CN103421761B

CN103421761B - Heatproof trehalose synthase fusion enzyme mutant gene and application thereof

Info

Publication number: CN103421761B
Application number: CN201310369255.0A
Authority: CN
Inventors: 韦航; 韦宇拓
Original assignee: ZHONGNUO BIOENGINEERING CO Ltd NANNING
Current assignee: Nanning baiou Bioengineering Co.,Ltd.
Priority date: 2013-08-23
Filing date: 2013-08-23
Publication date: 2014-11-26
Anticipated expiration: 2033-08-23
Also published as: CN103421761A

Abstract

The invention relates to a heatproof trehalose synthase fusion enzyme mutant gene and an application thereof. On the basis of optimized trehalose synthase of streptomyces roseus, an amino acid sequence capable of improving heat stability is linked to a C-end to construct to obtain a new fusion enzyme mutant; optimal reaction temperature of the trehalose synthase fusion enzyme mutant is increased from 20-25 DEG C to 50-55 DEG C, heat stability is also improved, yield of trehalose from catalytic maltose is 79% and higher than that, of 67%, of trehalose from Thermus thermophilus trehalose synthase, and the rehalose synthase fusion enzyme mutant is more beneficial for reducing production cost of the trehalose.

Description

A kind of thermostable mycose synthetic enzyme merges enzyme mutant gene and application thereof

Technical field

The present invention relates to genetically engineered and enzyme engineering field, specifically a kind of thermostable mycose synthetic enzyme merges enzyme mutant gene and application thereof, and the TreP of this genetic expression can be for the production of trehalose.

Background technology

Trehalose be by two molecule glucoses with α, α-1, the non-reducing disaccharide that 1 glycosidic link is connected and forms, is present in microorganism, shrimps, yeast saccharomyces cerevisiae, mushroom and various fungi, insect, plant etc. widely.Ectogenic trehalose has good non-specific provide protection to organism and biomacromolecule, microbial film equally; it can avoid the infringement causing due to after environmental change (as dehydration, height ooze) variation by Cell protection; be embodied in the effective protection to microbial film, protein and DNA etc., be therefore described as " sugar of life ".The content of natural middle trehalose is very low, and the past is mainly to extract from dry yeast, and because content is low, leaching process is more complicated, and cost is high.So expensive trehalose can only be confined to some special dimension as the application of the activity keeping of medical biotechnology goods, development and progress along with human society, the delicate flavour that hope can be applied to trehalose the conventional varieties of food items of everyday life keeps and improved texture, better life and the health level that improves people, therefore must find cheapness and efficient trehalose manufacture method as soon as possible.

TreP can be take maltose as substrate, by the α in maltose, α-1, the maltose that 4 glycosidic links connect is converted into α, α-1,1 glycosidic link, maltose is transformed into trehalose, maltose can generate by Starch Hydrolysis, thereby TreP has very strong competitive edge in suitability for industrialized production trehalose.

At present existing many documents with patent report the clone of trehalose synthesis related gene with separated; and the relevant enzyme of these trehalose synthesize enzyme genes is for the preparation of trehalose; but the route of synthesis of the synthetic relevant enzyme of these trehaloses is different; or bacterial classification source is different; caused DNA sequence dna and aminoacid sequence to differ greatly; the molecular weight of enzyme, enzymatic property and the enzyme transformation efficiency to substrate, and major part does not also obtain the application of industrial scale at present.

A reversible reaction when transforming maltose and generate trehalose due to trehalase, and along with the height of temperature of reaction, trehalose yield can reduce, and can increase the generation of by product glucose.At present clone's TreP can be divided into two large classes, and a kind of is the TreP of middle low temperature, as Thermobifida fusca and Pimelobacter sp.R48 are respectively 25 ℃ and 20 ℃ with its optimum temperuture.Be a resistant to elevated temperatures TreP, as the TreP from Thermus thermophilus, its optimal reactive temperature is 60-65 ℃.The advantage of the TreP of middle low temperature is compared with heat-resisting trehalose, when optimal reactive temperature, when reaction reaches balance, the yield of trehalose is higher, content of trehalose in the TreP reaction product of low temperature can reach more than 70%, and if the TreP from Pimelobacter sp.R48 is 25 ℃ time, the yield of trehalose is 76.7%, from Thermobifida fusca 20 ℃ time, the yield of trehalose is 68.8%, and in the time of 30 ℃, the yield of trehalose is 60.0%.And heat-resisting TreP, if the TreP from Thermus thermophilus is 65 ℃ time, trehalose yield is 61.6%, although when temperature of reaction is reduced to 20 ℃, can make trehalose yield bring up to 76.0%, but need speed of response greatly to lower.Although the TreP of middle low temperature have substrate conversion efficiency high aspect there is advantage, along with the rising of temperature, the transformation efficiency of its substrate can sharply decline, and can produce more by product, and the heat-retaining condition of low temperature is had relatively high expectations.The thermostability of thermostable mycose synthetic enzyme is higher, at high temperature can also keep good catalytic activity, can adapt to aborning the instability condition of production, the vigor of enzyme is difficult for inactivation under hot conditions, can keep long catalytic capability, be conducive to improve the utilising efficiency of enzyme.In addition thermostable enzyme can react at higher temperature, can reduce the required power consumption of cooling, but also can prevent to a certain extent the pollution of miscellaneous bacteria, and these advantage degree are conducive to reduce production costs.Although heat-resisting TreP has the feature of optimal reactive temperature height and Heat stability is good, but under the temperature of reaction condition higher, can increase the generation of the glucose of by product, thereby reduce the transformation efficiency of substrate, heat-resisting TreP also exists enzyme molecular weight large simultaneously, the feature that speed of response is slow.Therefore trehalose synthase heat-resisting and that trehalose yield is high reduces production costs and has great importance in the industrial production of trehalose.

