CN111944784B

CN111944784B - Maltogenic oligosaccharide based seaweed hydrolase mutant with improved heat stability and application thereof

Info

Publication number: CN111944784B
Application number: CN202010798479.3A
Authority: CN
Inventors: 吴敬; 宿玲恰; 陈春
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-08-11
Filing date: 2020-08-11
Publication date: 2022-02-01
Anticipated expiration: 2040-08-11
Also published as: CN111944784A

Abstract

The invention discloses a maltooligosyl seaweed hydrolase mutant with improved thermal stability and application thereof, belonging to the technical field of genetic engineering and enzyme engineering. The maltooligosyl trehalose hydrolase mutant of the invention obtains higher thermal stability compared with the parent maltooligosyl trehalose hydrolase by mutating two amino acid sites of the maltooligosyl trehalose hydrolase respectively. The half-life period of the two mutants of the maltooligosyl trehalose hydrolase at 60 ℃ is increased by 5min and 8min compared with that of the wild type, and the half-life period is respectively 1.3 times and 1.5 times of that of the wild type, and meanwhile, the conversion rate of trehalose is improved compared with that of the wild type.

Description

Maltogenic oligosaccharide based seaweed hydrolase mutant with improved heat stability and application thereof

Technical Field

The invention relates to a maltooligosyl seaweed hydrolase mutant with improved thermal stability and application thereof, belonging to the technical field of genetic engineering and enzyme engineering.

Background

Trehalose (trehalase), a disaccharide widely found in nature, is formed by two glucoses linked by α, α -1, 1-glycosidic bonds, and is widely used in food, agriculture, medicine, and cosmetics because of its unique high moisture retention, thermal acid stability, and safety. Since the 80 th 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.

At present, there are three main methods for producing trehalose, namely, an acidification enzyme method, a trehalose synthase single-enzyme method, an MTSase and an MTHase double-enzyme method. The method for producing the trehalose has the advantages of high conversion rate and fewer byproducts, and reduces the cost by using cheap starch as a substrate, so that the method is widely applied.

In the process of preparing trehalose by using a MTSase and MTHase double-enzyme method, starch is liquefied at high temperature and then added with pullulanase to react to generate maltodextrin; the maltooligosyl trehalose synthase acts on alpha, alpha-1, 4-glucoside at the reducing end of a substrate to generate intramolecular transglycosylation of alpha, alpha-1, 1-glycosidic bond to form an intermediate product maltooligosyl trehalose; the maltooligosyl trehalose hydrolase (MTHase) specifically cuts the alpha, alpha-1, 4-glycosidic bond connecting maltooligosyl with trehalose in the intermediate product, so as to decompose the maltooligosyl trehalose to generate trehalose and new maltooligose with two glucose units reduced, and the new maltooligose with two glucose units reduced is used as a new substrate to carry out the next reaction, thus repeatedly and alternately carrying out the two enzyme reactions to convert the maltooligose into products mainly comprising trehalose and a small amount of glucose, maltose and maltotriose.

Therefore, maltooligosyl trehalose hydrolase (MTHase) is one of the key enzymes for the two-enzyme process of trehalose production.

The maltooligosyl trehalose hydrolase has two types, namely high-temperature enzyme and moderate-temperature enzyme, wherein the high-temperature enzyme has poor expression in a host and relatively low specific activity compared with the moderate-temperature enzyme, but the moderate-temperature enzyme has poor stability and can not have too high conversion temperature, so that the reaction is easy to be infected with bacteria, and meanwhile, enzyme is added subsequently, so that the cost is high, and the method is particularly important for the thermal stability modification of the moderate-temperature enzyme.

Disclosure of Invention

Based on the current situation, the invention utilizes the means of genetic engineering and enzyme engineering to improve the thermal stability of the maltooligosyl trehalose hydrolase and create conditions for the industrial production of the maltooligosyl trehalose hydrolase.

The invention provides a maltooligosyl trehalose hydrolase mutant, which is obtained by mutating the 137 th amino acid of maltooligosyl trehalose hydrolase with the amino acid sequence shown as SEQ ID NO. 1;

or the mutant is obtained by mutating the 216 th amino acid of the maltooligosyl trehalose hydrolase whose amino acid sequence is shown as SEQ ID NO. 1. These mutants have improved thermostability compared to their parent maltooligosyl trehalose hydrolase.

In one embodiment of the present invention, the mutant is obtained by mutating the 137 th amino acid of maltooligosyl trehalose hydrolase, the amino acid sequence of which is shown in SEQ ID No.1, from leucine to methionine;

or the mutant is obtained by mutating the 216 th amino acid of the maltooligosyl trehalose hydrolase whose amino acid sequence is shown in SEQ ID NO.1 from alanine to threonine.

In one embodiment of the invention, the mutant is obtained by mutating the 137 th amino acid from leucine to methionine, and is named as L137M.

