CN105969713B

CN105969713B - Genetic engineering bacterium for high yield of maltooligosyl trehalose hydrolase and application thereof

Info

Publication number: CN105969713B
Application number: CN201610343218.6A
Authority: CN
Inventors: 吴敬; 宿玲恰; 吴世雄
Original assignee: Jiangnan University
Current assignee: LIYANG WEIXIN BIOLOGICAL SCIENCE & TECHNOLOGY Co.,Ltd.
Priority date: 2016-05-23
Filing date: 2016-05-23
Publication date: 2020-01-17
Anticipated expiration: 2036-05-23
Also published as: CN105969713A

Abstract

The invention discloses a genetic engineering bacterium for high yield of maltooligosyl trehalose hydrolase and application thereof, belonging to the technical field of genetic engineering and fermentation engineering. The invention takes E.coli Origami (DE3) as a host, and constructs a genetically engineered bacterium pET-32a (+) -treZ/E.coli Origami (DE3) for high-yield malto-oligosyl trehalose hydrolase. The strain is used as a production strain to produce enzyme in a fermentation tank by fermentation, and the enzyme activity can reach 204.0 U.mL^‑1About 4.9 times of that of shake flask fermentation. The invention reduces the production cost of the maltooligosyl trehalose hydrolase and lays a foundation for realizing the industrial production of trehalose.

Description

Genetic engineering bacterium for high yield of maltooligosyl trehalose hydrolase and application thereof

Technical Field

The invention relates to a genetic engineering bacterium for high yield of maltooligosyl trehalose hydrolase and application thereof, belonging to the technical field of genetic engineering and fermentation engineering.

Background

Trehalose (Trehalose) is a stable, non-reducing disaccharide with 3 optical isomers, namely, the α α, α β and β β forms, formed by linking two glucopyranose rings by 1, 1-glycosidic bonds.

Trehalose is a safe non-reducing disaccharide, widely exists in nature, has special biological functions of moisture retention, freezing resistance, drying resistance, thermal acid stability and the like, has a nonspecific protection effect on biological macromolecules, and has great application potential in the industries of medicine, agriculture, cosmetics, food and the like. Since the 80's of the 20 th century, studies on the physiological, biochemical and molecular biology of trehalose, which has now become one of the most recently developed oligosaccharides internationally, have been carried out in successive countries.

The production of trehalose by enzymatic conversion is a method which is gradually rising in the last 90 years, and the three methods mainly comprise a phosphorylase method, a trehalose synthase method and a double-enzyme method. Currently, trehalose is generated by taking starch as a substrate through the combined action of maltooligosyl trehalose synthetase and maltooligosyl trehalose hydrolase, namely trehalose is produced by a double-enzyme method, and the method has attracted extensive attention. The conversion rate of the trehalose produced by the method is up to more than 80 percent, the production cost of the trehalose is reduced to a certain extent, and the industrial production process of the trehalose is greatly promoted.

The maltooligosyl trehalose hydrolase (EC3.2.1.141) is encoded by the gene treZ and is one of the enzymes for the two-enzyme process of trehalose production. Starch is liquefied at high temperature and then added with pullulanase to generate maltodextrin, and the maltooligosyl trehalose synthase acts on alpha, alpha-1, 4-glucoside at the reducing end of a substrate to generate intramolecular transglycosylation of the alpha, alpha-1, 4-glucoside bond to the alpha, alpha-1, 1-glucoside bond, so as to form an intermediate product of maltooligosyl trehalose. The maltooligosyl trehalose hydrolase specifically cuts the alpha, alpha-1, 4-glycosidic bond connecting the maltooligosyl group and the trehalose in the intermediate product, so as to decompose the maltooligosyl trehalose to generate the trehalose and reduce the new maltooligose with two glucose units, and the new maltooligose with two glucose units is used as a new substrate to carry out the next reaction, and the two reactions are repeatedly and alternately carried out, so that the two enzyme reactions can convert the maltooligose into the trehalose as the main product and the products with a small amount of glucose, maltose and maltotriose.

The trehalose produced by the double-enzyme method takes starch as a substrate, the conversion rate is up to more than 80%, and the method has the advantage of low cost, but the maltooligosyl trehalose hydrolase gene has low soluble expression in host bacteria, generates more inclusion bodies, has low enzyme activity, and is not beneficial to industrial application.

Disclosure of Invention

The first purpose of the invention is to provide a genetic engineering bacterium for high yield of maltooligosaccharyl trehalose hydrolase, which is used for expressing the maltooligosaccharyl trehalose hydrolase from sulfobusacidocalamus ATCC 33909 by using a pET-32a (+) vector and E.coli Origami (DE3) as an expression host.

