CN104745562A - Preparation method and application of malto oligosaccharyl trehalose synthase mutant - Google Patents

Abstract

The invention discloses the preparation and application of a mutant of maltooligosaccharide-based trehalose synthase, and belongs to the fields of genetic engineering and enzyme engineering. The invention is to replace the amino acid residues related to the hydrophobic region on the surface of Sulfolobus acidocaldarius ATCC 33909 maltooligosaccharide-based trehalose synthetase. The obtained mutants achieved different degrees of increase in the soluble expression of maltooligosaccharide-based trehalose synthase, and the soluble expression of maltooligosaccharide-based trehalose synthase in the mutants L96T, L96H, M106K, M106H, I520H, I520R, and L96T/M106K were respectively Increased by 169.37%, 16.19%, 145.32%, 92.38%, 7.14%, 9.37%, 203.97%. The improvement of the soluble expression of the mutant is beneficial to its industrial application.

Description

Preparation and application of a maltooligosaccharide-based trehalose synthase mutant

technical field

The invention relates to the preparation and application of a mutant of maltooligosaccharide-based trehalose synthase and belongs to the fields of genetic engineering and enzyme engineering.

Background technique

Trehalose (Trehalose) is composed of two glucopyranose linked by 1,1-glycosidic bonds. It is a stable non-reducing disaccharide. It has three optical isomers, namely αα type and αβ type. and ββ-type.

Trehalose is a safe non-reducing disaccharide that widely exists in nature. It has special biological functions such as moisture retention, anti-freeze and anti-drying properties, and thermal-acid stability. It has non-specific protective effects on biological macromolecules , so it has great application potential in medicine, agriculture, cosmetics, food and other industries. Since the 1980s, countries have successively carried out studies on the physiology, biochemistry and molecular biology of trehalose, and the sugar has now become one of the main oligosaccharides recently developed in the world.

Enzymatic conversion to produce trehalose is a method that gradually emerged in the 1990s in the world. There are mainly three methods: phosphorylase method, trehalose synthase method and double enzyme method. At present, starch is used as a substrate to generate trehalose through the joint action of maltooligosaccharide-based trehalose synthase and maltooligosaccharide-based trehalose hydrolase, that is, the dual-enzyme production of trehalose has attracted widespread attention. The conversion rate of trehalose produced by the method is as high as over 80%, which reduces the production cost of trehalose to a certain extent and greatly promotes the industrial production process of trehalose.

Maltooligosaccharide-based trehalose synthase is one of the enzymes for the double-enzyme production of trehalose. After the starch is liquefied at high temperature, pullulanase is added to generate maltodextrin, and maltooligosaccharide-based trehalose synthase acts on the α,α-1,4-glucoside at the reducing end of the substrate to produce α,α-1,4 Intramolecular transglycosidation of -glycosidic bond to α,α-1,1-glycosidic bond forms the intermediate product maltooligosaccharide-based trehalose, and maltooligosaccharide-based trehalose hydrolase specifically cuts the malt in the intermediate product The α,α-1,4-glucosidic bond between the oligosaccharide base and the trehalose makes it decompose to produce trehalose and reduce the new maltooligosaccharide of two glucose units, and reduce the new maltooligosaccharide of two glucose units as the new base The next round of reaction is carried out, and the two enzyme reactions are repeated alternately to convert maltooligosaccharides into products mainly consisting of trehalose and a small amount of glucose, maltose, and maltotriose.

Double-enzyme production of trehalose takes starch as the substrate, and the conversion rate is as high as 80%, which has the advantage of low cost, but the soluble expression of the maltooligosaccharide-based trehalose synthase gene in the host bacteria is very low, resulting in more inclusions Body, low enzyme activity, unfavorable for its industrial application. Therefore, the present invention uses genetic engineering and enzyme engineering means to improve its soluble expression without affecting the enzymatic properties and conversion rate of maltooligosaccharyl trehalose synthetase, thereby increasing the enzyme activity and creating conditions for its industrial production.