In one embodiment of the invention, the mutant is obtained by mutating the 216 th amino acid from alanine to threonine and is named a 216T.

In one embodiment of the invention, the amino acid sequence of the mutant L137M is shown as SEQ ID NO. 2.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the mutant L137M is shown in SEQ ID NO. 4.

In one embodiment of the invention, the amino acid sequence of the mutant A216T is shown in SEQ ID NO. 3.

In one embodiment of the invention, the nucleotide sequence of the gene encoding the mutant A216T is shown in SEQ ID NO. 5.

In one embodiment of the invention, the source of maltooligosyl trehalose hydrolase is mycobacterium (Arthrobacter ramosus).

The invention also provides a gene for coding the maltooligosyl trehalose hydrolase mutant.

The invention also provides a recombinant plasmid carrying the gene.

In one embodiment of the present invention, the vector of the recombinant plasmid is a pUC plasmid vector, a pET plasmid vector, or a pGEX plasmid vector.

In one embodiment of the present invention, the vector of the recombinant plasmid is a pET24a plasmid vector.

The invention also provides a host cell carrying the gene or the recombinant plasmid.

In one embodiment of the invention, the host cell is a bacterium or a fungus.

In one embodiment of the invention, the host cell is E.coli.

The invention also provides a preparation method of the maltooligosyl trehalose hydrolase mutant, which comprises the following steps:

(1) designing a mutation primer of site-directed mutation according to the determined mutation site, and carrying out site-directed mutation by taking a vector carrying the maltooligosyl trehalose hydrolase gene as a template; constructing a plasmid vector containing a gene encoding the mutant;

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

(3) and (3) selecting the positive cloning host cells for fermentation culture, after the fermentation is finished, centrifuging fermentation liquor obtained by fermentation, collecting cells, and obtaining cell wall breaking supernatant fluid which is crude enzyme liquid of the mutant maltooligosyl trehalose hydrolase.

The invention also provides the application of the maltooligosyl trehalose hydrolase mutant or the gene or the recombinant plasmid or the host cell or the preparation method in the production of trehalose.

The present invention also provides a method for producing trehalose using the maltooligosyl trehalose hydrolase mutant according to claim 1 or 2.

In one embodiment of the invention, rice starch is used as a substrate, alpha-amylase is added into a rice starch solution, the addition amount is 5U/g starch, and the rice starch solution is liquefied at high temperature of 90-100 ℃ until the DE value is 16, so as to obtain a liquefied rice starch solution; respectively adding pullulanase, cyclodextrin glucosyltransferase, 4-alpha glycosyltransferase and maltooligosyl trehalose synthetase into the liquefied rice starch solution according to the addition amounts of 5U/g starch, 2U/mL, 0.5U/mL and 6.0U/mL, and reacting for 36h at 60 ℃ and 150r/min to obtain a reaction solution 1; adding the maltooligosyl trehalose hydrolase mutant L137M and/or A216T into the reaction solution 1 according to the total addition amount of 6.0U/mL, and reacting for 36h at 60 ℃ under the condition of 150r/min to obtain a reaction solution 2; trehalose was isolated from the reaction solution 2.

Has the advantages that:

(1) the invention constructs a meaningful maltooligosyl trehalose hydrolase mutant, and realizes the improvement of the thermal stability of the maltooligosyl trehalose hydrolase;

(2) half-lives of the maltooligosyl trehalose hydrolase mutants L137M and A216T at 60 ℃ are respectively increased by 5min and 8min compared with the wild type, and are respectively 1.3 times and 1.5 times of the wild type, so that the stability of the maltooligosyl trehalose hydrolase mutants is respectively improved by 1.3 times and 1.5 times compared with the wild type;

(3) the conversion rates of the wild type, L137M and A216T are 51.3, 52.6 and 55.7 respectively under the condition of the same enzyme adding amount of 6.0U/mL when the maltooligosyl trehalose hydrolase mutant is used for producing trehalose, which shows that the conversion rates of the maltooligosyl trehalose hydrolase mutants L137M and A216T are improved while the stability is improved.

Drawings

FIG. 1: thermal stability at 60 ℃ of wild-type MTHase, MTHase mutant L37M, and MTHase mutant A216T.

FIG. 2: optimum temperatures for wild-type MTHase, MTHase mutant L137M, and MTHase mutant a 216T.

FIG. 3: the pH optimum of the wild type MTHase, MTHase mutant L137M, and MTHase mutant A216T.

FIG. 4: trehalose conversion of wild-type MTHase, MTHase mutant L137M, and MTHase mutant a 216T.

Detailed Description

The following examples relate to alpha-amylase, maltooligosyl trehalose synthase, pullulanase, cyclodextrin glucosyl transferase, glycosyltransferase from novicent.

