CN102660515A

CN102660515A - Glutamine transaminase with improved enzymatic activity and thermal stability

Info

Publication number: CN102660515A
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: 2012-09-12
Anticipated expiration: 2032-05-10
Also published as: CN102660515B

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 (protein-L-glutamic acid-Transglutaminase 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 cell that protein itself and protein adheres to, tissue etc. and the characteristic of functional property, improves proteinic nutritive value.Therefore, MTG has a wide range of applications in fields such as food, weaving, bio-pharmaceuticals.But because some defectives of MTG self have limited the range of application of MTG as than factors such as enzyme work, thermostabilitys.Therefore based on the expression platform of the MTG that has obtained in intestinal bacteria, utilize aminoacid deletion and saturation mutation, MTG is carried out molecular modification, in the hope of obtaining the MTG that zymologic property is more suitable for industrial application.

Summary of the invention

Alive lower for improving MTG than enzyme, the present situation of poor heat stability, this research lacks through the N terminal amino acid to MTG, thereby reduces the resistance that combines in substrate and MTG active site, and raising MTG is to the affinity of substrate.Continue to utilize saturation mutation to obtaining than the mutant strain that enzyme live to improve, change the polarity effect between the MTG internal amino acid, improve then 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 and transforms efficient, and obtained living and the thermally-stabilised mutant strain that all improves 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, through gene clone method; Obtain MTG gene order and upstream and downstream sequence thereof, contained promotor and the terminator (Genebank:EU477523) of MTG self, and realized MTG and its proenzyme district (Liu S of efficiently expressing in intestinal bacteria; 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 lack and saturation mutation MTG maturing enzyme N terminal amino acid, have obtained zymologic property mutant strain preferably.

The used substratum of the present invention:

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 among the present invention:

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

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.

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

The present invention serves as to transform platform with MTG efficiently expressing in intestinal bacteria, and MTG maturing enzyme N terminal amino acid is lacked and saturation mutation, has obtained zymologic property mutant strain preferably, lives than enzyme 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.

Description of drawings

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

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

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

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

2, the correct plasmid that will check order, Transformed E .coli BL 21 selects transformant and is inoculated in the LB liquid nutrient medium, and 37 ℃, cultivate 12h, be transferred in the TB substratum, inoculum size is 3%.Thalline is long to OD ₆₀₀Be 2 o'clock, add IPTG and 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 the His-ni-sepharose purification, the purifying protein electrophoresis is as shown in Figure 2.

4, the work of ratio enzyme and the Km value of purified proteins are measured, the result is as shown in table 1.Experimental result shows Del 1, than enzyme work raising is arranged, but Del 1-2 enzyme lives and obviously to reduce, when the enzyme work that lacks Del 1-3, Del 1-4 is improved significantly, and lack Del 1-5, Del 1-6 enzyme is lived and is reduced, and then detects less than enzyme alive to Del 1-7.To the sequence analogue crystals structure in swiss-model after the disappearance, as shown in Figure 3, owing to be the disappearance of N terminal amino acid; The TGase structure there is not change basically, but bigger for the position influence of N end loop structure, the N end of Del 1, Del 1-3, Del 1-4, Del 1-5 all has the upper reaches, active site to transfer to a side; The loop of Del 1-2 is then still at the upper reaches, active site; And more near the active site, detect the Km value simultaneously, the Km value of Del 1-2 is bigger than normal; Explain that disappearance N end 1-2 has influenced the binding ability of TGase and substrate; And that the Km value of Del 1-3, Del 1-4 is compared with contrast is less than normal, explains that the N end by transferring to a side in the upper reaches, active site, helps 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, be the sudden change starting strain based on the Del 1-4 that obtains among the embodiment 2, continue to hold the 5th amino acid to carry out saturation mutation the N of MTG, the correct son that obtains is carried out Transformed E .coli BL 21, fermentation condition is the same.The fermentation supernatant is carried out the enzyme biopsy survey,, and fermented liquid carried out the thermostability bigness scale, handle 10min (Fig. 3 C) for 55 ℃ like Fig. 3.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, the thermostability of mutant strain TG-Del 1-4E5D (4 amino acid of disappearance N end, and be mutated into D with five amino acid E) and live than enzyme all is significantly increased.

Table 2 MTG mutant strain zymologic property

Claims

1. the glutamine transaminage that improves of enzymic activity and thermostability is characterized in that aminoacid sequence is shown in SEQ ID NO.1.

2. the described glutamine transaminage of claim 1 is characterized in that and can also sport D by E for primary amino acid.

3. the method for preparing the said glutamine transaminage of claim 1; It is characterized in that with the carrier that contains S.hygroscopicus TGase be material; Utilize the rite-directed mutagenesis test kit to obtain the aminoacid sequence shown in the SEQ ID NO.1; Carrier transformed into escherichia coli after the sudden change obtains the glutamine transaminage that described enzymic activity of claim 1 and thermostability improve behind the fermentation purifying.