wild-type lysyl endonuclease.
The artificially designed lysyl endonuclease also comprises the following situations: one or more of the lysines at positions 30, 49, 106 and 155 of the wild-type lysyl endonuclease are mutated to arginine. Thus, the artificially designed lysyl endonuclease has stronger stability than the wild-type lysyl endonuclease.
The artificially designed lysyl endonuclease is preferably prepared by mutating lysine at position 30, 49, 106 and 155 in wild-type lysyl endonuclease into arginine.
The combination of mutations at position 167, 193 and 207 in the artificially designed lysyl endonuclease is preferably as follows: V167I, S193A, G V; V167I, S193A, G L; V167I, S193G, G V; V167L, S193A, G V; V167I, S193G, G L; V167L, S193A, G L; V167L, S193G, G V; V167L, S193G; V167L; or V167L, S193G, G L.
The artificially designed lysyl endonuclease is preferably a lysyl endonuclease (SEQ ID NO: 1) with the amino acid sequence shown as follows:
GVSGSCNIDVVCPEGDGRRDIIRAVGAYSRSGTLACTGSLVNNTANDRRMYFLTAHHCGMGTASTAASIVVYWNYQNSTCRAPNTPA SGANGDGSMSQTQSGSTVRATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAERRISNSTSPTSFIAWGGGAGTTHL NVQWQPSGGVTEPGASGSPIYSPEKRVLVQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP。
the nucleotide sequence for coding the artificially designed lysyl endonuclease is obtained according to the codon principle; preferably a nucleotide sequence derived from a host cell according to codon bias; more preferably as shown below (SEQ ID NO: 2):
ggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctcgtt
ctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtcgtatgtacttcctgaccgctcaccactgcggtatggg
taccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaac
ggtgacggttctatgtctcagacccagtctggttctaccgttcgtgctacctacgctacctctgacttcaccctgctggaactgaacaacg
ctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgt
tgctgaacgtcgtatctctaactctacctctccgacctctttcatcgcttggggtggtggtgctggtaccacccacctgaacgttcagtgg
cagccgtctggtggtgttaccgaaccgggtgcttctggttctccgatctactctccggaaaaacgtgttctggttcagctgcacggtggtc
cgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga。
an artificially designed lysyl endoenzyme is prepared from signal peptide, N-terminal leader peptide and said artificially designed lysyl endoenzyme through sequentially connecting.
The amino acid sequence of the signal peptide is as follows: MKKTAIAIAVALAGFATVAQA.
The amino acid sequence of the N-terminal leader peptide is as follows:
APASRPAAFDYANLSSVDKVALRTMPAVDVAKAKAEDLQRDKRGDIPRFALAIDVDMTPQNSGAWEYTADGQFAVWRQRVRSEKALS LNFGFTDYYMPAGGRLLVYPATQAPAGDRGLISQYDASNNNSARQLWTAVVPGAEAVIEAVIPRDKVGEFKLRLTKVNHDYVGFGPLARRL AAASGEK。
the amino acid sequence of the artificially designed lysyl incision zymogen is preferably as follows:
MKKTAIAIAVALAGFATVAQAAPASRPAAFDYANLSSVDKVALRTMPAVDVAKAKAEDLQRDKRGDIPRFALAIDVDMTPQNSGAWEYTADGQFAVWRQRVRSEKALSLNFGFTDYYMPAGGRLLVYPATQAPAGDRGLISQYDASNNNSARQLWTAVVPGAEAVIEAVIPRDKVGEFKLRLTKVNHDYVGFGPLARRLAAASGEKGVSGSCNIDVVCPEGDGRRDIIRAVGAYSRSGTLACTGSLVNNTANDRRMYFLTAHHCGMGTASTAASIVVYWNYQNSTCRAPNTPASGANGDGSMSQTQSGSTVRATYATSDFTLLELNNAANPAFNLFWAGWDRRDQNYPGAIAIHHPNVAERRISNSTSPTSFIAWGGGAGTTHLNVQWQPSGGVTEPGASGSPIYSPEKRVLVQLHGGPSSCSATGTNRSDQYGRVFTSWTGGGAAASRLSDWLDPASTGAQFIDGLDSGGGTP。
the nucleotide sequence for coding the artificially designed lysyl incision zymogen is obtained according to a codon principle; preferably a nucleotide sequence derived from a host cell according to codon bias; more preferably as shown below (SEQ ID NO: 3):
atgaaaaaaaccgctatcgctatcgctgttgctctggctggtttcgctaccgttgctcaggctgctccggcttctcgtccggctgctttcgactacgctaacctgtcttctgttgacaaagttgctctgcgtaccatgccggctgttgacgttgctaaagctaaagctgaagacctgcagcgtgacaaacgtggtgacatcccgcgtttcgctctggctatcgacgttgacatgaccccgcagaactctggtgcttgggaatacaccgctgacggtcagttcgctgtttggcgtcagcgtgttcgttctgaaaaagctctgtctctgaacttcggtttcaccgactactacatgccggctggtggtcgtctgctggtttacccggctacccaggctccggctggtgaccgtggtctgatctctcagtacgacgcttctaacaacaactctgctcgtcagctgtggaccgctgttgttccgggtgctgaagctgttatcgaagctgttatcccgcgtgacaaagttggtgaattcaaactgcgtctgaccaaagttaaccacgactacgttggtttcggtccgctggctcgtcgtctggctgctgcttctggtgaaaaaggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctcgttctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtcgtatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcagacccagtctggttctaccgttcgtgctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgttgctgaacgtcgtatctctaactctacctctccgacctctttcatcgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggtgcttctggttctccgatctactctccggaaaaacgtgttctggttcagctgcacggtggtccgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga。
the recombinant vector for expressing the artificially designed lysyl incision zymogen is obtained by recombining the nucleotide sequence for encoding the artificially designed lysyl incision zymogen on an expression vector.