The following examples refer to the following media:

LB liquid medium:

0.5g of yeast extract, 1.0g of tryptone and 1.0g of NaCl were added per 100mL of sterile water.

LB solid medium:

2.0g of agar powder is added into 100mL of LB liquid culture medium.

TB liquid fermentation medium:

adding 5.0g glycerol, 24.0g yeast powder, 12.0g peptone, 7.5g glycine, 16.4g K into 1L sterile water₂HPO₄·3H₂O and 2.3g KH₂PO₄。

The detection methods referred to in the following examples are as follows:

the method for detecting the enzymatic activity of the maltooligosyl trehalose hydrolase comprises the following steps:

preheating: 1.9mL of 0.4% maltotriosyl trehalose solution (pH 6.0 phosphate buffer) was placed in a stoppered tube and preheated in a 50 ℃ water bath for 10 min.

Reaction: adding 0.1mL of diluted crude enzyme solution in fermentation cells, uniformly oscillating, accurately timing for 10min, adding 3mLDNS, uniformly oscillating, and stopping reaction; boiling for 7min, and cooling.

Measurement: adding distilled water into the reaction system, fixing the volume to 15mL, and uniformly mixing; the absorbance was measured at a wavelength of 540nm and the enzyme activity was calculated.

Definition of enzyme activity: the unit enzyme activity corresponds to the amount of enzyme required to convert one micromole maltotriosyl trehalose to glucose per minute.

The enzyme stability detection method comprises the following steps:

diluting the purified enzyme with 20mM phosphate buffer solution with pH6.0 to protein content of 0.25mg/mL and pH6.0, placing in constant temperature water bath at 60 deg.C, sampling every 5min, measuring residual enzyme activity, and comparing stability.

The detection method of the optimum temperature and the optimum pH of the enzyme comprises the following steps:

diluting the purified enzyme with 20mM phosphate buffer solution with pH6.0 to protein content of 0.25mg/mL and pH6.0, and measuring enzyme activity in constant temperature water bath with gradient change at 40, 45, 50, 55, 60, 65 deg.C to obtain optimum temperature; the purified enzyme was diluted with 20mM phosphate buffer pH6.0 to a protein content of 0.25mg/mL, and the enzyme activity was measured in a 50 ℃ water bath with gradient changes in pH4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 to obtain the optimum pH.

The method for detecting the residual enzyme activity comprises the following steps:

The trehalose conversion rate detection method comprises the following steps:

and (3) diluting and precipitating the reaction product, measuring the content of trehalose in the reaction product by using High Performance Liquid Chromatography (HPLC), and calculating the conversion rate.

Calculated formula trehalose mass/rice starch mass 100%

HPLC detection conditions: mobile phase (acetonitrile: water 80: 20); flow rate: 0.8mL/min, column temperature 40 deg.C, NH2 column (APS-2HYPERSIL, Thermo Scientific), differential refractometer detector (RID).

Example 1: expression of wild type maltooligosyl trehalose hydrolase

Synthesizing a gene treZ which has a nucleotide sequence shown as SEQ ID NO.10 and codes the maltooligosyl trehalose hydrolase; the gene treZ is subjected to double enzyme digestion by HindIII and NdeI and then is connected with an expression vector pET24a, and is transferred into escherichia coli BL21(DE3), so that BL21(DE3)/pET24a-treZ is obtained.

After the obtained bacterial liquid of the recombinant bacterium BL21(DE3)/pET24a-treZ is cultured on an LB solid culture medium by streaking, a single colony is selected and inoculated in an LB liquid culture medium (containing 100mg/L kanamycin) to grow for 10 hours to obtain a seed liquid, the obtained seed liquid is inoculated into a TB liquid fermentation culture medium (containing 100mg/L kanamycin) according to the inoculation amount of 5% (v/v), after the seed liquid is cultured for 2 hours at 37 ℃, IPTG (isopropylthio-beta-D galactoside) with the final concentration of 0.1mmol/L is added for induction, and the seed liquid is continuously cultured and fermented for 24 hours at 32 ℃ by a shaking table to obtain a fermentation liquid; centrifuging the fermentation liquid at 4 deg.C and 12000rpm for 10min, removing supernatant, collecting thallus, resuspending thallus precipitate with 20mmol/L phosphate buffer solution of pH6.0, and mixing; and (3) crushing cell walls of the thallus suspension by using an ultrasonic cell crusher (the working condition of the ultrasonic cell crusher: psi 6 working probe, working time 10min, working 2s stopping 3s, and working power 20%), then centrifuging at 12000rpm for 10min, and obtaining supernatant after centrifugation, namely the wild type maltooligosyl trehalose hydrolase fermentation intracellular crude enzyme liquid.