This research group has identified a plurality of trehalose synthesize enzyme genes from rose streptomycete and other streptomycete, and it is high to find that TreP from rare streptomycete has substrate conversion efficiency, the feature that speed of response is fast, under the reaction conditions of 20-25 ℃, the conversion of substrate reaches more than 80%, see a kind of Streptosporangium roseum trehalose synthase gene of Chinese patent and application thereof, application number 201210011457.3.

Summary of the invention

The object of the invention is to provide a kind of thermostable mycose synthetic enzyme and merges enzyme mutant gene and application thereof, by a plurality of trehalose synthesize enzyme genes, carry out homology compare of analysis, optimization design new trehalose and synthetic enzyme, again and the structure homology modeling analysis of conjugated protein, 415 amino-acid residues of TreP C-terminal from extreme thermophile bacteria (Thermus thermophilus ATCC13939) are connected to the C-terminal that newly-designed trehalose merges enzyme, obtain new algae sugar synthetic enzyme and merge enzyme mutant TreS-Tt Δ, optimal reactive temperature and thermostability that TreP merges enzyme mutant are improved than protoenzyme.

The inventor is in the process of research TreP, from multiple streptomyces gene group Sequence Identification a plurality of trehalose synthesize enzyme genes, it is fast that these TrePs from streptomycete have speed of response, the feature that trehalose yield is high, particularly from the TreP of rose streptomycete, under the suitableeest temperature of reaction condition, react 2 hours content of trehalose and just can reach the highest more than 81%, be to report at present the highest TreP of trehalose yield.In research, also find to there is from 415 amino-acid residues of TreP C-terminal of extreme thermophile bacteria Thermus thermophilus ATCC13939 the function that improves TreP thermostability.Therefore our general solution homology modeling analysis is connected to 415 amino-acid residues of the TreP C-terminal of Thermus thermophilus ATCC13939 the C-terminal of the TreP of the streptomycete through optimizing, obtains a new thermostable mycose synthetic enzyme and merges enzyme mutant.

The present invention compared with prior art, has outstanding substantive distinguishing features and significant advantage:

1. although the TreP from rose streptomycete has trehalose yield height and the fast feature of speed of response, but its optimal reactive temperature is lower, thermostability is reported to the leadship after accomplishing a task, 40 ℃ are incubated 1 hour, the residual activity of enzyme only has an appointment 70%, while surpassing 45 ℃, be incubated 1 hour, the vigor of enzyme completely loses substantially.The TreP that the present invention builds merges TreP substrate conversion efficiency and the fast feature of speed of response that enzyme mutant has retained rose streptomycete, optimal reactive temperature is from 20～25 ℃, bring up to 50～55 ℃, be more conducive to produce in trehalose and improve temperature of reaction in application; Simultaneously the thermally-stabilised of enzyme also improved greatly, can keep catalytic capability more of a specified duration, is conducive to improve the utilizing effect of enzyme and reduces and produce old.

2. the TreP of thermophile bacteria has the advantages that thermostability is high, but it exists trehalose yield lower, the deficiency that by product glucose is more, the TreP that the present invention builds merges enzyme mutant to be compared with the TreP of thermophile bacteria, there is higher transformation efficiency, speed of response faster, react content of trehalose in 2 hours after products and can reach the highest by 78%, and the TreP of thermophile bacteria will react, 5 hours content of trehalose are the highest just reaches 67%, this is favourable enhances productivity and the utilization ratio of substrate, be conducive to reduce production costs.

Accompanying drawing explanation

Fig. 1 is the optimal reactive temperature comparative result figure of three kinds of TrePs.

Fig. 2 is the thermal stability analysis result figure of three kinds of TrePs.