The expression vector is pET9a, pET28a-c (+), pET32a-c (+), pET30a-c (+), or pET33b (+); preferably pET9a or pET28a.
The recombinant vector for expressing the artificially designed lysyl incision zymogen is obtained through the following steps:
(1) Adding an NdeI enzyme cutting site to the 5 'end of the nucleotide sequence for coding the artificially designed lysyl incision zymogen, and adding a BamHI enzyme cutting site to the 3' end to obtain a sequence containing NdeI and BamHI double enzyme cutting sites;
(2) Double-enzyme cutting the sequences obtained in the step (1) and the expression vector by using NdeI and BamHI restriction enzymes respectively; the sequence after double enzyme digestion is connected with the expression vector after double enzyme digestion to obtain the recombinant vector for expressing the artificially designed lysyl incision zymogen.
A strain expressing an artificially designed lysyl incision zymogen is obtained by transforming a recombinant vector of the artificially designed lysyl incision zymogen into a host strain.
The host strain is Escherichia coli BL21 (DE 3), BL21 (DE 3) pLysS, BL21star (DE 3) or BL21star (DE 3) pLysS; coli BL21 (DE 3) is preferred.
The fermentation method of the strain for expressing the artificially designed lysyl incision zymogen comprises the following steps: the strain expressing the artificially designed lysyl incision zymogen is inoculated into a fermentation medium for fermentation.
The fermentation conditions are preferably as follows: the temperature is controlled between 35 ℃ and 37 ℃, the pH value is controlled between 6.5 and 7.0, the stirring rotating speed is controlled between 150rpm and 700rpm, the air flow rate is controlled between 200L/h and 1600L/h, the Dissolved Oxygen (DO) is controlled at the maximum oxygen saturation of 10-50 percent, and the addition amount of inducer IPTG is 0.1mmol/L to 0.6mmol/L (final concentration in a fermentation system); when the carbon source is exhausted, feeding is started, and the specific growth rate mu is controlled to be 0.03-0.15h by adopting index feeding -1 Between them.
The composition of the fermentation medium is as follows: each liter contains 2 to 5g of yeast extract, 3 to 8g of peptone, 1 to 2g of sodium chloride, 2 to 5g of monopotassium phosphate, 2 to 5g of disodium hydrogen phosphate, 0.01 to 0.02g of calcium chloride dihydrate, 1 to 2g of magnesium sulfate, 4 to 7g of glycerin, 5 to 7g of ammonium sulfate and 0.875mL of microelements; water is used for constant volume to 1L, and the pH value is 6.5-7.0; the following are preferred: each liter contains 3g of yeast extract, 5g of peptone, 1g of sodium chloride, 3g of potassium dihydrogen phosphate, 3.25g of disodium hydrogen phosphate, 0.014g of calcium chloride dihydrate, 1g of magnesium sulfate, 4.125g of glycerin, 6g of ammonium sulfate and 0.875mL of trace elements; the volume is fixed to 1L by water, and the pH value is 6.5-7.0.
The trace elements comprise the following components: each liter contains 20 to 30g of ferrous chloride tetrahydrate, 1 to 3g of zinc chloride, 2 to 4g of cobalt chloride hexahydrate, 2 to 4g of sodium molybdate dihydrate, 1 to 2g of calcium chloride dihydrate, 1 to 2g of copper chloride dihydrate, 0.4 to 0.6g of boric acid, 2 to 3g of manganese sulfate monohydrate, 100mL of concentrated hydrochloric acid with the concentration of 37 mass percent, and water is used for fixing the volume to 1L; the following are preferred: each liter contains 22.87g of ferrous chloride tetrahydrate, 1.31g of zinc chloride, 2g of cobalt chloride hexahydrate, 2g of sodium molybdate dihydrate, 1g of calcium chloride dihydrate, 1.25g of copper chloride dihydrate, 0.5g of boric acid, 2.17g of manganese sulfate monohydrate and 100mL of concentrated hydrochloric acid with the concentration of 37 mass percent, and the volume is fixed to 1L by water.
The composition of the feed is as follows: each liter contains 250-500 g glycerin, and the balance is water; the following are preferred: each liter contains 500g of glycerin, the balance being water.
The addition amount of IPTG is preferably 0.3mmol/L calculated as the final concentration.
The enzyme activity of the artificially designed lysyl endonuclease reaches over 8000U/L (the enzyme activity is defined as 1U when the enzyme activity is used for catalyzing a substrate to generate 1 mu mol of paranitroaniline per minute at 30 ℃).