Example 2: preparation and expression of maltooligosyl trehalose hydrolase single mutant

(1) Preparation of mutants

According to the gene sequence of the gene treZ which has the nucleotide sequence shown as SEQ ID No.10 and codes the maltooligosyl trehalose hydrolase and is obtained in the example 1, primers which introduce L37M and A216T mutations are respectively designed and synthesized, site-directed mutagenesis is carried out on the maltooligosyl trehalose hydrolase gene TreZ, and the coding gene of the maltooligosyl trehalose hydrolase mutant is respectively sequenced to confirm whether to be correct; and introducing the vector carrying the mutant gene into escherichia coli for expression to obtain the single mutation maltooligosyl trehalose hydrolase.

PCR amplification of site-directed mutant coding gene: the rapid PCR technology is utilized, and an expression vector pET-24a (+) -TreZ carrying a gene coding the wild type maltooligosyl trehalose hydrolase is taken as a template.

Site-directed mutagenesis primers for introducing the L37M mutation were:

a forward primer: 5'-GGAATTAATGCCAGTTAATGCGTTTAATGGT-3' (SEQ ID NO. 6);

reverse primer: 5'-CTGGCATTAATTCCACAGCATCAACGC-3' (SEQ ID NO. 7);

the site-directed mutagenesis primers for introducing the A216T mutation were:

a forward primer: 5'-CCTGGGGGGATACCTTAAATCTGGATGG-3' (SEQ ID NO. 8);

reverse primer: 5'-CCATCCAGATTTAAGGTATCCCCCCAGG-3' (SEQ ID NO. 9);

the PCR system is as follows: mu.L of each of 20. mu.M forward and reverse primers, 0.5. mu.L of dNTPmix, 10. mu.L of 5 XPS Buffer, 0.5. mu.L of PrimeStar polymerase (2.5U/. mu.L), 0.5. mu.L of template, and 50. mu.L of double distilled water.

The PCR conditions were: pre-denaturation at 94 ℃ for 4min, followed by 25 cycles (94 ℃ for 10s, 55 ℃ for 5s, 72 ℃ for 7min for 40s) of extension at 72 ℃ for 10 min; finally, preserving the heat at 4 ℃; the PCR product was detected by electrophoresis on a 1% agarose gel.

Adding Dpn I into the PCR product which is verified to be correct for digestion, then transforming E.coli JM109 competent cells to obtain a transformation product, coating the transformation product in an LB solid culture medium containing 100mg/L kanamycin, culturing overnight at 37 ℃, selecting positive clones, inoculating the positive clones into an LB liquid culture medium for culturing for 8 hours, extracting plasmids and sequencing, wherein the sequencing is correct: plasmids carrying the mutants L37M and A216T are extracted and sequenced correctly, and transformed to express host escherichia coli BL21(DE3) competent cells to obtain recombinant strains BL21(DE3)/pET24a-treZ 1 and BL21(DE3)/pET24a-treZ 2 capable of expressing the mutants L37M and A216T respectively.

(2) Expression of the mutant

Respectively streaking the BL21(DE3)/pET24a-treZ 1 and BL21(DE3)/pET24a-treZ 2 obtained in the step (1) on an LB solid culture medium to culture, respectively selecting a single colony to be inoculated on an LB liquid culture medium (containing 100mg/L kanamycin) to culture for 10 hours to obtain a seed solution, inoculating the seed solution into a TB liquid fermentation culture medium (containing 100mg/L kanamycin) according to the inoculation amount of 5% (v/v), culturing for 2 hours at 37 ℃, adding IPTG (isopropylthio-beta-D galactoside) with the final concentration of 0.1mmol/L to induce, and continuously culturing and fermenting for 24 hours at 32 ℃ by a shaking table to obtain a fermentation liquid; centrifuging the obtained fermentation liquid at 4 deg.C and 12000rpm for 10min, removing supernatant, collecting thallus, resuspending the collected thallus with 20mmol/L phosphate buffer solution of pH6.0, and mixing; and (3) crushing cell walls of the thallus suspension by using an ultrasonic cell crusher (the working condition of the ultrasonic cell crusher: psi 6 working probe, working time of 10min, working 2s stopping for 3s, and working power of 20%) to obtain cell crushing liquid, then centrifuging the cell crushing liquid at 12000rpm for 10min, and obtaining supernatant after centrifugation, namely the crude enzyme liquid in the fermentation cells of the maltooligosyl trehalose hydrolase mutants L37M and A216T.

Example 3: activity analysis of maltooligosyl trehalose hydrolase

The enzyme activities of the crude enzyme solution in the fermentation cells of the wild-type maltooligosyl trehalose hydrolase obtained in example 1 and the crude enzyme solutions in the fermentation cells of the maltooligosyl trehalose hydrolase mutants L37M and A216T obtained in example 2 were measured, and the results were as follows: the enzyme activities of the wild type maltooligosyl trehalose hydrolase (WT) and the crude enzyme liquid in the fermentation cells of the mutants L137M and A216T are 354U/mL, 335U/mL and 346U/mL respectively.

