Galactosidase mutant and application thereof in production of human milk oligosaccharide

Abstract

The invention relates to the technical field of gene engineering and enzyme engineering, in particular to a galactosidase mutant and application thereof in production of human milk oligosaccharide. According to the galactosidase mutant, threonine at the 192nd position of galactosidase which is derived from pseudoclostridium thermosuccinate and has an amino acid sequence shown as SEQ ID NO: 1 is changed into tryptophan, or aspartic acid at the 193rd position of the galactosidase is changed into proline, or methionine at the 194th position of the galactosidase is changed into alanine, or methionine at the 195th position of the galactosidase is changed into threonine, or aspartic acid at the 193rd position of the galactosidase is changed into proline. Or the aspartic acid at the 237 position is changed into asparagine. The galactosidase mutant obtained by the invention not only has efficient enzymatic activity, but also can maintain the enzymatic activity at a relatively high temperature, and is suitable for catalyzing the synthesis of human milk oligosaccharide.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering and enzyme engineering, in particular to a galactosidase mutant and application thereof in the production of human milk oligosaccharides. Background Art

[0002] Human milk oligosaccharides (HMOs) are increasingly important food additives. European and American countries and China have begun to review and approve the listing of related human milk oligosaccharide products. Among them, galactosidase with glycosyl transfer function is one of the key enzymes in the biosynthesis of human milk oligosaccharides. It can catalyze the synthesis of lacto-N-triose and its derivatives and the relatively cheap raw material nitro-o-nitrophenyl-β-pyranogalactoside (oNPG) from lacto-N-tetraose and more complex human milk oligosaccharides. The temperature is generally high in industrial production, and enzymes with excellent heat resistance are required.

[0003] Extracting human milk oligosaccharides directly from human milk is costly and the source is insufficient, making it difficult to produce on a large scale. Since chemical synthesis requires the addition of multiple chemical reagents, it is easy to cause food contamination and unpredictable toxic side effects. Therefore, the use of synthetic biology technology for the biosynthesis of human milk oligosaccharides is the main production method internationally. Since the temperature is generally high in industrial production, enzymes with excellent heat resistance are required, but there is a lack of enzymes with both high catalytic activity and high thermal stability on the market.

[0004] To this end, in response to the problems raised in the above background technology, those skilled in the art have developed an enzyme for the production of human milk oligosaccharides that has both high catalytic activity and high thermal stability. Summary of the invention

[0005] The object of the present invention is to provide a galactosidase mutant and its application in the production of human milk oligosaccharides to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A β-galactosidase mutant, the amino acid sequence of the β-galactosidase mutant is shown in SEQ ID NO: 1;

[0008] The galactosidase mutant is a galactosidase derived from Pseudomonas thermophila succinicus with an amino acid sequence as shown in SEQ ID NO: 3, wherein the threonine at position 192 is changed to tryptophan, and / or the aspartic acid at position 193 is changed to proline, and / or the methionine at position 194 is changed to alanine, and / or the methionine at position 195 is changed to threonine, and / or the aspartic acid at position 237 is changed to asparagine.

[0009] The DNA sequence encoding the β-galactosidase derived from Pseudoclostridium thermosuccinicum is shown in SEQ ID NO:2.

[0010] Application of the above-mentioned galactosidase mutant in the production of human milk oligosaccharides.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The enzyme activity of the galactosidase mutant obtained by the present invention before heat treatment is not much different from that of the thermogenic succinic pseudoclostridium galactosidase, which proves that the mutant has efficient enzyme activity under normal circumstances. At the same time, the galactosidase mutant is kept at 55°C, 60°C and 65°C for 24 hours, 12 hours and 4 hours, and the activity can be retained by 70%, 61% and 52% respectively, which proves that the mutant can maintain enzyme activity for a long time at a higher temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The test diagram of the enzyme activity of the galactosidase mutant and the galactosidase of thermogenic Clostridium succinicum in the present invention at different temperatures (the figure shows the initial enzyme activity of the galactosidase mutant and the galactosidase of thermogenic Clostridium succinicum, and the enzyme activity after incubation at 55°C for 24 hours, at 60°C for 12 hours, and at 65°C for 4 hours, respectively). DETAILED DESCRIPTION

[0014] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] See also Figure 1 , the present invention provides:

[0016] A beta-galactosidase mutant, whose amino acid sequence SEQ ID NO:1 is as follows:

[0017] MSKFEISGTDFLYDGKPVKILSGAIHYFRIVPEYWKDRLLKLKACGFNTVETYIAWNMHEPREGEFCFEGICDIERFVRTAGELGLFVIVRPSPYICAEWEFGGLPAWLLSYPDMKLRCNYKPYLDKVDAYYDVLIPKLLPYLCTNGGPIIAMQIENEYGSYGADKEYLQHIKEALKKRGVDVLLFTSDGPWPATLQGGTLPDV FKTVNFGSRHEEAFAKLKEYQNDKPLMCTEFWDGWFDHWGEEHHTRGHEDTVREFDGMLKYGASVNFYMFHGGTNFGFMNGANHSDSYEPTVTSYDYDAPLNESGDPTPKYYAVRDVLSKYVEIDHSIKVEPSLKKSYGEARMTQQADLFDSLDKLSEPMERVCPEPMEKLGQNYGFILYRTQLKGYK GHLPLEIKDVHDRALIFLNGKYVDVIYRNDKDKRVFLDLSGGSSTLDILVENMGRVNYGPFLKDHKGITEHVRLDYHFLFGWTIYPLPMDDLSKLCFDDAKVPRTTSFYRGTFNVDEMADTFLSLDGWVKGAVWINGFNIGRYWNVGPQKTLYVPAPLLKKSENEIIIFEVHEPQNLTVKFTDKPDLG.

[0018] Example 1. Construction of β-galactosidase mutants

[0019] (1) Construction of expression vector for β-galactosidase mutant

[0020] The galactosidase mutant of the present invention synthesizes the gene of the galactosidase derived from Pseudoclostridium thermosuccinogenes and is constructed into a pET22b vector using Ned1 and Xho1 as restriction sites. The galactosidase gene is fused with the XhoI restriction site and the His tag on the vector for expression. The vector expresses a fusion protein, which is composed of galactosidase, Leu, Glu and 6 His from the N-terminus.

[0021] The DNA sequence of the β-galactosidase of the thermogenic Pseudoclostridium succinate described above is as follows:

[0022]

[0023] (2) Construction of β-galactosidase mutant

[0024] The amino acid sequence of the β-galactosidase of Pseudomonas thermophila succinate is as follows:

[0025] ;

[0026] Through bioinformatics cluster analysis of existing gene libraries and structural biology research on enzyme and substrate recognition mechanisms, combined with artificial intelligence design technology, we screened out the galactosidase expressed by the galactosidase gene from Pseudoclostridium thermosuccinicum, and on this basis, we designed mutations at position 192 (threonine to tryptophan), position 193 (aspartic acid to proline), position 194 (methionine to alanine), position 195 (methionine to alanine) and position 237 (aspartic acid to asparagine). The above mutations are referred to as T192W, D193P, M194A, M195T, and N237D mutations, respectively.

[0027] The T192W mutation corresponds to a codon change in the gene sequence of the galactosidase of Pseudomonas thermosuccinicus from ACC to TGG;

[0028] The codon change corresponding to the D193P mutation in the gene sequence of the galactosidase of Pseudomonas thermosuccinicus is GAT—CCA / CCT / CCC / CC;

[0029] The codon change corresponding to the M194A mutation in the gene sequence of the galactosidase of Pseudomonas thermosuccinicus is ATG-CCA / GCT / GCC / GCG;

[0030] The codon change corresponding to the M195T mutation in the gene sequence of the galactosidase of Pseudomonas thermosuccinicus is ATG-ACA / ACT / ACC / ACG;

[0031] The codon change corresponding to the N237D mutation in the gene sequence of the galactosidase of Pseudomonas thermosuccinicus is AAC-GAT / GAC;

[0032] The galactosidase gene sequence of Pseudoclostridium thermosuccinate was used as a template to amplify the gene by PCR reaction. The reaction system is as follows:

[0033]

[0034] The PCR program is as follows:

[0035]

[0036] Add 1 μl of DpnI enzyme to 50 μl of PCR product, digest at 37°C for 3 hours, take 10 μl of the digested mixture and directly transform it into DH5α competent cells, spread it on LB plates containing 100 μg / ml ampicillin, invert it in a 37°C incubator and culture it for 12 hours, pick a single clone from the plate, inoculate it in 5 ml of LB medium containing 100 μg / ml ampicillin, culture it at 37°C for more than 8 hours, extract the plasmid, and transform the plasmid with successful mutation confirmed by gene sequencing into the Escherichia coli expression strain BL21 (DE3);

[0037] First, a single mutation is performed, and then mutation of another site is superimposed on the basis of the single mutant until all five sites are mutated. The mutation method is the same as the above method.