Compared with the prior art, the invention has the following advantages and effects:
(1) The inventor combines the conserved sequence of the lysyl endonuclease and the amino acid sequence of the active center of a substrate, and provides the artificially designed lysyl endonuclease in the aspects of taking the catalytic activity, the structural stability of the protein and the like into consideration. The enzyme has the advantages of high enzyme activity and high stability.
(2) The invention provides a nucleotide sequence for coding an artificially designed achromobacter lysyl incision zymogen. The nucleotide sequence is obtained through optimization, and is suitable for expression in escherichia coli.
(3) The invention provides a fermentation method of a strain for expressing artificially designed achromobacter lysyl incision zymogen, wherein a fermentation culture medium consists of an organic nitrogen source, a carbon source and inorganic salt, and the culture medium is suitable for expressing genetically engineered bacteria for producing lysyl incision enzymes. The enzyme activity of the lysyl endonuclease obtained by the fermentation method reaches more than 8000U/L.
Drawings
FIG. 1 is a diagram showing the alignment of amino acids of an artificially designed lysyl endonuclease with amino acid sequences of wild-type Achromobacter lysyl endonuclease.
FIG. 2 is a SDS-PAGE diagram of engineering bacterium BL21 (DE 3)/pET 28a-LyC fermentation broth supernatant before and after induction respectively; lane M is protein Marker, each band size: 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa, 18.0kDa, 14.4kDa; lane 1 is the sample 5h before induction, lane 2 is the sample 2h before induction, lane 3 is the sample 0h after induction, lane 4 is the sample 6h after induction, lane 5 is the sample 12h after induction, and the arrow indicates the mature peptide.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Summary of the experimental procedure of the invention:
1. artificial design and screening of lysyl endonucleases
Lysyl endonucleases belong to the serine protease family, and the enzyme activity of the enzymes under alkaline conditions is 5-6 times that under neutral conditions. In lysyl endonuclease-producing wild-type bacteria, an inactive precursor protein is first synthesized. The precursor protein is called as a zymogen lysyl endonuclease, and comprises a signal peptide sequence, an N-terminal leader peptide, a mature peptide and a C-terminal extension peptide, and the zymogen is led through the whole peptide chain to enter a cell membrane by the signal peptide, and then the signal peptide is cut off. Lysyl endonucleases cleave the N-terminal and C-terminal peptide chains by self-cleavage, forming mature active lysyl endonucleases.
In the self-cleavage process of the lysyl endonuclease, the N-terminal leader peptide plays an important role, and can promote the correct folding modification of mature peptide of the lysyl endonuclease. The role of the C-terminal extension peptide relative to the N-terminal leader peptide is not clear. The inventors obtained a preferred signal peptide (MKKTAIAIAVALAGFATVAQA, shown as SEQ ID NO: 4) by screening, replacing the lysyl endonuclease self signal peptide.
Researchers of the invention analyze the catalytic mechanism of lysyl endonuclease-substrate binding, and in consideration of the catalytic activity of the substrate and the characteristic of the substrate entering the active pocket of the lysyl endonuclease, the mutant is capable of assisting the lysyl endonuclease to bind the substrate more efficiently and promote the rotation of the substrate by mutating a plurality of amino acid residues with relatively low hydrophilicity or hydrophobicity near the active pocket of the wild-type lysyl endonuclease (shown as SEQ ID NO: 5) to amino acid residues with relatively large hydrophobic side chains. Through experimental analysis, one or more of Val167, ser193 and Gly207 of the wild lysyl endonuclease are mutated into Val, leu, IIe or Ala; preferably Val167 IIe, ser193 Ala, gly207 Val, the activity of the resulting artificially designed lysyl endonuclease is increased. The researchers of the invention comprehensively consider the structural characteristics and the enzyme catalysis mechanism of the lysyl endonuclease, and mutate one or more of the 30 th, 49 th, 106 th and 155 th lysines into arginine, so that the enzyme activity stability of the obtained artificially designed lysyl endonuclease is further enhanced. The artificially designed lysyl endonuclease with the highest enzyme activity obtained by screening is named as LyC, and the amino acid sequence of the artificially designed lysyl endonuclease is shown as SEQ ID NO. 1.
The sequence of the wild type lysyl endonuclease is as follows:
the artificially designed lysyl endonuclease has 97% homology with the amino acid sequence of Achromobacter lysyl endonuclease (on-line NCBI Blast analysis), and the amino acid sequence comparison is shown in figure 1.
2. Construction of recombinant expression vectors
The pET series vector developed by Novagen company can efficiently drive the expression of a target gene by using a T7 promoter with strong promoter capability, and is one of the most commonly used prokaryotic expression vectors.
The carrier pET28a (+) adopted by the invention uses a T7lac composite promoter, so that the expression of genes can be freely closed and opened, the genes are not basically expressed before induction, the load of host bacteria is greatly reduced, a large amount of target proteins can be rapidly expressed after induction, and the obtained recombinant expression carrier is named pET28a-LyC.