Example 4: analysis of thermostability of maltooligosyl trehalose hydrolase mutants

The crude enzyme liquid in the fermentation cells obtained in the example 1 and the example 2 is purified by the following method: the crude enzyme solution is sequentially subjected to ammonium sulfate precipitation, redissolution and dialysis (the dialysate is buffer A, and 20mM sodium phosphate buffer solution pH7.0), and finally purified by using an AKTA-FPLC system. The conditions are as follows: the MonoQ 10/100 column was equilibrated for 1h with buffer A and the protein to be purified was loaded onto the MonoQ 10/100 column. The purified enzyme was obtained by gradient elution using buffer A and buffer B (20mM sodium phosphate buffer, with the addition of sodium chloride at a final concentration of 1M, pH7.0), and collecting the eluate.

The purified enzyme was subjected to stability testing:

as shown in FIG. 1, it can be seen that at 60 ℃, the half-lives of the pure enzymes of the wild type and the two mutants L137M, A216T are 15.5, 20.5 and 23.3min, respectively, and the t1/2 of the pure enzymes of L137M and A216T are 1.3 and 1.5 times higher than that of the wild type, respectively.

Example 5: optimum temperature and optimum pH analysis of maltooligosyl trehalose hydrolase mutant

The pure enzymes of the wild type and the two mutants L137M, A216T obtained in example 4 were subjected to temperature-optimum and pH-optimum analyses.

As shown in FIGS. 2 and 3, the optimum temperatures of the wild type and the two mutants, L137M and A216T, are both 50 ℃, and the optimum pH values of the wild type and the two mutants, L137M and A216T, are both 6.0.

Example 6: analysis of enzyme addition amount and trehalose conversion rate of maltooligosyl trehalose hydrolase mutant

Adding alpha-amylase into a rice starch solution by taking rice starch as a substrate, wherein the adding amount of the alpha-amylase is 5U/g of starch, and liquefying at the high temperature of 90 ℃ to obtain a liquefied rice starch solution with a DE value of 16; respectively adding pullulanase, cyclodextrin glucosyltransferase, 4-alpha glycosyltransferase and maltooligosyl trehalose synthetase into the liquefied rice starch solution according to the addition amounts of 5U/g starch, 2U/mL, 0.5U/mL and 6.0U/mL, and reacting for 36h at 60 ℃ and 150r/min to obtain a reaction solution 1; adding pure enzyme of the wild type obtained in example 4 and two mutants L137M and A216T into the reaction solution 1 according to the addition amount of 6.0U/mL respectively, and reacting for 36h under the conditions of 60 ℃ and 150r/min to obtain a reaction solution 2; trehalose was isolated from the reaction solution 2.

Trehalose conversion in reaction solution 2 prepared using pure enzymes of wild type and two mutants, L137M, a216T, respectively, was examined.

As shown in FIG. 4, the trehalose conversion rates of reaction mixture 2 obtained by using the pure enzymes of wild type and two mutants, L137M and A216T, were 51.3%, 52.6% and 55.7%, respectively, and the conversion rates of both mutants were improved as compared with that of the wild type.

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.

SEQUENCE LISTING

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Asp Pro Ala Arg Tyr Ala Trp Gly Asp Asp Gly Trp Arg Gly Arg Asp

