CN102660515B

CN102660515B - Glutamine transaminase with improved enzymatic activity and thermal stability

Info

Publication number: CN102660515B
Application number: CN2012101451790A
Authority: CN
Inventors: 陈坚; 陈康康; 刘松; 张东旭; 马建龙; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-05-10
Filing date: 2012-05-10
Publication date: 2013-12-11
Anticipated expiration: 2032-05-10
Also published as: CN102660515A

Abstract

The invention provides glutamine transaminase with improved enzymatic activity, wherein the amino acid sequence is shown as SEQ ID NO.1. According to the invention, amino acid at the N end of MTG (Microbial Transglutaminase) maturase is subjected to deficiency and saturation mutation by taking the efficient expression of MTG in colibacillus as an improvement platform, and a mutant strain with good enzymatic property is obtained. The enzyme activity is improved by 1.85 times, and the thermal stability is improved by 2.7 times. The modified enzyme is more suitable for industrial applications, the production cost can be lowered, and the production efficiency can be improved.

Description

The glutamine transaminage that a kind of enzymic activity and thermostability improve

Technical field

The present invention relates to a kind of glutamine transaminage, the glutamine transaminage that particularly a kind of enzymic activity and thermostability improve.

Background technology

Glutamine of microbe transaminase (EC2.3.2.13, Microbial Transglutaminase, EC2.3.2.13 is called for short MTG) its biological function is directly to change the structure of protein itself and the accompanying cell of protein, tissue etc. and the characteristic of functional property, improves the nutritive value of protein.Therefore, MTG has a wide range of applications in fields such as food, weaving, bio-pharmaceuticals.But as than factors such as enzyme work, thermostabilitys, limited the range of application of MTG due to some defects of MTG self.Therefore the expression platform of MTG in intestinal bacteria based on having obtained, utilize aminoacid deletion and saturation mutation, and MTG is carried out to molecular modification, is more suitable for the MTG of industrial application to obtaining zymologic property.

Summary of the invention

Alive lower than enzyme for improving MTG, the present situation of poor heat stability, this research is lacked by the N terminal amino acid to MTG, thus reduce substrate and MTG active centre in conjunction with resistance, the affinity of raising MTG to substrate.The mutant strain that obtains live improving than enzyme is continued to utilize saturation mutation, changes the polarity effect between the MTG internal amino acid, then improve MTG than enzyme alive and thermostability.This research provides a kind of new transformation thinking, in conjunction with design and rational and saturation mutation, improves transformation efficiency, and has obtained living and the thermally-stabilised mutant strain all improved than enzyme.

In early-stage Study, this research department filters out the bacterial strain (Streptomyces hygroscopicus CCTCC NO.M203062) of the new product Transglutaminase EC2.3.2.13 of a strain, pass through gene clone method, MTG gene order and upstream and downstream sequence thereof have been obtained, promotor and terminator (Genebank:EU477523) containing MTG self, and MTG and high efficient expression (the Liu S of its proenzyme district in intestinal bacteria have been realized, Zhang D, Wang M, Cui W, Chen K, Liu Y, Du G, Chen J, Zhou Z (2011) The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli.FEMS Microbiol Lett 324 (2): 98-105).Based on the intestinal bacteria construction platform, we are lacked and saturation mutation MTG maturing enzyme N terminal amino acid, have obtained zymologic property mutant strain preferably.

The present invention's substratum used:

The LB substratum: Tryptones 10g/L, yeast powder 5g/L, NaCl 10g/L, pH 7.0;

TB substratum: peptone 12g/L, yeast extract paste 24g/L, glycerine 8g/L, 17mmol/L KH ₂pO ₄, 72mmol/LK ₂hPO ₄.

The mensuration of Transglutaminase EC2.3.2.13 vigor in the present invention:

The colorimetric method for determining enzyme is lived: take N-α-CBZ-GLN-GLY as the effect substrate, the mono-Hydroxylamine HCL of Pidolidone-γ is done typical curve.The Transglutaminase EC2.3.2.13 enzyme work of 1 unit is defined as: during 37C, per minute catalysis forms the enzyme amount (U/mL) of the mono-Hydroxylamine HCL of 1 μ mol Pidolidone-γ.

N α-CBZ-GLN-GLY of reagent A: 100mg is dissolved in the NaOH solution of 2mL 0.2moL/L, adds the Tris-HC damping fluid 4mL of 0.2mol/L pH6.0,0.1mol/L azanol 2mL, and the reduced glutathion 2mL of 0.01mol/L, and regulate pH to 6.0.

The HCL of reagent B:3mol/L, 12%TCA, 5%FeCL ₃pressing 1:1:1 mixes.

Pidolidone-γ of preparation 0-4 μ mol/mL-mono-hydroxamic acid standardized solution.Get the Pidolidone-γ of 1mL reagent A and 0.4mL different concns-mono-hydroxamic acid standardized solution and mix, 37 ℃ of water-baths 10 minutes.Add 0.4mL reagent B termination reaction, in the 525nm colorimetric, draw out typical curve.Replace standardized solution with 0.4mL through the enzyme liquid of suitably dilution, insulation and colorimetric, obtain enzyme from typical curve and live under the same conditions.Supernatant liquor after the 100C of take heating 10 minutes centrifugal is blank.Enzyme activity (u/mL)=(6.8548 * OD ₅₂₅-0.0164) * extension rate

The present invention efficiently is expressed as the transformation platform with MTG in intestinal bacteria, and MTG maturing enzyme N terminal amino acid is lacked and saturation mutation, has obtained zymologic property mutant strain preferably, than enzyme, lives and improves 1.85 times, and thermostability improves 2.7 times.Improved enzyme is more suitable for industrial application, can reduce production costs, and enhances productivity.