[0038] Example 2. Expression and purification of galactosidase mutants

[0039] The β-galactosidase mutant plasmid was transformed into the Escherichia coli expression strain BL21 (DE3), and an LB plate containing 100 μg / ml ampicillin was coated and inverted at 37°C for 12 hours. A single clone was picked and inoculated into 100 ml LB (100 μg / ml ampicillin) medium, and cultured at 37°C overnight. Then, the overnight cultured bacterial solution was inoculated into a large bottle containing 800 ml LB (100 μg / ml ampicillin) at a ratio of 1:100, and cultured at 37°C and 200 rpm for about 3 hours until the OD600 reached 0.6-0.8, and an inducer IPTG was added to a final concentration of 1 mM, induced at 37°C for 4 hours, and centrifuged at 4000 rpm for 30 minutes to collect the bacteria;

[0040] The collected bacteria were ultrasonically disrupted (power 200 W, 3s on, 8s off, 100 times), the buffer was 20mM Tris-HCl solution containing 200mM NaCl, pH8.0; the ultrasonic disruption product was centrifuged at 16000rpm, 4 degrees for 50min, the supernatant was purified by affinity chromatography using nickel gel, the supernatant after centrifugation of the disruption solution was combined with Ni-NTA beads at 4°C for 1 hour, eluted with different concentrations of imidazole elution solution, and a relatively pure target protein was obtained, and the protein was stored in 20mM phosphate buffer (pH 7.0) for subsequent activity measurement experiments.

[0041] Example 3: Enzyme activity and thermal stability detection of galactosidase mutants

[0042] For enzyme activity detection, prepare the activity detection reaction system according to the following concentrations:

[0043] Prepare an activity test reaction solution containing 5mM oNPG (o-nitrophenyl-β-D-galactopyranoside) and 50mM citrate buffer (pH 5.0), dilute the β-galactosidase to an appropriate concentration (e.g. 0.1mg / mL), take 225μL of the activity test reaction solution, add 25μL of the diluted β-galactosidase solution, mix well and let it react at 55°C for 10 minutes, add 750μL of 2M sodium carbonate to the system to terminate the reaction, and the oNP (o-nitrobenzene) generated by the reaction is detected by measuring the absorbance of the reaction solution at 410nm using a spectrophotometer. Unit enzyme activity is defined as the amount of enzyme required to catalyze the production of 1μmol of oNP within 1 minute under the above conditions;

[0044] The method for detecting the thermal stability of the enzyme is to take a certain amount of purified enzyme and keep it at 55°C, 60°C and 65°C for 24 hours, 12 hours and 4 hours respectively, then dilute the heat-treated enzyme to an appropriate concentration range and perform enzyme activity detection according to the above method.

[0045] The enzyme activity was calculated as follows:

[0046]

[0047] Vt: Total volume of reaction solution 1000 μl

[0048] ε: The extinction of oNP at 420nm is 4500M-1cm-1

[0049] Ve: Enzyme solution volume 25μl

[0050] d: Colorimetric cup light diameter (1cm)

[0051] C: Enzyme concentration (mg / mL)

[0052] The specific test results are shown in Table 1 below:

[0053]

[0054] The enzyme activity of the galactosidase mutant obtained by the present invention before heat treatment is not much different from that of the thermogenic Pseudoclostridium succinate galactosidase, which are 18.0 U / mg and 17.6 U / mg respectively, which are comparable to the reported galactosidase with similar functions; however, the thermal stability of the mutant enzyme has been significantly improved. The activity of the galactosidase of the thermogenic Pseudoclostridium succinate is lost by more than half after 24 hours at 55°C, while the activity of the galactosidase mutant can be retained by 70%, 61% and 52% respectively after being kept at 55°C, 60°C and 65°C for 24 hours, 12 hours and 4 hours, as shown in the following figure. Figure 1 shown.

[0055] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A β-galactosidase mutant, characterized in that: The amino acid sequence of the β-galactosidase mutant is shown in SEQ ID NO: 1; The β-galactosidase mutant is a β-galactosidase derived from Pseudoclostridium thermosuccinate whose amino acid sequence is shown in SEQ ID NO: 3, in which the threonine at position 192 is changed to tryptophan; and / or, changing the aspartic acid at position 193 to proline; and / or, changing the methionine at position 194 to alanine; and / or, changing the methionine at position 195 to threonine; and / or, converting the aspartic acid at position 237 to asparagine.

2. Use of the β-galactosidase mutant as claimed in claim 1 in the production of human milk oligosaccharides.

Images