The invention also selects pET9a as an expression vector, which has an independent T7 promoter and can start the expression of a target gene under the condition of no inducer. The zymogen gene of lysyl endonuclease is expressed as zymogen of lysyl endonuclease in host bacteria first, and then becomes mature enzyme with biological activity through a series of transportation, cleavage and modification. Therefore, the excessive expression of the exogenous protein may form inclusion bodies, so that more mature enzyme cannot be processed, and meanwhile, the excessive accumulation of the protease such as lysyl endonuclease in the escherichia coli may seriously influence the normal metabolism of the escherichia coli and inhibit the growth of the thalli. Therefore, the constitutive expression mode can regulate the synthesis of protein and raise the enzyme producing capacity of host bacteria. Similarly, lyC was cloned into vector pET9a using restriction sites NdeI and BamHI, and this was achieved, giving a recombinant expression vector designated pET9a-LyC.
3. Establishment of recombinant lysyl endonuclease
The recombinant expression vector is introduced into an escherichia coli expression host by a calcium chloride method to construct the lysyl endonuclease recombinant strain.
4. Fermentation culture of engineering bacteria
The invention discloses a fermentation medium, which consists of an organic nitrogen source, a carbon source and inorganic salt, and is suitable for expressing genetically engineered bacteria for producing lysyl endonuclease. Experiments prove that the feeding mode adopts index feeding to be suitable, and the specific growth rate mu is controlled to be 0.03-0.15h -1 Between them.
In the invention, the following components are added:
the primers used for point mutation are shown in Table 1:
TABLE 1 primer sequences for point mutations
Fermentation pilot experiment steps:
(1) 20. Mu.L of the recombinant strain was inoculated into 50ml of a resistant liquid medium and cultured in a shaker at 28℃and 250rpm for 16 hours, thereby activating the strain. Then inoculating 50ml of activated strain into 400ml of resistant liquid culture medium, continuously culturing for 3 hours at 28 ℃ and 250rpm to obtain seed culture, and controlling the bacterial concentration OD600 of the seed culture to be between 0.8 and 1.2.
(2) The fermentation medium formulations shown in tables 2 and 3 were fed using a 20L stirred tank fermenter with a feed volume of 8L. Fermentation conditions are strictly controlled: the temperature is controlled between 35 ℃ and 37 ℃, the pH is controlled between 6.5 and 7.0, the fermentation rotating speed is controlled between 150rpm and 700rpm (regulated according to the change of DO), the air flow rate is controlled between 200L/h and 1600L/h (regulated according to the change of DO), and the Dissolved Oxygen (DO) is controlled between 10 and 50 percent of the maximum oxygen saturation. Feeding is started when the carbon source is exhausted (the feeding culture medium contains 500g of glycerol per L and is fixed to 1L by water), feeding is started when the carbon source is exhausted, exponential feeding is adopted, and the specific growth rate mu is controlled to be 0.12h -1 The method comprises the steps of carrying out a first treatment on the surface of the When the culture was carried out to a cell concentration OD 600. Apprxeq.30, the addition of IPTG to a final concentration of 0.3mmol/L was started, and induction was started.
TABLE 2 fermentation Medium formulation table
TABLE 3 microelement recipe
After 12h of fermentation induction, 1ml of fermentation broth is taken, 12000rmp is centrifuged for 1min, and the supernatant is taken, and the lysyl endonuclease enzyme activity in the supernatant of the fermentation broth is detected.
The enzyme activity detection method of the lysyl endonuclease comprises the following steps:
1450. Mu.L of substrate solution (180 mmol/L Tris-HCl (pH 9.2), 0.25mmol/L Bz-Lys-pNA (Na-benzoyl-DL-lysine-p-nitrophenyl, as shown in formula I)) was taken.
Heating in a water bath at 30 ℃ for 5min, adding 50 mu L of fermentation broth supernatant diluted by a proper multiple, reacting at 30 ℃ for 5min, immediately adding 500 mu L of 45% (V/V) acetic acid to terminate the reaction, diluting with purified water for 3 times, and measuring the absorbance of the reaction solution at 405 nm. The enzyme activity is defined as: the amount of enzyme catalyzing the substrate to 1. Mu. Mol of p-nitroaniline per minute at 30℃is defined as 1U.
The composition of the LB liquid medium is as follows: 10g/L peptone, 5g/L yeast powder, 5g/L sodium chloride and pH 7.0-7.5;
the composition of the LB solid culture medium is 15g/L of LB liquid culture medium plus agar powder.
Resistant liquid medium: LB liquid medium containing 50. Mu.g/ml kanamycin.
Resistant solid medium: LB solid medium containing 50. Mu.g/ml kanamycin.
EXAMPLE 1 study of specific Activity of lysyl endonucleases
1. Construction of the mutant vector
1.1 site-directed mutagenesis primers were designed using site-directed mutagenesis techniques as shown in Table 1.
1.2 wild type lysyl endonuclease zymogen is obtained by connecting a preferable signal peptide, a lysyl endonuclease N-terminal leader peptide and a wild type mature peptide in sequence, and by optimizing nucleotide sequence codons of the wild type lysyl endonuclease zymogen, a corresponding nucleotide sequence is obtained according to a codon usage database of escherichia coli, and the optimized nucleotide sequence of the wild type lysyl endonuclease zymogen is shown as SEQ ID NO. 6. To facilitate the ligation of the gene to the vector, a restriction enzyme site NdeI (CATATG) was added to the 5 'end of the nucleotide sequence, a restriction enzyme site BamHI (GGATCC) was added to the 3' end, the sequence was designated as LyC0, and submitted to Nanjing Jinshi Biotechnology Co., ltd for synthesis, and the LyC sequence synthesized was ligated to the vector pMD18T by Nanjing Jinshi Biotechnology Co., ltd, and the recombinant vector obtained was designated as pMD18T-LyC0.