85 90 95

Leu Thr Gly Ala Val Ile Tyr Glu Leu His Val Gly Thr Phe Thr Pro

100 105 110

Glu Gly Thr Leu Asp Ser Ala Ile Arg Arg Leu Asp His Leu Val Arg

115 120 125

Leu Gly Val Asp Ala Val Glu Leu Leu Pro Val Asn Ala Phe Asn Gly

130 135 140

Thr His Gly Trp Gly Tyr Asp Gly Val Leu Trp Tyr Ala Val His Glu

145 150 155 160

Pro Tyr Gly Gly Pro Glu Ala Tyr Gln Arg Phe Val Asp Ala Cys His

165 170 175

Ala Arg Gly Leu Ala Val Val Gln Asp Val Val Tyr Asn His Leu Gly

180 185 190

Pro Ser Gly Asn His Leu Pro Asp Phe Gly Pro Tyr Leu Gly Ser Gly

195 200 205

Ala Ala Asn Thr Trp Gly Asp Thr Leu Asn Leu Asp Gly Pro Leu Ser

210 215 220

Asp Glu Val Arg Arg Tyr Ile Ile Asp Asn Ala Val Tyr Trp Leu Arg

225 230 235 240

Asp Met His Ala Asp Gly Leu Arg Leu Asp Ala Val His Ala Leu Arg

245 250 255

Asp Ala Arg Ala Leu His Leu Leu Glu Glu Leu Ala Ala Arg Val Asp

260 265 270

Glu Leu Ala Gly Glu Leu Gly Arg Pro Leu Thr Leu Ile Ala Glu Ser

275 280 285

Asp Leu Asn Asp Pro Lys Leu Ile Arg Ser Arg Ala Ala His Gly Tyr

290 295 300

Gly Leu Asp Ala Gln Trp Asp Asp Asp Val His His Ala Val His Ala

305 310 315 320

Asn Val Thr Gly Glu Thr Val Gly Tyr Tyr Ala Asp Phe Gly Gly Leu

325 330 335

Gly Ala Leu Val Lys Val Phe Gln Arg Gly Trp Phe His Asp Gly Thr

340 345 350

Trp Ser Ser Phe Arg Glu Arg His His Gly Arg Pro Leu Asp Pro Asp

355 360 365

Ile Pro Phe Arg Arg Leu Val Ala Phe Ala Gln Asp His Asp Gln Val

370 375 380

Gly Asn Arg Ala Val Gly Asp Arg Met Ser Ala Gln Val Gly Glu Gly

385 390 395 400

Ser Leu Ala Ala Ala Ala Ala Leu Val Leu Leu Gly Pro Phe Thr Pro

405 410 415

Met Leu Phe Met Gly Glu Glu Trp Gly Ala Arg Thr Pro Trp Gln Phe

420 425 430

Phe Thr Ser His Pro Glu Pro Glu Leu Gly Glu Ala Thr Ala Arg Gly

435 440 445

Arg Ile Ala Glu Phe Ala Arg Met Gly Trp Asp Pro Ala Val Val Pro

450 455 460

Asp Pro Gln Asp Pro Ala Thr Phe Ala Arg Ser His Leu Asp Trp Ser

465 470 475 480

Glu Pro Glu Arg Glu Pro His Ala Gly Leu Leu Ala Phe Tyr Thr Asp

485 490 495

Leu Ile Ala Leu Arg Arg Glu Leu Pro Val Asp Ala Pro Ala Arg Glu

500 505 510

Val Asp Ala Asp Glu Ala Arg Gly Val Phe Ala Phe Ser Arg Gly Pro

515 520 525

Leu Arg Val Thr Val Ala Leu Arg Pro Gly Pro Val Gly Val Pro Glu

530 535 540

His Gly Gly Leu Val Leu Ala Tyr Gly Glu Val Arg Ala Gly Ala Ala

545 550 555 560

Gly Leu His Leu Asp Gly Pro Gly Ala Ala Ile Val Arg Leu Glu

565 570 575

<210> 4

<211> 1728

<212> DNA

<213> Artificial sequence

<400> 4

atgaatcgtc gctttcctgt gtgggcacca caggccgcac aggtgaccct ggttgtgggt 60

cagggtcgcg cagaattacc tctgacccgc gatgaaaacg gatggtgggc attacagcag 120

ccttgggatg gcggcccaga cttagttgat tatggctatc tggttgatgg taaaggtccg 180

tttgcagatc ctcgtagctt acgtcagcca cgcggtgttc atgaattagg tcgcgaattt 240

gatccggcac gctatgcttg gggcgatgat ggctggcgcg gtcgcgatct gacgggtgcg 300

gttatctatg aactgcatgt tggcacgttt acaccggaag gtacactgga tagcgcaatt 360

cgtcgcttag atcacttggt tcgcttaggc gttgatgctg tggaattaat gccagttaat 420

gcgtttaatg gtacacatgg ttggggctat gatggtgttc tgtggtatgc tgttcatgaa 480

ccgtatggcg gcccagaagc atatcagcgc tttgtggatg cttgtcatgc tcgcggtctg 540

gcagttgttc aggatgttgt gtataatcac ctcggcccga gcggtaatca tctgccagat 600

tttggtccat atctgggctc aggcgccgca aatacctggg gggatgcctt aaatctggat 660

ggcccactga gcgatgaagt gcgtcgctat attatcgata atgctgtgta ttggttacgc 720

gatatgcacg ccgatggcct gcgcctggat gctgtccacg cattacgcga tgcacgcgca 780

ttacatctgt tagaagaatt agccgcccgc gttgacgagt tagcaggcga attaggccgt 840

ccactgaccc tgattgccga aagcgacctg aatgatccaa aactgattcg ctctcgtgct 900

gcccacggct atggcctgga tgctcagtgg gatgatgatg ttcatcatgc agttcacgcc 960