The accompanying drawing explanation

The MTG crystalline structure simulation of Fig. 1 S.hygroscopicus source

Fig. 2 TGase N end disappearance fermentation supernatant (A) and purifying protein SDS-PAGE (B)

Fig. 3 saturation mutation strain fermentation supernatant enzyme is lived and heat stability test

Table 1MTG fermentation supernatant enzyme is lived and the TGase zymologic property

Table 2MTG mutant strain zymologic property

Embodiment

Embodiment 1: the MTG crystalline structure simulation of streptomyces hygroscopicus source

The TGase crystalline structure of the S.mobaraensis that reported of take is template, and in the swiss-model website (http: the ∥ swissmodel.expasy.org/) crystalline structure of simulation S.hygroscopicus TGase, as shown in Figure 1.

Embodiment 2: the acquisition of high reactivity mutant strain (Del1-4)

1, utilize rite-directed mutagenesis test kit (TaKaRa), lack respectively 7 amino acid (Asp 1, Ala 2, Ala 3, Asp 4, Glu 5, Arg 6, Val 7) of N end, lack an amino acid called after Del 1, by that analogy, seven amino acid called after Del1-7 before disappearance, transformant is given birth to work by Shanghai and is checked order.

2, the correct plasmid that will check order, Transformed E .coli BL 21, select transformant and be inoculated in the LB liquid nutrient medium, and 37 ℃, cultivate 12h, be transferred in the TB substratum, inoculum size is 3%.Thalline grows to OD ₆₀₀be 2 o'clock, add IPTG to induce, and culture temperature is dropped to 20 ℃, cultivate 48h.

3, collect the fermentation supernatant, detect fermentation supernatant enzyme and live, and sample is carried out to the His-ni-sepharose purification, the purifying protein electrophoresis as shown in Figure 2.

4, the ratio enzyme of the albumen of purifying is lived and the Km value is measured, result is as shown in table 1.Experimental result shows Del 1, than enzyme work, is improved, and the obvious reduction but Del 1-2 enzyme is lived, when the enzyme work of disappearance to Del 1-3, Del 1-4 is improved significantly, and disappearance can't detect enzyme to Del 1-7 and lives to Del 1-5, the reduction alive of Del 1-6 enzyme.To the sequence analogue crystals structure in swiss-model after disappearance, as shown in Figure 3, due to the disappearance that is the N terminal amino acid, the TGase structure there is not change substantially, but the position influence for N end loop structure is larger, Del 1, Del 1-3, Del 1-4, the N end of Del 1-5 has the upstream, active centre to transfer to a side, the loop of Del 1-2 is still in the upstream, active centre, and more close active centre, detect the Km value simultaneously, the Km value of Del 1-2 is bigger than normal, illustrate that disappearance N end 1-2 has affected the binding ability of TGase and substrate, and Del 1-3, the Km value of Del 1-4 is compared less than normal with contrast, illustrate that N end transfers to a side by the upstream, active centre, contribute to the combination of substrate.

Table 1 MTG fermentation supernatant enzyme is lived and the TGase zymologic property

Embodiment 3: the acquisition of high reactivity and thermally-stabilised mutant strain (TG-Del 1-4E5D)

Utilize rite-directed mutagenesis, the Del 1-4 based on obtaining in embodiment 2 is the sudden change starting strain, continues the 5th amino acid of the N of MTG end is carried out to saturation mutation, and the correct son obtained is carried out to Transformed E .coli BL 21, and fermentation condition is the same.The fermentation supernatant is carried out to enzyme biopsy survey, as Fig. 3, and fermented liquid is carried out to the thermostability bigness scale, process 10min(Fig. 3 C for 55 ℃).Obtain 4 strains under enzyme is lived undiminished situation, the mutant strain that thermostability improves.To the mutant strain purifying, detect zymologic property, as shown in table 2,4 amino acid of mutant strain TG-Del 1-4E5D(disappearance N end, and five amino acid E is mutated into to D) thermostability and live and all be significantly increased than enzyme.

Table 2 MTG mutant strain zymologic property

Claims

1. the glutamine transaminage that an enzymic activity and thermostability improve, is characterized in that its aminoacid sequence is for only to sport by E the aminoacid sequence that D obtains by aminoacid sequence the first amino acids shown in SEQ ID NO.1.

2. the method for preparing the described glutamine transaminage of claim 1, the carrier that it is characterized in that containing streptomyces hygroscopicus (Streptomyces hygroscopicus) TGase is material, utilize the rite-directed mutagenesis test kit to obtain the aminoacid sequence of the described glutamine transaminage of claim 1, carrier after sudden change transforms intestinal bacteria, obtains the glutamine transaminage that enzymic activity claimed in claim 1 and thermostability improve after the fermentation purifying.