The nucleotide sequence of the maltooligosyl trehalose hydrolase derived from Sulfolobus acidocaldarius ATCC 33909 is shown as SEQ ID NO. 1.

The second purpose of the invention is to provide a construction method of the genetic engineering bacteria, which comprises the following steps: connecting the maltooligosyl trehalose hydrolase gene with the nucleotide sequence of SEQ ID NO.1 to an expression vector pET-32a (+), then transforming the gene into Escherichia coli E.coli Origami (DE3), and screening a correct transformant to obtain the genetically engineered bacterium pET-32a (+) -treZ/E.coli Origami (DE 3).

The third purpose of the invention is to provide a method for producing the maltooligosyl trehalose hydrolase by fermenting the genetically engineered bacteria.

The method comprises the following steps: (1) a batch fermentation stage: inoculating the seed liquid into a fermentation tank by 8-10% of inoculation amount, controlling the temperature to be 36-38 ℃, the initial rotation speed to be 180-200rpm, the initial ventilation volume to be 7L/min, the dissolved oxygen to be 28-32% and the pH value to be 6.8-7.2; (2) and (3) a feeding fermentation stage: when the dissolved oxygen is increased to 80-100%, the specific growth rate mu is controlled to be 0.2h^-1The fed-batch culture is carried out in an exponential fed-batch mode, the temperature is controlled to be 36-38 ℃, the dissolved oxygen is 28-32 percent, and the pH value is 6.8-7.2; (3) and (3) an induction culture stage: when the cell concentration of the bacteria reaches OD₆₀₀At a flow rate of 30-50, at a flow rate of 0.1-0.8 g.L^-1·h^-1Adding lactose to induce enzyme production, controlling temperature at 25-35 deg.C, dissolved oxygen at 28-32% and pH at 6.8-7.2, and inducing for 0-27 h.

In one embodiment of the present invention, the induction culture stage is performed when the cell concentration of the bacterial cells reaches OD₆₀₀At 40, the enzyme production was induced by the initial feeding of lactose.

In one embodiment of the invention, the induction culture phase is at a flow rate of 0.2 g.L^-1·h^-1And (3) feeding lactose to induce enzyme production.

In one embodiment of the invention, the induction culture stage is controlled at 30 ℃.

The time for inducing enzyme production in the present invention is not particularly limited, and is measured from the first time of adding the inducer.

The invention has the beneficial effects that:

(1) the invention constructs genetically engineered bacterium pET-32a (+) -treZ/E.coli Origami (DE3) by fusion expression of maltooligosyl trehalose hydrolase from Sulfolobus acidocaldarius ATCC 33909 and Thioredoxin (Trx)). Compared with a control pET-24a (+) -treZ/E.coli BL21(DE3), the soluble expression of the fusion protein in host bacteria is improved to a certain degree, and the shake flask fermentation enzyme activity of the fusion protein Trx-MTHase is 42.0 U.mL^-1It is 4.0 times of the control (MTHase 10.4U. mL)^-1). After the N-terminal Trx protein was cleaved with Enterokinase (Enterokinase), the MTHase enzyme activity was 1.31 times that before cleavage, which was 5.2 times that of the control.

(2) The genetically engineered bacterium pET-32a (+) -treZ/E. coli Origami (DE3) constructed by the invention has the property of high yield of maltooligosyl trehalose hydrolase, is used for fermentation tank production, and has the highest enzyme activity of 204.0 U.mL^-1The method can solve the problems of low enzyme activity and high production cost in the existing fermentation process, improve the soluble expression of the maltooligosyl trehalose hydrolase on the basis of not influencing the enzymological properties and the conversion rate of the maltooligosyl trehalose hydrolase as much as possible, and create conditions for the industrial production of the maltooligosyl trehalose hydrolase.

Drawings

FIG. 1 and 5 SDS-PAGE (shaking flask) electrophoresis charts of fermentation wall-breaking supernatant and wall-breaking precipitate of OD recombinant bacteria.

M is molecular weight standard protein; 1, E.coli BL21(DE3)/pET-24a (+) -treZ cell wall breaking supernatant; coli Origami (DE3)/pET-32a (+) -treZ cell wall breaking supernatant; e.coli BL21(DE3)/pET-24a (+) -treZ cell wall breaking sediment; coli Origami (DE3)/pET-32a (+) -treZ cell wall breaking pellet.

FIG. 2 shows fermentation enzyme production curves of recombinant bacteria in 3.6L fermentation tank at different induction times.

FIG. 3 shows fermentation enzyme production curves of recombinant bacteria in 3.6L fermentation tank at different induction temperatures.