Contents of the invention

The present invention provides a mutant of maltooligosaccharide-based trehalose synthase, which is obtained by substituting amino acid residues related to the hydrophobic region of the protein surface of the maltooligosaccharide-based trehalose synthase derived from Sulfolobusacidocaldarius ATCC33909. Compared with the maltooligosaccharide-based trehalose synthase of its parent, the soluble expression of the mutant in the host bacteria is improved to a certain extent, and the enzyme activity of the maltooligosaccharide-based trehalose synthase in the host bacteria is improved.

The amino acid sequence of the maltooligosaccharide-based trehalose synthase derived from Sulfolobus acidocaldarius ATCC33909 is shown in SEQ ID NO.1.

In one embodiment of the present invention, the mutant is mutated from leucine (Leu) at position 96 to threonine (Thr), and the mutant is named L96T.

In one embodiment of the present invention, the mutant is mutated from leucine (Leu) at position 96 to histidine (His), and the mutant is named L96H.

In one embodiment of the present invention, the mutant is mutated from 106th methionine (Met) to lysine (Lys), and the mutant is named M106K.

In one embodiment of the present invention, the mutant is mutated from methionine (Met) at position 106 to histidine (His), and the mutant is named M106H.

In one embodiment of the present invention, the mutant is mutated from proline (Pro) at position 374 to glutamic acid (Glu), and the mutant is named P374E.

In one embodiment of the present invention, the mutant is mutating proline (Pro) at position 374 to histidine (His), and the mutant is named P374H.

In one embodiment of the present invention, the mutant is mutated from isoleucine (Ile) at position 520 to histidine (His), and the mutant is named I520H.

In one embodiment of the present invention, the mutant is mutated from isoleucine (Ile) at position 520 to arginine (Arg), and the mutant is named I520R.

In one embodiment of the present invention, the mutant is to mutate the 96th leucine (Leu) to threonine (Thr), and simultaneously mutate the 106th methionine (Met) to lysine acid (Lys), the mutant was named L96T/M106K.

Another technical problem to be solved by the present invention is to provide a method for preparing a maltooligosaccharide-based trehalose synthase mutant, comprising the following steps:

(1) According to the determined mutation site, design mutation primers for site-directed mutation, and use the vector carrying the maltooligosaccharide-based trehalose synthase gene as a template to perform site-directed mutation; construct a plasmid vector containing the gene encoding the mutant;

(2) transforming the mutant plasmid into the host cell;

(3) Select positive clones for fermentation and culture, collect cells by centrifugation, and the supernatant of broken cell walls is the crude enzyme solution of maltooligosaccharide-based trehalose synthase mutant.

The plasmid vector is any one of pUC series, pET series, or pGEX.

The host cells are bacterial or fungal cells.

The bacteria are Gram-negative bacteria or Gram-positive bacteria.

The invention provides a series of maltooligosaccharide-based trehalose synthase mutants with enhanced soluble expression intensity in host bacteria. Under appropriate culture conditions, the enzyme activity of mutant L96T, L96H, M106K, M106H, I520H, I520R, L96T/M106K maltooligosaccharyl trehalose synthase was 2.69 times, 1.16 times, 2.45 times, 1.92 times that of the wild enzyme, respectively. times, 1.07 times, 1.09 times, 3.04 times.

Description of drawings

Figure 1 Determination of enzyme activity of wild-type maltooligosaccharyl trehalose synthase and mutants (L96T, L96H, M106K, M106H, I520H, I520R, L96T/M106K) in shake flasks

Detailed ways

Example 1 Preparation of wild maltooligosaccharide-based trehalose synthase

(1) Construction of maltooligosaccharide-based trehalose synthase recombinant bacteria