aatgtgacag gcgaaacagt gggctattat gcagattttg gcggcttagg tgccttagtt 1020

aaagtgtttc agcgcggctg gtttcatgat ggaacatgga gtagctttcg tgaacgccat 1080

catggccgtc ctttagaccc agatattccg tttcgtcgct tagttgcgtt tgcacaggat 1140

catgatcagg ttggtaatcg tgcagtgggc gatcgtatgt cagctcaggt tggtgaaggt 1200

agcttagcag cggcagccgc cttagtgtta ttaggcccgt tcacaccaat gctttttatg 1260

ggtgaagaat ggggcgcccg caccccttgg cagtttttca cctcacatcc ggaaccggaa 1320

ctgggtgaag caacggcccg cggtcgtatt gccgaatttg cccgtatggg ctgggaccca 1380

gccgttgttc cagatccaca ggatccagct acctttgctc gtagtcactt ggattggagc 1440

gaaccggaac gtgaaccaca tgcgggtctg ttagcctttt ataccgatct gatcgcactg 1500

cgtcgtgaac tgccagttga tgctcctgct agagaagtgg atgccgatga agcacgcggc 1560

gtttttgcct tttctcgcgg tccgttacgg gtgacggttg ctctgcgtcc gggtcctgtg 1620

ggtgttccgg aacatggcgg cttagttctg gcttatggcg aagtgcgtgc gggcgccgcc 1680

ggtttacatc tggatggtcc gggtgctgca atcgttcgtc tggaataa 1728

<210> 5

<211> 1728

<212> DNA

<213> Artificial sequence

<400> 5

atgaatcgtc gctttcctgt gtgggcacca caggccgcac aggtgaccct ggttgtgggt 60

cagggtcgcg cagaattacc tctgacccgc gatgaaaacg gatggtgggc attacagcag 120

ccttgggatg gcggcccaga cttagttgat tatggctatc tggttgatgg taaaggtccg 180

tttgcagatc ctcgtagctt acgtcagcca cgcggtgttc atgaattagg tcgcgaattt 240

gatccggcac gctatgcttg gggcgatgat ggctggcgcg gtcgcgatct gacgggtgcg 300

gttatctatg aactgcatgt tggcacgttt acaccggaag gtacactgga tagcgcaatt 360

cgtcgcttag atcacttggt tcgcttaggc gttgatgctg tggaattact gccagttaat 420

gcgtttaatg gtacacatgg ttggggctat gatggtgttc tgtggtatgc tgttcatgaa 480

ccgtatggcg gcccagaagc atatcagcgc tttgtggatg cttgtcatgc tcgcggtctg 540

gcagttgttc aggatgttgt gtataatcac ctcggcccga gcggtaatca tctgccagat 600

tttggtccat atctgggctc aggcgccgca aatacctggg gggatacctt aaatctggat 660

ggcccactga gcgatgaagt gcgtcgctat attatcgata atgctgtgta ttggttacgc 720

gatatgcacg ccgatggcct gcgcctggat gctgtccacg cattacgcga tgcacgcgca 780

ttacatctgt tagaagaatt agccgcccgc gttgacgagt tagcaggcga attaggccgt 840

ccactgaccc tgattgccga aagcgacctg aatgatccaa aactgattcg ctctcgtgct 900

gcccacggct atggcctgga tgctcagtgg gatgatgatg ttcatcatgc agttcacgcc 960

aatgtgacag gcgaaacagt gggctattat gcagattttg gcggcttagg tgccttagtt 1020

aaagtgtttc agcgcggctg gtttcatgat ggaacatgga gtagctttcg tgaacgccat 1080

catggccgtc ctttagaccc agatattccg tttcgtcgct tagttgcgtt tgcacaggat 1140

catgatcagg ttggtaatcg tgcagtgggc gatcgtatgt cagctcaggt tggtgaaggt 1200

agcttagcag cggcagccgc cttagtgtta ttaggcccgt tcacaccaat gctttttatg 1260

ggtgaagaat ggggcgcccg caccccttgg cagtttttca cctcacatcc ggaaccggaa 1320

ctgggtgaag caacggcccg cggtcgtatt gccgaatttg cccgtatggg ctgggaccca 1380

gccgttgttc cagatccaca ggatccagct acctttgctc gtagtcactt ggattggagc 1440

gaaccggaac gtgaaccaca tgcgggtctg ttagcctttt ataccgatct gatcgcactg 1500

cgtcgtgaac tgccagttga tgctcctgct agagaagtgg atgccgatga agcacgcggc 1560

gtttttgcct tttctcgcgg tccgttacgg gtgacggttg ctctgcgtcc gggtcctgtg 1620

ggtgttccgg aacatggcgg cttagttctg gcttatggcg aagtgcgtgc gggcgccgcc 1680

ggtttacatc tggatggtcc gggtgctgca atcgttcgtc tggaataa 1728

<210> 6

<211> 31

<212> DNA

<213> Artificial sequence

<400> 6

ggaattaatg ccagttaatg cgtttaatgg t 31

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence

<400> 7

ctggcattaa ttccacagca tcaacgc 27

<210> 8

<211> 28

<212> DNA

<213> Artificial sequence

<400> 8

cctgggggga taccttaaat ctggatgg 28

<210> 9

<211> 28

<212> DNA

<213> Artificial sequence

<400> 9

ccatccagat ttaaggtatc cccccagg 28

<210> 10

<211> 1728

<212> DNA

<213> Artificial sequence

<400> 10