FIG. 4 shows fermentation enzyme production curves of 3.6L fermentation tanks for recombinant bacteria at different lactose feeding speeds.

FIG. 5 shows SDS-PAGE electrophoresis of the 3L tank fermentation wall-broken supernatant of the recombinant bacteria, and the underlined part is the target protein.

M is molecular weight standard protein; 1, inducing the cell wall breaking supernatant for 0 h; inducing the cell wall breaking supernatant for 3-27h at 2-10.

Detailed Description

Culture medium:

(1) the composition of the LB solid medium is: 9-11g/L of molecular peptone, 4-6g/L of molecular yeast powder, 9-11g/L of NaCl and 1.5-2% of agar powder.

(2) The composition of LB liquid medium is: 9-11g/L of molecular peptone, 4-6g/L of molecular yeast powder, 9-11g/L of NaCl and 6.8-7.2 of pH.

(3) The TB fermentation medium comprises the following components: 4-6g/L of glycerol, 23-25g/L of industrial yeast powder, 11-13g/L of industrial peptone and KH₂PO₄2.2-2.4g/L，K₂HPO₄16-17g/L。

(4) Seed culture medium: (industrial grade peptone 10g/L, industrial grade yeast powder 5g/L, NaCL 10g/L, 100mg/L kanamycin, pH 7.00)

(5) The fermentation medium is as follows: glycerol 8g/L, peptone 12g/L, yeast powder 24g/L, (NH)₄)₂HPO₄4.0g/L，KH₂PO₄13.5g/L, citric acid 1.7g/L, MgSO₄·7H₂O1.4 g/L and trace element liquid 10 mL/L. Kanamycin (100. mu.g/mL) may also be added.

(6) The microelement liquid comprises: FeSO₄·7H₂O 10.0g/L，ZnSO₄·7H₂O 5.3g/L，CuSO₄·5H₂O3.0g/L，MnSO₄·4H₂O 0.5g/L，Na₂B₄O₇·10H₂O 0.23g/L，CaCl₂2.0g/L，(NH₄)₆Mo₇O₂₄0.1g/L。

(7) The supplementary culture medium comprises: peptone 12g/L, yeast powder 24g/L, MgSO₄·7H₂O15 g/L and glycerol 300 g/L.

Example 1: construction of pET-32a (+) -treZ/E.coli Origami (DE3)

The gene treZ (nucleotide sequence is SEQ ID NO.1) of maltooligosyl trehalose hydrolase is amplified by PCR, and the obtained treZ gene fragment is connected with a vector pET-32a (+) which is purified by enzyme digestion of EcoR I and Hind III at the temperature of T4DNAligase4 ℃ for 16 h. Transformation of the ligation product cloning host E.coli JM109 plated with LB solid Medium (containing 100. mu.g.mL)^-1Ampicillin), culturing at 37 deg.C for 8 hr, selecting single colony, and culturing in LB liquid medium (containing 100 μ g. multidot.mL)^-1Ampicillin) at 37 deg.C for 8 hr, collecting thallusExtracting plasmid, enzyme digestion verification, and then determining a DNA sequence of the recombinant plasmid with correct verification to obtain the recombinant plasmid pET-32a (+) -treZ. The pET-32a (+) vector contains a thioredoxin (TrxA) fusion tag, a small molecule protein with conserved sequence, and a molecular weight of about 12 kDa.

Coli Origami (DE3) as a host E.coli Origami, which was transformed by (+) -treZ transformation of recombinant plasmid pET-32a, and spread on LB solid medium (containing 100. mu.g. mL)^-1Ampicillin), culturing at 37 deg.C for 12 hr, picking single colony, and culturing in LB liquid medium (containing 100. mu.g. mL)^-1Ampicillin) was cultured at 37 ℃ for 8 hours, and then the glycerol-retaining strain was obtained.

Primers used for PCR amplification of treZ gene:

F:5’-CGGAATTCATGTTTAGCTTTGGCGGCAA-3’28bp

R:5’-CCAAGCTTATTCCAGTTGATACACGC-3’26bp

EXAMPLE 2 Shake flask fermentation for enzyme production

1. Recombinant bacteria pET-24a (+) -treZ/E.coli BL21(DE3) and pET-32a (+) -treZ/E.coli Origi (DE3) constructed by using pET-24a (+) as a vector and E.coli BL21(DE3) as a host are respectively inoculated to 10mLLB liquid culture medium (added with 30 mug. multidot.mL of bacterial liquid with final concentration) by sucking 10 mul of bacterial liquid by using a pipette gun^-1Kanamycin or 100. mu.g/mL^-1Ampicillin), put in a constant temperature shaking table at 37 ℃ for 200 r.min^-1Culturing for 8-10h to obtain seed liquid.