The nucleic acid sequence of the optimized wild-type lysyl endonuclease zymogen:
atgaaaaaaaccgctatcgctatcgctgttgctctggctggtttcgctaccgttgctcaggctgctccggcttctcgtccggctgctttcgactacgctaacctgtcttctgttgacaaagttgctctgcgtaccatgccggctgttgacgttgctaaagctaaagctgaagacctgcagcgtgacaaacgtggtgacatcccgcgtttcgctctggctatcgacgttgacatgaccccgcagaactctggtgcttgggaatacaccgctgacggtcagttcgctgtttggcgtcagcgtgttcgttctgaaaaagctctgtctctgaacttcggtttcaccgactactacatgccggctggtggtcgtctgctggtttacccggctacccaggctccggctggtgaccgtggtctgatctctcagtacgacgcttctaacaacaactctgctcgtcagctgtggaccgctgttgttccgggtgctgaagctgttatcgaagctgttatcccgcgtgacaaagttggtgaattcaaactgcgtctgaccaaagttaaccacgactacgttggtttcggtccgctggctcgtcgtctggctgctgcttctggtgaaaaaggtgtttctggttcttgcaacatcgacgttgtttgcccggaaggtgacggtcgtcgtgacatcatccgtgctgttggtgcttactctaaatctggtaccctggcttgcaccggttctctggttaacaacaccgctaacgaccgtaaaatgtacttcctgaccgctcaccactgcggtatgggtaccgcttctaccgctgcttctatcgttgtttactggaactaccagaactctacctgccgtgctccgaacaccccggcttctggtgctaacggtgacggttctatgtctcagacccagtctggttctaccgttaaagctacctacgctacctctgacttcaccctgctggaactgaacaacgctgctaacccggctttcaacctgttctgggctggttgggaccgtcgtgaccagaactacccgggtgctatcgctatccaccacccgaacgttgctgaaaaacgtatctctaactctacctctccgacctctttcgttgcttggggtggtggtgctggtaccacccacctgaacgttcagtggcagccgtctggtggtgttaccgaaccgggttcttctggttctccgatctactctccggaaaaacgtgttctgggtcagctgcacggtggtccgtcttcttgctctgctaccggtaccaaccgttctgaccagtacggtcgtgttttcacctcttggaccggtggtggtgctgctgcttctcgtctgtctgactggctggacccggcttctaccggtgctcagttcatcgacggtctggactctggtggtggtaccccgtaatga。
double digestion was performed on the recombinant vector pMD18T-LyC using restriction enzymes NdeI and BamHI from TaKaRa, 5. Mu.L of the digested product was analyzed by mass-to-volume ratio 1% agarose gel electrophoresis, after confirming that the digestion was complete, the whole digested product was subjected to mass-to-volume ratio 0.8% agarose gel electrophoresis, and the gel containing 1.4kb DNA fragment was excised, and the DNA fragment in the gel was purified into 30. Mu.L of deionized water using TIANGEN agarose gel recovery kit to obtain a cohesive end gene fragment. Similarly, plasmid pET28a (+) was double digested with NdeI and BamHI, and the digested plasmid fragment was purified into 20. Mu.L deionized water. The gene fragment and the plasmid fragment were ligated overnight at 16℃using the DNA Ligation Kit 2.0 from TaKaRa. 10. Mu.L of overnight ligation product was taken and added to 80. Mu.L of CaCl 2 Coli Top10F' (purchased from the company Invitrogen) prepared by the method (third edition of molecular cloning laboratory Manual published by Cold spring harbor laboratory, U.S.A.) was treated at 42℃for 90s, 300. Mu.L of LB liquid medium preheated at 37℃was rapidly added, and cultured for 45min with shaking (150-200 rpm) at low speed in a shaking table at 37℃and 100. Mu.L of the culture was then coated with the resistant solid medium. The plate is inversely cultured in a 37 ℃ incubator for about 18 hours until single colony is grown, partial single colony is randomly picked for bacterial liquid PCR identification, and the conditions of the PCR reaction are as follows: 94 ℃ for 5min; 30 cycles at 94℃for 30s, 55℃for 30s, 72℃for 30 s; 72 ℃ for 5min; the primer is as follows: t7 upstream primer 5'-TAATACGACTCACTATAGGG-3', T7 downstream primer 5'-GCTAGTTATTGCTCAGCGG-3'. The positive clone obtained by preliminary screening is extracted from the plasmid, and is identified by digestion with NdeI and BamHI, so that a 1.4kb gene fragment and a 5.3kb vector fragment clone are obtained, namely a clone containing LyC0 fragment. Clones identified as containing the LyC0 fragment were sent to Invitrogen for sequencing. The monoclonal which has no base mutation and frame shift is inoculated into 50ml of resistant liquid culture medium, cultured for 18h at 37 ℃ and 250rpm, and the obtained culture is subjected to small-scale extraction and mass test by TIANGEN company plasmidThe kit extracts the plasmid (operating according to the instructions) and the recombinant expression plasmid obtained is named pET28a-LyC0.