atgaatcgtc gctttcctgt gtgggcacca caggccgcac aggtgaccct ggttgtgggt 60

cagggtcgcg cagaattacc tctgacccgc gatgaaaacg gatggtgggc attacagcag 120

ccttgggatg gcggcccaga cttagttgat tatggctatc tggttgatgg taaaggtccg 180

tttgcagatc ctcgtagctt acgtcagcca cgcggtgttc atgaattagg tcgcgaattt 240

gatccggcac gctatgcttg gggcgatgat ggctggcgcg gtcgcgatct gacgggtgcg 300

gttatctatg aactgcatgt tggcacgttt acaccggaag gtacactgga tagcgcaatt 360

cgtcgcttag atcacttggt tcgcttaggc gttgatgctg tggaattact gccagttaat 420

gcgtttaatg gtacacatgg ttggggctat gatggtgttc tgtggtatgc tgttcatgaa 480

ccgtatggcg gcccagaagc atatcagcgc tttgtggatg cttgtcatgc tcgcggtctg 540

gcagttgttc aggatgttgt gtataatcac ctcggcccga gcggtaatca tctgccagat 600

tttggtccat atctgggctc aggcgccgca aatacctggg gggatgcctt aaatctggat 660

ggcccactga gcgatgaagt gcgtcgctat attatcgata atgctgtgta ttggttacgc 720

gatatgcacg ccgatggcct gcgcctggat gctgtccacg cattacgcga tgcacgcgca 780

ttacatctgt tagaagaatt agccgcccgc gttgacgagt tagcaggcga attaggccgt 840

ccactgaccc tgattgccga aagcgacctg aatgatccaa aactgattcg ctctcgtgct 900

gcccacggct atggcctgga tgctcagtgg gatgatgatg ttcatcatgc agttcacgcc 960

aatgtgacag gcgaaacagt gggctattat gcagattttg gcggcttagg tgccttagtt 1020

aaagtgtttc agcgcggctg gtttcatgat ggaacatgga gtagctttcg tgaacgccat 1080

catggccgtc ctttagaccc agatattccg tttcgtcgct tagttgcgtt tgcacaggat 1140

catgatcagg ttggtaatcg tgcagtgggc gatcgtatgt cagctcaggt tggtgaaggt 1200

agcttagcag cggcagccgc cttagtgtta ttaggcccgt tcacaccaat gctttttatg 1260

ggtgaagaat ggggcgcccg caccccttgg cagtttttca cctcacatcc ggaaccggaa 1320

ctgggtgaag caacggcccg cggtcgtatt gccgaatttg cccgtatggg ctgggaccca 1380

gccgttgttc cagatccaca ggatccagct acctttgctc gtagtcactt ggattggagc 1440

gaaccggaac gtgaaccaca tgcgggtctg ttagcctttt ataccgatct gatcgcactg 1500

cgtcgtgaac tgccagttga tgctcctgct agagaagtgg atgccgatga agcacgcggc 1560

gtttttgcct tttctcgcgg tccgttacgg gtgacggttg ctctgcgtcc gggtcctgtg 1620

ggtgttccgg aacatggcgg cttagttctg gcttatggcg aagtgcgtgc gggcgccgcc 1680

ggtttacatc tggatggtcc gggtgctgca atcgttcgtc tggaataa 1728

Claims

1. A maltooligosyl trehalose hydrolase mutant characterized in that,

the mutant is obtained by mutating the 137 th amino acid of the maltooligosyl trehalose hydrolase with the amino acid sequence shown as SEQ ID NO.1 from leucine to methionine;

2. A gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. The recombinant plasmid of claim 3, wherein the vector of the recombinant plasmid is a pUC plasmid vector, a pET plasmid vector, or a pGEX plasmid vector.

5. An engineered cell carrying the gene of claim 2, or the recombinant plasmid of claim 3 or 4.

6. The engineered cell of claim 5, wherein the host cell is a bacterium or a fungus.

7. A method for preparing the mutant of claim 1, comprising the steps of:

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

(3) and selecting positive cloning engineering cells for fermentation culture, centrifuging fermentation liquor obtained by fermentation after fermentation is finished, collecting cells, and obtaining cell wall breaking supernatant as crude enzyme liquid of mutant maltooligosyl trehalose hydrolase.

8. Use of the maltooligosyl trehalose hydrolase mutant according to claim 1, or the gene according to claim 2, or the recombinant plasmid according to claim 3 or 4, or the engineered cell according to claim 5 or 6, or the preparation process according to claim 7 for the production of trehalose.

9. A method for producing trehalose, which comprises using the maltooligosyl trehalose hydrolase mutant according to claim 1.