According to the treY amino acid sequence on NCBI (NCBI number: WP_015385613.1), the treY gene sequence on NCBI (NCBI number: NC_020247.1) was codon-optimized, and maltooligosaccharyl trehalose synthase was synthesized by chemical total synthesis The gene sequence treY. The plasmid used to construct the expression vector in E. coli is pET24a(+). The pET24a(+) plasmid and the plasmid with the treY gene were subjected to Nde Ⅰ and Hind Ⅲ double-enzyme digestion respectively. After the digestion products were recovered by gel, they were ligated with T4 ligase overnight, and the ligated products were transformed into Escherichia coli JM109 competent cells, transformed into The product was spread on an LB plate containing 100mg/L kanamycin, and cultured overnight at 37°C. Three single colonies were picked from the plate and inserted into LB liquid medium. After 8 hours, the plasmid was extracted for verification. The result was correct and the enriched Set the treY/pET24a plasmid. Transform the plasmid treY/pET24a into Escherichia coli BL21(DE3) competent cells, pick the transformant and culture it in LB liquid medium (containing 100mg/L kanamycin) at 37°C overnight, save the glycerol tube, and name it treY/pET24a /BL21(DE3).

(2) Expression of maltooligosaccharide-based trehalose synthase

Inoculate treY/pET24a/BL21 (DE3) from glycerol tubes and grow in LB liquid medium (containing 100mg/L kanamycin) for 8h, and insert the seeds into TB liquid fermentation medium (containing 100mg/L kanamycin) according to the inoculum size of 5%. Namycin). After Escherichia coli was cultured at 37°C for 2h, it was induced by adding 0.01mM final concentration of IPTG (isopropylthio-β-D-galactoside), and continued to culture and ferment on a shaker at 25°C for 24h, and a certain volume of fermentation broth Centrifuge at 4°C, 12000r·min ^-1 for 10min to remove the supernatant, collect the bacteria, resuspend the bacteria pellet with 50mmol·L ^-1 pH8.5Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer, and mix well. The cell wall of broken bacterium suspension with ultrasonic cell pulverizer (the working condition of supersonic cell pulverizer: Working probe, working time 5min, working 3s and stopping 3s, working power is 20%), then centrifuged at 12000r·min ⁻¹ for 10min, the supernatant after centrifugation is fermented intracellular crude enzyme liquid.

Example 2 Preparation and expression of maltooligosaccharyl trehalose synthase mutant

(1) Preparation of mutants

Eight mutant enzymes L96T, L96H, M106K, M106H, P374E, P374H, I520H, and I520R derived from Sulfolobus acidocaldarius ATCC33909 maltooligosaccharide-based trehalose synthase: according to the gene sequence of Sulfolobus acidocaldariusATCC33909 maltooligosaccharide-based trehalose synthase , respectively designed and synthesized primers introducing L96T, L96H, M106K, M106H, P374E, P374H, I520H, and I520R mutations, carried out site-directed mutation on the maltooligosaccharide-based trehalose synthase gene, determined the DNA coding sequence, and identified the 96th position respectively The Leu codon at position 1 becomes Thr codon and His codon, the Met codon at position 106 becomes Lys codon and His codon, the Pro codon at position 374 becomes Glu codon and His codon, and the 520 position The Ile codon at the position becomes a His codon and an Arg codon. The mutant gene is placed in an appropriate expression vector and introduced into Escherichia coli for expression to obtain a single mutant maltooligosaccharide-based trehalose synthetase. Site-directed mutagenesis of single mutations L96T, L96H, M106K, M106H, P374E, P374H, I520H, and I520R: Using rapid PCR technology, the expression vector treY/pET24a(+) was used as a template.