1.3 construction of mutant plasmids
(1) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by IIe, the 193 rd Ser is replaced by Ala, and the 207 th Gly is replaced by Val through a primer V167I-F, V167I-R, S193A-F, S A-R, G207V-F, G207V-R, the obtained gene is named LyC1, and the obtained mutant plasmid is named pET28a-LyC1.
(2) The 207 th Gly of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Leu through primers G207L-F and G207L-R, the obtained gene is named LyC2, and the obtained mutant plasmid is named pET28a-LyC2.
(3) The Ser at position 193 of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Gly and the Gly at position 207 is replaced by Leu by primers S193G-F, S193G-R, G L-F and G207L-R, the obtained gene is named LyC3, and the obtained mutant plasmid is named pET28a-LyC3.
(4) The gene obtained by replacing Val at 167 th position with Leu and Gly at 207 th position with Leu of the mature peptide gene of the zymogen of lysyl endonuclease on recombinant plasmid pET28a-LyC0 by primers V167L-F, V167L-R, G L-F and G207L-R was named LyC4, and the obtained mutant plasmid was named pET28a-LyC4.
(5) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by Leu, the 193 rd Ser is replaced by Gly, the 207 th Gly is replaced by Leu by the primer V167L-F, V167L-R, S193G-F, S G-R, G207L-F and G207L-R, the obtained gene is named LyC, and the obtained mutant plasmid is named pET28a-LyC5.
(6) The Ser at position 193 of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Gly through a primer S193G-F, S193G-R, the obtained gene is named LyC6, and the obtained mutant plasmid is named pET28a-LyC6.
(7) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Leu through a primer V167L-F, V167L-R, the obtained gene is named LyC7, and the obtained mutant plasmid is named pET28a-LyC7.
(8) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Leu, the 193 rd Ser is replaced by Gly through a primer V167L-F, V167L-R, S193G-F, S193G-R, the obtained gene is named LyC, and the obtained mutant plasmid is named pET28a-LyC8.
(9) The 193 rd Gly of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Val through a primer G193V-F, G193V-R, the obtained gene is named LyC9, and the obtained mutant plasmid is named pET28a-LyC9.
(10) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by Leu, the 193 rd Ser is replaced by Gly, the 207 th Gly is replaced by Val by the primer V167L-F, V167L-R, S193G-F, S G-R, G207V-F and G207V-R, the obtained gene is named LyC10, and the obtained mutant plasmid is named pET28a-LyC10.
(11) The Ser at position 193 of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Ala through a primer S193A-F, S193A-R, the obtained gene is named LyC11, and the obtained mutant plasmid is named pET28a-LyC11.
(12) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by Leu, the 193 rd Ser is replaced by Ala, the 207 th Gly is replaced by Leu by the primer V167L-F, V167L-R, S193A-F, S A-R, G207L-F and G207L-R, the obtained gene is named LyC12, and the obtained mutant plasmid is named pET28a-LyC12.
(13) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by IIe through a primer V167I-F, V167I-R, the obtained gene is named LyC13, and the obtained mutant plasmid is named pET28a-LyC13.
(14) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by IIe, the 193 rd Ser is replaced by Gly, the 207 th Gly is replaced by Leu through the primers V167I-F, V167I-R, S193G-F, S G-R, G207L-F and G207L-R, the obtained gene is named LyC14, and the obtained mutant plasmid is named pET28a-LyC14.
(15) The Ser at position 193 of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Ala, gly at position 207 is replaced by Val by primers S193A-F, S193A-R, G V-F and G207V-R, the obtained gene is named LyC15, and the obtained mutant plasmid is named pET28a-LyC15.
(16) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by Leu, the 193 rd Ser is replaced by Ala, the 207 th Gly is replaced by Val by the primer V167L-F, V167L-R, S193A-F, S A-R, G207V-F and G207V-R, the obtained gene is named LyC16, and the obtained mutant plasmid is named pET28a-LyC16.
(17) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by IIe, the 207 th Gly is replaced by Val by the primers V167I-F, V167I-R, G V-F and G207V-R, the obtained gene is named LyC17, and the obtained mutant plasmid is named pET28a-LyC17.
(18) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC0 is replaced by IIe, the 193 rd Ser is replaced by Gly, the 207 th Gly is replaced by Val by the primers V167I-F, V167I-R, S193G-F, S G-R, G207V-F and G207V-R, the obtained gene is named LyC18, and the obtained mutant plasmid is named pET28a-LyC18.
(19) The 167 th Val of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by IIe, the 193 rd Ser is replaced by Ala, the obtained gene is named LyC19, and the obtained mutant plasmid is named pET28a-LyC19 through a primer V167I-F, V167I-R, S193A-F, S193A-R.
(20) The gene obtained by substituting the 167 th Val of the mature peptide gene of the zymogen of lysyl endonuclease on the recombinant plasmid pET28a-LyC0 with IIe, the 193 rd Ser with Ala and the 207 th Gly with Leu through the primers V167I-F, V167I-R, S193A-F, S A-R, G207L-F and G20LV-R is named LyC, and the obtained mutant plasmid is named pET28a-LyC20.