Fig. 3 is the comparative result figure of the differential responses time trehalose yield of three kinds of TrePs.

Embodiment

By the following examples technical scheme of the present invention is described further, described in following experiment and example, content belongs to main contents of the present invention, but be not restricted to these contents, although some is that apparent expansion content does not occur in patent specification to those skilled in the art, also should belong to patent request scope of the present invention.

1. the synthetic and expression of trehalase syzygy variant TreS-Tt gene

By the TreP of streptomycete is carried out, homology compare of analysis is optimized and design in conjunction with homology modeling analysis the DNA sequence dna that trehalase merges enzyme mutant TreS-Tt Δ gene, and sequence is as SEQ ID NO:1.Adopt the method for full total gene synthesis to obtain the DNA sequence dna of TreS-Tt Δ gene, then take carrier pSE380 as expression vector, import e. coli strain bl21 and carry out high efficient expression, the purifying that trehalose merges enzyme TreS-Tt Δ, after purifying, trehalase is for transforming the experiment of maltose.

2. trehalase syzygy variant TreS-Tt Δ transforms the performance of maltose

Take from the low temperature TreP TreS-Ros of rose streptomycete and be contrast from the high temperature resistant TreP Tre-Tt of extreme thermophile bacteria Thermus thermophilus, carry out the performance analysis that trehalase syzygy variant TreS-Tt Δ transforms maltose.The optimal reaction pH value of three kinds of trehaloses is all 6.5-70, there is no notable difference, therefore mainly compares the optimal reactive temperature of three kinds of TrePs, the yield of thermostability and trehalose.By after the marine alga synthetic enzyme quantitative and qualitative analysis of the different sources preparing, 10% the maltose (pH7.0) of take is substrate, under identical reaction system condition, carry out the optimal reactive temperature of enzyme, the thermostability of enzyme, the transformation efficiency analysis of enzyme substrate under optimum reaction conditions.

As shown in Figure 1, insulation reaction 1 hour under 10 ℃ of-80 ℃ of different reaction temperature respectively, then use the content of trehalose in HPLC analytical reaction product, with the highest content of trehalose value, be made as 100, calculate different enzymes relative content of trehalose in product when differential responses temperature, result shows that the optimal reactive temperature of TreP TreS-Ros is 25 ℃, the optimal reactive temperature of high temperature resistant TreP Tre-Tt is 60-65 ℃, and the optimal reactive temperature of trehalose fusion enzyme mutant TreS-Tt Δ is 50-55 ℃.The optimal reactive temperature that shows trehalose fusion enzyme mutant TreS-Tt Δ has improved 30 ℃ than protoenzyme.

As shown in Figure 2, take respectively uninsulated enzyme as contrast, and be made as 100, different marine alga enzymes are incubated after 1 hour under differing temps, taking-up carrys out the thermostability of enzyme analysis at the residual enzyme vigor of the optimum reaction conditions mensuration enzyme of enzyme.Result shows that the thermostability of TreP TreS-Ros is poor, and 40 ℃ are incubated 1 hour, and the residual activity of enzyme only has an appointment 70%, while surpassing 45 ℃, is incubated 1 hour, and the vigor of enzyme completely loses substantially.High temperature resistant TreP Tre-Tt's is best, and 70 ℃ of following insulations 1 hour, residual enzyme vigor can reach more than 90%.Trehalose merge enzyme mutant TreS-Tt Δ thermostability than protoenzyme TreS-Ros, be also improved, 50 ℃ of following insulations 1 hour, residual enzyme vigor can reach more than 90%.

As shown in Figure 3, take 10% maltose as substrate, respectively by TreS-Ros at 25 ℃, TreS-Tt Δ is at 50 ℃, Tre-Tt is at 60 ℃ of reaction different times, and the percentage composition that then in assaying reaction product, content of trehalose accounts for total reducing sugar carrys out speed of response and the transformation efficiency of enzyme analysis.Result shows that the transformation efficiency of low temperature TreP TreS-Ros is the highest, speed of response is fast, react 2 hours content of trehalose and just can reach the highest by 81%, and high temperature resistant TreP Tre-Tt transformation efficiency is lower than the TreP of low temperature, speed of response is also slower, react 5 hours content of trehalose and reach the highest by 67%, trehalose merges enzyme mutant TreS-Tt Δ and has retained the fast feature of the high speed of response of low temperature TreP TreS-Ros transformation efficiency, react after 2 hours with regard to content of trehalose and can reach the highest by 78%, transformation efficiency a little less than TreS-Ros.

Claims

1. TreP merges an enzyme mutant gene, it is characterized in that, its nucleotide sequence is as shown in sequence table SEQ ID NO.1.

2. the TreP of coded by said gene according to claim 1, its aminoacid sequence is as shown in sequence table SEQ ID NO.2.