The site-directed mutagenesis primers for introducing the L96T mutation are:

Forward primer: 5'-GGTTAACAGC ACG AATTGGCGC-3' (the underline is the mutated base)

Reverse primer: 5'-GCGCCAATT CGT GCTGTTAACC-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the L96H mutation are:

Forward primer: 5'-GGTTAACAGC CAC AATTGGCGC-3' (underline is the mutated base)

Reverse primer: 5'-GCGCCAATT GTG GCTGTTAACC-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the M106K mutation are:

Forward primer: 5'-ATGTTCTGAAG AAG GGTAAGAA-3' (underlined for mutant bases)

Reverse primer: 5'-TTCTTACC CTT CTTCAGAACAT-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the M106H mutation are:

Forward primer: 5'-ATGTTCTGAAG CAC GGTAAGAA-3' (the underline is the mutated base)

Reverse primer: 5'-TTCTTACC GTG CTTCAGAACAT-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the P374E mutation are:

Forward primer: 5'-CCAAACGTAAT GAG GAAGCGTA-3' (the underline is the mutated base)

Reverse primer: 5'-TACGCTTC CTC ATTACGTTTGG-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the P374H mutation are:

Forward primer: 5'-CCAAACGTAAT CAC GAAGCGTA-3' (the underline is the mutated base)

Reverse primer: 5'-TACGCTTC GTG ATTACGTTTGG-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the I520H mutation are:

Forward primer: 5'-TGAAGCGAAA CAC AATACGAGC-3' (the underline is the mutated base)

Reverse primer: 5'-GCTCGTATTCTTT GAG GCTTCA-3' (the underline is the mutated base)

The site-directed mutagenesis primers for introducing the I520R mutation are:

Forward primer: 5'-TGAAGCGAAA CGG AATACGAGC-3' (the underline is the mutated base)

Reverse primer: 5'-GCTCGTATT CCG TTTCGCTTCA-3' (the underline is the mutated base)

The PCR reaction system is: 5×PS buffer 10 μL, dNTPs Mix (2.5mM) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, PrimerStar HS (5U/μL) 0.5 μL, add double Distill water to 50 μL.

The PCR amplification conditions were: pre-denaturation at 94°C for 4min; followed by 30 cycles (98°C for 10s, 55°C for 5s, 72°C for 7min30s); 72°C for 10min.

The PCR product was digested with Dpn Ⅰ, transformed into competent Escherichia coli JM109, and the competent cells were cultured in LB solid medium (containing 100mg/L ampicillin) overnight, and cloned in LB liquid medium (containing 100mg/L ampicillin) After culturing, the plasmids were extracted, and the mutant plasmids were transformed into competent cells of the expression host Escherichia coli BL21 (DE3), and all the mutant plasmids were sequenced correctly.

(2) Expression of mutant enzymes

The expression process of the mutants was as described in Example 1.

Example 3 Enzyme activity analysis of maltooligosaccharide-based trehalose synthase

(1) Definition of enzyme activity unit

When using 3,5-dinitrosalicylic acid method (DNS method) to measure the enzyme activity of maltooligosaccharide-based trehalose synthase, the amount of enzyme needed to convert 1 μmol of maltopentaose into maltotriosyl trehalose per minute is taken as a vitality unit.

(2) Enzyme Activity Determination Steps

Preheating: Take 0.5mL of 1% maltopentose solution (50mM pH5.5 phosphate buffer) in a test tube and place it in a 60°C water bath to preheat for 10min.

Reaction: Add 0.05mL fermented intracellular crude enzyme solution, oscillate evenly, accurately time 10min, boil in a boiling water bath for 10min to terminate the reaction, and cool down. Add 1mL DNS and shake evenly, boil in a boiling water bath for 7min, and cool.

Measurement: add distilled water to the above reaction system and adjust the volume to 10mL, and mix well. The absorbance was measured at a wavelength of 540nm and the enzyme activity was calculated.

The 24h OD _600nm and enzyme activities of wild-type maltooligosaccharyl trehalose synthase and mutant enzymes in shake flask culture are listed in Table 1. Among them, L96T and M106K have the most significant effects, and their enzyme activities are 2.69 times and 2.45 times that of wild enzymes, respectively. In addition, the results of enzymatic qualitative experiments showed that the enzymatic properties of each mutant were similar to wild enzymes.