2. The obtained mutant plasmid was confirmed by sequencing, and then the above mutant vector was transformed into competent E.coli BL21 (DE 3) according to the calcium chloride method provided in the third edition of molecular cloning laboratory Manual published in Cold spring harbor laboratory, U.S.A., to construct a recombinant strain. Then, a small fermentation experiment was performed, and after 12 hours of induction, the enzyme activity of lysyl endonuclease in the supernatant of the fermentation broth was detected, and the results of the study are shown in Table 4.
TABLE 4 relative enzyme activities of lysyl endonucleases at different mutation sites
| Gene | V167 mutation | S193 mutation | G207 mutation | Relative enzyme activity% |
| LyC0 | - | - | - | 100 |
| LyC1 | V167I | S193A | G207V | 332.2 |
| LyC2 | - | - | G207L | 113.6 |
| LyC3 | - | S193G | G207L | 131.5 |
| LyC4 | V167L | - | G207L | 133.5 |
| LyC5 | V167L | S193G | G207L | 200.3 |
| LyC6 | - | S193G | - | 180.1 |
| LyC7 | V167L | - | - | 210.9 |
| LyC8 | V167L | S193G | - | 226.9 |
| LyC9 | - | - | G207V | 170.8 |
| LyC10 | V167L | S193G | G207V | 223.6 |
| LyC11 | - | S193A | - | 187.0 |
| LyC12 | V167L | S193A | G207L | 219.9 |
| LyC13 | V167I | - | - | 169.1 |
| LyC14 | V167I | S193G | G207L | 214.3 |
| LyC15 | - | S193A | G207V | 137.9 |
| LyC16 | V167L | S193A | G207V | 274.4 |
| LyC17 | V167I | - | G207V | 164.1 |
| LyC18 | V167I | S193G | G207V | 300.7 |
| LyC19 | V167I | S193A | - | 173.1 |
| LyC20 | V167I | S193A | G207L | 306.9 |
As is clear from Table 4, the enzyme activities were improved by substituting the 167 th amino acid Val, the 193 rd Ser and the 207 th Gly of the wild-type lysyl endonuclease with Val, leu, IIe, gly or Ala, respectively, wherein the optimal combinations are Val167 IIe, ser193 Ala and Gly207 Val.
EXAMPLE 2 stability Studies of lysyl endonucleases at different mutation sites
1. Construction of the mutant vector
1.1 site-directed mutagenesis primers were designed using site-directed mutagenesis techniques as shown in Table 1.
1.2 construction of mutants pET28a-LyC1 as template according to the instructions of the TaKaRa point mutation kit:
(1) The 49 th Lys of the mature peptide gene of the zymogen of the lysyl endonuclease on the recombinant plasmid pET28a-LyC is replaced by Arg, the 106 th Lys is replaced by Arg, the 155 th Lys is replaced by Arg, the obtained gene is named LyC21, and the obtained mutant plasmid is named pET28a-LyC21 through primers Arg49-F, arg49-R, arg106-F, arg106-R, arg155-F, arg 155-R.
(2) The Lys at position 30 of the mature peptide gene of the zymogen of lysyl endonuclease on recombinant plasmid pET28a-LyC is replaced by Arg, the Lys at position 155 is replaced by Arg, the obtained gene is named LyC22, and the obtained mutant plasmid is named pET28a-LyC22 by primers Arg30-F, arg-R, arg155-F and Arg 155-R.
(3) The Lys at position 30 of the mature peptide gene of the zymogen of lysyl endonuclease on recombinant plasmid pET28a-LyC1 is replaced by Arg through primers Arg30-F and Arg30-R, the obtained gene is named LyC, and the obtained mutant plasmid is named pET28a-LyC23.
(4) The lysyl endonuclease zymogen mature peptide gene on recombinant plasmid pET28a-LyC1 was replaced by Arg at position 30, arg at position 49, arg at position 106, arg at position 155, and designated by Arg at position 155 by primers Arg30-F, arg, 30-R, arg, F, arg, 49-R, arg, 106-F, arg, R, arg, 155-F, arg, and the resulting gene was designated by LyC, and the resulting mutant plasmid was designated by pET28a-LyC.
2. The resulting mutant plasmid was determined by sequencing, and then the recombinant plasmid constructed as described above was transformed into competent E.coli BL21 (DE 3) according to the calcium chloride method provided in the third edition of molecular cloning laboratory Manual published in Cold spring harbor laboratory, U.S.A., to construct a recombinant strain. And (5) carrying out a fermentation pilot experiment on the obtained recombinant bacteria. After fermentation (induction for 12 hours and fermentation period for 30 hours), 5ml of fermentation broth supernatant was collected and subjected to enzyme activity stability study, the fermentation broth supernatant was placed at room temperature, and samples were taken at 24h intervals to detect enzyme activity, and the study results are shown in Table 5:
TABLE 5 stability studies of lysyl endonucleases at different mutation sites
As can be seen from Table 5, the studies showed that there was no significant change in enzyme activity but a significant difference in enzyme stability after mutation of one or more of K30R, K49R, K106R and K155R. The lysine in the wild type lysyl endonuclease is replaced by arginine, so that the stability of the lysyl endonuclease can be obviously improved, and the lysyl endonuclease has specific lysyl residue cutting activity, so that not only can other proteins be hydrolyzed, but also the lysyl endonuclease can hydrolyze the lysyl endonuclease, and the stability of the lysyl endonuclease gradually decreases along with the increase of mature peptide lysine residues of the lysyl endonuclease.