The cultured seed solution was inoculated into 40mL of TB medium (to which a final concentration of 30. mu.g/mL was added) at an inoculum size of 4%^-1Kanamycin or 100. mu.g/mL^-1Ampicillin), placing in a constant temperature shaking table at 37 ℃ and the rotating speed of 200 r.min^-1Culturing for 2 h; adding the mixture to the reaction kettle to a final concentration of 0.05-0.4 mmol.L^-1IPTG 25 ℃, rotation speed of 200 r.min-¹And culturing for 24-48 h. Centrifuging the fermentation liquor to remove supernatant, collecting thallus, and using the same volume of 20 mmol.L^-1And (3) redissolving with a Tris-HCl buffer solution with the pH value of 8.0, crushing cells by an ultrasonic cell crusher, centrifuging and taking supernate to obtain a crude enzyme solution.

2. Activity analysis of maltooligosyl trehalose hydrolase

(1) Definition of enzyme Activity Unit

When the enzyme activity of the maltotriosyl trehalose synthase is measured by a 3, 5-dinitrosalicylic acid method (DNS method), the enzyme amount required for converting maltotriosyl trehalose to produce 1. mu. mol trehalose per minute is taken as an activity unit.

(2) Enzyme activity determination procedure

Preheating: 0.49mL of a 1% maltopentaose solution (50mM phosphate buffer pH 5.5) was placed in a test tube and preheated in a 60 ℃ water bath for 10 min.

Reaction: adding 0.05mL of maltooligosyl trehalose synthase enzyme solution, oscillating uniformly, reacting at 60 ℃ for 1h, boiling in boiling water bath for 10min to terminate the reaction, and cooling; adding 0.05mL of maltooligosyl trehalose hydrolase to ferment intracellular crude enzyme solution, oscillating uniformly, timing for 10min accurately, adding 1mL of DNS, oscillating uniformly, boiling in boiling water bath for 7min, and cooling.

Measurement: adding distilled water into the reaction system, fixing the volume to 10mL, and uniformly mixing. The absorbance was measured at a wavelength of 540nm and the enzyme activity was calculated.

3. FIG. 1 is SDS-PAGE protein electrophoresis diagram of recombinant cell wall-broken supernatant obtained after 24h of shake flask fermentation, and protein electrophoresis results show that a band is obvious at about 59kDa and 71kDa, and the band is consistent with the molecular weight of theoretical maltooligosyl trehalose hydrolase (MTHase) and fusion expression maltooligosyl trehalose hydrolase (Trx-MTHase). Through enzyme activity detection, the intracellular enzyme activity of the fusion expression maltooligosyl trehalose hydrolase (Trx-MTHase) is 42.0 U.mL-¹(no enzyme activity was detected extracellularly), which is 4.0 times that of the unfused expression maltooligosyl trehalose hydrolase (MTHase). When the N-terminal Trx protein is cut off by Enterokinase (Enterokinase), the enzyme activity of the maltooligosyl trehalose hydrolase is improved to 1.31 times that before cutting off and 5.2 times that of the enzyme activity in non-fusion expression. In this case, the enzymatic activity of maltooligosyl trehalose hydrolase was 55.0 U.mL^-1。

EXAMPLE 3 Effect of different Induction times on enzyme production by recombinant fermentation

1. Seed culture: inoculating pET-32a (+) -treZ/E. coli Origami (DE3) strain stored in-80 deg.C glycerol tube into seed culture medium, culturing with constant temperature shaker at 37 deg.C and 200rpm for 8 h.

2. And (3) fermenting to produce enzyme:

inoculating the seed liquid into a fermentation medium at an inoculation amount of 8%. The dissolved oxygen was maintained at 30% by controlling the stirring speed and the ventilation rate, the temperature was controlled at 37 ℃ and the pH was controlled at 7.0 by feeding 25% (v/v) aqueous ammonia. And after the initial glycerol is consumed, the dissolved oxygen is increased to 80-100%, and the batch fermentation culture is finished. At a specific growth rate of 0.2h^-1The fed-batch culture medium is supplemented in an exponential feeding mode. Selecting in OD₆₀₀Starting at 0.4 g.L when reaching 30, 40 and 50^-1·h^-1Lactose was added at constant flow rate for induction. Inducing enzyme production at 30 deg.C, maintaining dissolved oxygen at 30% and controlling pH at about 7.0. Inducing for about 24 h. After the fermentation culture, OD₆₀₀When the enzyme is induced under the condition of 40, the activity of the maltooligosyl trehalose hydrolase is the highest and reaches 184.0 U.mL^-1(FIG. 2).