Table 1 Shake flask OD _600nm and enzyme activity of wild-type maltooligosaccharide-based trehalose synthase and mutant enzyme

Example 4 Preparation of double mutants of maltooligosaccharyl trehalose synthase

From Example 3, it was found that the soluble expression of mutant L96T and mutant M106K was significantly increased in many mutants, so the Leu96 site and Met106 site of the maltooligosaccharide-based trehalose synthase were double-mutated, and the mutant L96T/ M106K.

Site-directed mutation of the double mutation L96T/M106K: using the rapid PCR technique and using the expression vector L96T/pET24a(+) as a template.

The site-directed mutagenesis primers for introducing the M106K mutation are:

Forward primer: 5'-ATGTTCTGAAG AAG GGTAAGAA-3' (underlined for mutant bases)

Reverse primer: 5'-TTCTTACC CTT CTTCAGAACAT-3' (the underline is the mutated base)

The PCR reaction system, reaction conditions and the sequencing method of the mutant gene are the same as those for the single mutant.

Example 5 Enzyme activity analysis of maltooligosaccharyl trehalose synthase double mutant L96T/M106K

The enzyme activity assay method is as described in Example 3.

The 24h OD _600nm and enzyme activities of wild-type maltooligosaccharyl trehalose synthase and double mutant enzymes in shake flask culture are listed in Table 2. The results of enzymatic qualitative experiments show that the enzymatic properties of double mutants are similar to wild enzymes.

Table 2 Shake flask OD _600nm and enzyme activity of wild-type maltooligosaccharide-based trehalose synthase and double mutant enzyme

The enzyme activities of mutants L96T, L96H, M106K, M106H, I520H, I520R, and L96T/M106K maltooligosaccharide-based trehalose synthase were 2.69 times, 1.16 times, 2.45 times, 1.92 times, 1.07 times, 1.09 times that of the wild enzyme, respectively , 3.04 times.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. a malt oligosaccharide based mycose synthetase mutant, is characterized in that, obtains after being replaced by the protein surface hydrophobic region relevant amino acid residue of malt oligosaccharide based mycose synthetase; The aminoacid sequence of described malt oligosaccharide based mycose synthetase is as shown in SEQ ID NO.1; Described replacement is that the 96th leucine is sported Threonine or Histidine, or the 106th methionine(Met) is sported Methionin or Histidine, or the 374th proline(Pro) is sported L-glutamic acid or Histidine, or be Histidine or arginine by the 520th isoleucine mutation, or while the 96th leucine is sported Threonine, the 106th methionine(Met) is sported Methionin.

2. mutant according to claim 1, is characterized in that, described mutant is that the 96th leucine is sported Threonine.

3. mutant according to claim 1, is characterized in that, described mutant is that the 96th leucine is sported Histidine.

4. mutant according to claim 1, is characterized in that, described mutant is that the 106th methionine(Met) is sported Methionin.

5. mutant according to claim 1, is characterized in that, described mutant is that the 106th methionine(Met) is sported Histidine.

6. mutant according to claim 1, is characterized in that, described mutant is that the 96th leucine is sported Threonine, the 106th methionine(Met) is sported Methionin simultaneously.

7. prepare a method for the arbitrary described malt oligosaccharide based mycose synthetase mutant of claim 1-6, it is characterized in that, comprise the steps:

(1) according to the mutational site determined, the mutant primer of design rite-directed mutagenesis, with the carrier carrying malt oligosaccharide based mycose synthetase gene for template carries out rite-directed mutagenesis; Build the plasmid vector of the gene containing encode mutant;

(2) mutant plasmid is transformed into host cell;

(3) select positive colony and carry out fermentation culture, centrifugal collecting cell, cell wall breaking supernatant is the crude enzyme liquid of malt oligosaccharide based mycose synthetase mutant.

8. the gene of the arbitrary described mutant of coding claim 1-6.

9. carry carrier or the reconstitution cell of gene described in claim 8.

10. the arbitrary described mutant of claim 1-6 is preparing the application in trehalose.