Example 3
Referring to a recombinant vector construction and competent escherichia coli preparation and transformation method of a third edition of molecular cloning experiment guidelines, a recombinant strain BL21 (DE 3)/pET 28a-LyC is obtained, and lysyl endonuclease gene engineering bacteria BL21 (DE 3)/pET 28a-LyC are subjected to a fermentation small test. The induction time is 12h, and the fermentation period is 30h. After fermentation, the enzyme activity in the supernatant of the fermentation broth reaches 8000U/L.
Electrophoresis analysis of lysyl endonucleases: 5ml of the fermentation broths of 5h before induction, 2h before induction, 0h before induction, 6h after induction and 12h after induction were centrifuged at 12000rmp for 1min, and the supernatants of the fermentation broths were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE, concentration of separating gel of 12% and concentration of concentrating gel of 5%) to give the results shown in FIG. 2. As can be seen, the molecular weight of the lysyl endonuclease of the present invention is in accordance with the theoretical size, about 30kDa; the lysyl endonuclease gene engineering bacteria BL21 (DE 3)/pET 28a-LyC have no expression of target proteins before induction, which shows that the gene engineering bacteria have no background expression, the induction is carried out for 6 hours and 12 hours, the target proteins are detected in the supernatant of the fermentation broth, and the expression of the target proteins is obviously improved along with the extension of the induction time.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. An artificially designed lysyl endonuclease, characterized in that: the artificially designed lysyl endonuclease is optionally selected from the following:
mutation of serine at position 193 in wild type lysyl endonuclease to glycine or alanine;
or, mutating the 207 th glycine in the wild type lysyl endonuclease to leucine or valine;
or, the mutant is obtained by combining the mutation of serine at 193 rd position in wild lysyl endonuclease into glycine and the mutation of glycine at 207 th position into leucine;
or, the mutant is obtained by combining the mutation of serine at 193 rd position to alanine and the mutation of glycine at 207 th position to valine in the wild type lysyl endonuclease;
the amino acid sequence of the wild type lysyl endonuclease is shown as SEQ ID NO. 5.
2. A polynucleotide encoding the artificially designed lysyl endonuclease of claim 1.
3. An artificially designed lysyl endo zymogen, characterized in that: consists of a signal peptide, an N-terminal leader peptide and the artificially designed lysyl endonuclease as defined in claim 1;
the amino acid sequence of the signal peptide is shown as SEQ ID NO. 4;
the amino acid sequence of the N-terminal leader peptide is shown in SEQ ID NO. 8.
4. A polynucleotide encoding the artificially designed lysyl incision zymogen according to claim 3.
5. A recombinant vector for expressing an artificially designed lysyl incision zymogen, characterized in that: a polynucleotide according to claim 4, wherein said polynucleotide is recombinant on an expression vector.
6. The recombinant vector according to claim 5, wherein: the expression vector is pET9a, pET28a-c (+), pET32a-c (+), pET30a-c (+), or pET33b (+).
7. A strain expressing an artificially designed lysyl incision zymogen, characterized in that: transforming the recombinant vector of claim 5 or 6 into a host strain; the host strain is escherichia coli.
8. The fermentation method of artificially designed lysyl incision zymogen expression strain according to claim 7, comprising the steps of: inoculating the strain expressing the artificially designed lysyl incision zymogen in the fermentation medium for fermentation;
the fermentation conditions were as follows: the temperature is controlled between 35 ℃ and 37 ℃, the pH value is controlled between 6.5 and 7.0, the stirring rotating speed is controlled between 150rpm and 700rpm, the air flow rate is controlled between 200L/h and 1600L/h, the dissolved oxygen is controlled at the maximum oxygen saturation of 10 to 50 percent, and the addition amount of the inducer IPTG is calculated to be between 0.1mM and 0.6mM according to the final concentration; when the carbon source is exhausted, feeding is started, and the specific growth rate mu is controlled to be 0.03-0.15h by adopting index feeding -1 Between them;
the composition of the fermentation medium is as follows: each liter contains 2 to 5g of yeast extract, 3 to 8g of peptone, 1 to 2g of sodium chloride, 2 to 5g of monopotassium phosphate, 2 to 5g of disodium hydrogen phosphate, 0.01 to 0.02g of calcium chloride dihydrate, 1 to 2g of magnesium sulfate, 4 to 7g of glycerin, 5 to 7g of ammonium sulfate and 0.875mL of microelements; water is used for constant volume to 1L, and the pH value is 6.5-7.0;
the trace elements comprise the following components: each liter contains 20 to 30g of ferrous chloride tetrahydrate, 1 to 3g of zinc chloride, 2 to 4g of cobalt chloride hexahydrate, 2 to 4g of sodium molybdate dihydrate, 1 to 2g of calcium chloride dihydrate, 1 to 2g of copper chloride dihydrate, 0.4 to 0.6g of boric acid, 2 to 3g of manganese sulfate monohydrate, 100mL of concentrated hydrochloric acid with the concentration of 37 mass percent, and water is used for fixing the volume to 1L;
the composition of the feed is as follows: each liter contains 250-500 g glycerin, and the balance is water.
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