EXAMPLE 4 Effect of different Induction temperatures on enzyme production by recombinant fermentation

2. And (3) fermenting to produce enzyme:

inoculating the seed liquid into a fermentation medium at an inoculation amount of 8%. The dissolved oxygen was maintained at 30% by controlling the stirring speed and the ventilation rate, the temperature was controlled at 37 ℃ and the pH was controlled at 7.0 by feeding 25% (v/v) aqueous ammonia. And after the initial glycerol is consumed, the dissolved oxygen is increased to 80-100%, and the batch fermentation culture is finished. At a specific growth rate of 0.2h^-1The fed-batch culture medium is supplemented in an exponential feeding mode. When the cell concentration reached OD₆₀₀When the ratio is 40, the ratio is 0.4 g.L^-1·h^-1Lactose is fed at constant flow rate for induction at 35 deg.C, 30 deg.C, 25 deg.C, dissolved oxygen is maintained at 30%, and pH is controlled at about 7.0. Inducing for about 24 h. After the fermentation culture is finished, the fermentation culture is induced for 24 hours at 30 ℃, and the enzyme activity of the maltooligosyl trehalose hydrolase is the highest and reaches 193.0 U.mL^-1(FIG. 3).

Example 5 Effect of different lactose feeding rates on recombinant fermentation enzyme production

2. And (3) fermenting to produce enzyme:

inoculating the seed liquid into a fermentation medium at an inoculation amount of 8%. The dissolved oxygen was maintained at 30% by controlling the stirring speed and the ventilation rate, the temperature was controlled at 37 ℃ and the pH was controlled at 7.0 by feeding 25% (v/v) aqueous ammonia. And after the initial glycerol is consumed, the dissolved oxygen is increased to 80-100%, and the batch fermentation culture is finished. At a specific growth rate of 0.2h^-1The fed-batch culture medium is supplemented in an exponential feeding mode. When the cell concentration reached OD₆₀₀When the molecular weight is 40, 0.1 g.L is used^-1·h^-1，0.2g·L^-1·h^-1，0.4g·L^-1·h^-1，0.8g·L^-1·h^-1Lactose was added at constant flow rate for induction. The dissolved oxygen is maintained at 30% and the pH is controlled at about 7.0. Inducing for about 24 h. When the lactose feeding rate is 0.2 g.L^-1·h^-1When the enzyme activity of the maltooligosyl trehalose hydrolase reaches the maximum value of 204 U.mL^-1(FIG. 4).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A genetic engineering bacterium for high yield of maltooligosyl trehalose hydrolase is characterized in that a pET-32a (+) vector is used, and E.coli Origami (DE3) is used as an expression host to express the maltooligosyl trehalose hydrolase; the maltooligosyl trehalose hydrolase is derived from Sulfolobus acidocaldarius; the nucleotide sequence of the gene for coding the maltooligosyl trehalose hydrolase is shown as SEQ ID NO. 1.

2. A method for constructing the genetically engineered bacterium of claim 1, wherein the maltooligosyl trehalose hydrolase gene having the nucleotide sequence of SEQ ID No.1 is ligated to an expression vector pET-32a (+), and then transformed into E.coli Origami (DE3) to obtain the genetically engineered bacterium pET-32a (+) -treZ/E.coli Origami (DE 3).

3. A method for producing malto-oligosyl trehalose hydrolase by fermentation using the genetically engineered bacterium of claim 1, comprising: (1) a batch fermentation stage: inoculating the seed liquid into a fermentation tank by 8-10% of inoculation amount, controlling the temperature to be 36-38 ℃, the initial rotation speed to be 180-200rpm, the initial ventilation volume to be 7L/min, the dissolved oxygen to be 28-32% and the pH value to be 6.8-7.2; (2) and (3) a feeding fermentation stage: when the dissolved oxygen is increased to 80-100%, the specific growth rate mu is controlled to be 0.2h^-1The fed-batch culture is carried out in an exponential fed-batch mode, the temperature is controlled to be 36-38 ℃, the dissolved oxygen is 28-32 percent, and the pH value is 6.8-7.2; (3) and (3) an induction culture stage: when the cell concentration of the bacteria reaches OD₆₀₀At 40 f, the flow rate is 0.2 g.L^-1·h^-1Adding lactose to induce enzyme production, controlling temperature at 30 deg.C, dissolved oxygen at 28-32%, pH at 6.8-7.2, and inducing for 3-27 h.

4. The use of the genetically engineered bacterium of claim 1 in the production of trehalose.