阅读说明：本技术 酶活提高的麦芽寡糖基海藻糖合成酶突变体 (Maltooligosyl trehalose synthetase mutant with improved enzyme activity ) 是由 吴敬 宿玲恰 姚锴琳 于 2017-09-13 设计创作，主要内容包括：本发明公开了酶活提高的麦芽寡糖基海藻糖合成酶突变体,属于基因工程和酶工程技术领域。本发明提供了一系列酶活提高的麦芽寡糖基海藻糖合成酶突变体。突变体G586D、F284V/G586D、F284V/T439A/G586D麦芽寡糖基海藻糖合成酶的酶活分别为野生酶的1.4倍、2.4倍、3.4倍。(The invention discloses a maltooligosyl trehalose synthase mutant with improved enzyme activity, belonging to the technical field of genetic engineering and enzyme engineering. The invention provides a series of maltooligosyl trehalose synthetase mutants with improved enzyme activity. The enzyme activities of the mutants G586D, F284V/G586D and F284V/T439A/G586D maltooligosyl trehalose synthetase are respectively 1.4 times, 2.4 times and 3.4 times of that of the wild enzyme.)

1. A mutant of maltooligosaccharyl trehalose synthase which is characterized by changing glycine (Gly) at position 586 of maltooligosaccharyl trehalose synthase derived from Sulfolobus acidocaldarius into aspartic acid (Asp);

or, the mutant is obtained by changing phenylalanine (Phe) at position 284 into valine (Val) and simultaneously changing glycine (Gly) at position 586 into aspartic acid (Asp), thus obtaining F284V/G586D;

or, the mutant is that glycine (Gly) at position 432 is changed into aspartic acid (Asp), and glycine (Gly) at position 586 is changed into aspartic acid (Asp), so as to obtain G432D/G586D;

or, the mutant is obtained by changing phenylalanine (Phe) at position 284 to valine (Val), threonine (Thr) at position 439 to alanine (Ala) and glycine (Gly) at position 586 to aspartic acid (Asp), and F284V/T439A/G586D is obtained.

2. A gene encoding the mutant of claim 1.

3. A vector or recombinant cell carrying the gene of claim 2.

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

(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 synthase gene as a template; constructing a plasmid vector containing a gene encoding the mutant;

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

(3) selecting positive clones, carrying out fermentation culture, centrifuging and collecting cells, wherein the cell wall-broken supernatant is the crude enzyme solution of the maltooligosyl trehalose synthase mutant.

5. Use of the mutant of claim 1 for the production of trehalose.

6. Use of the gene of claim 2 for the production of trehalose.

7. Use of the vector or recombinant cell of claim 2 for the production of trehalose.

8. An enzyme preparation for producing trehalose, comprising the mutant of claim 1.

Technical Field

The invention relates to a maltooligosyl trehalose synthase mutant with improved enzyme activity, belonging to the technical field of genetic engineering and enzyme engineering.

Background

Trehalose (trehalase) is a stable non-reducing oligosaccharide formed by connecting two glucopyranose molecules through alpha, alpha-1, 1-glycosidic bonds, has high safety and good stability, and is widely applied to the fields of medicine, food, cosmetics, agriculture and the like. Since 1995, trehalose was approved by japan, the united states, and the european union, etc. to be used as a food additive. In 2005, trehalose was officially approved as a new resource food by the ministry of health in China.

In 1993, Japanese forest primary research institute firstly discovers that maltooligosyl trehalose synthetase (MTSase) and maltooligosyl trehalose hydrolase (MTHase) take liquefied starch as a substrate, the two enzymes act synergistically to generate trehalose, and industrial production of trehalose is realized first. At present, the existing companies in China begin to produce trehalose, but certain differences exist between product performance and yield and imported products, and the production cost is always high. More seriously, the price of trehalose is continuously reduced to meet the market demand, which brings great challenge and pressure to trehalose production. Therefore, how to improve the productivity of the trehalose and realize the low-cost large-scale preparation of the trehalose makes the trehalose to be a hotspot concerned by the academic world and the industrial world.

Currently, trehalose is produced industrially mainly by two methods: firstly, trehalose is generated by taking trehalose synthetase and maltose as a substrate through intramolecular transglycosylation; ② the trehalose is produced by taking starch liquefied liquid as a substrate and carrying out the synergistic action of maltooligosyl trehalose synthetase (MTSase) and maltooligosyl trehalose hydrolase (MTHase). The trehalose yield of both methods was about 80%. However, in view of production cost and cycle, the process for preparing trehalose by using starch as a substrate is simpler and the cost is lower. Therefore, the synergistic effect of the maltooligosyl trehalose synthetase (MTSase) and the maltooligosyl trehalose hydrolase (MTHase) to produce trehalose is more advantageous.

The domestic and foreign two-enzyme process for producing trehalose includes medium and low temperature enzyme systems (trehalose conversion: Arthrobacter sp. Q36: 80%, Arthrobacter ramosus S34: 66%, Brevibacterium helolum: 70.4%) and high temperature enzyme systems (Sulfolobus solfataricus KM 1: 81.5%, Sulfolobus acidocaldarius ATCC 33909: 80.2%). The high-temperature enzyme system generally has higher trehalose conversion rate and good thermal stability, can convert starch at higher temperature to generate trehalose, and is not easy to pollute mixed bacteria in the production process. However, the high temperature enzyme system has the disadvantages of low protein expression level and low specific enzyme activity compared with the medium and low temperature enzyme system, and is not favorable for industrial application.

Disclosure of Invention

Based on the current situation, the invention utilizes the means of gene engineering and enzyme engineering to improve the enzyme activity of the maltooligosyl trehalose synthase and create conditions for the industrial production of the maltooligosyl trehalose synthase.

The present invention provides mutants of maltooligosaccharyl trehalose synthase having improved enzymatic activity, including mutants obtained by substituting one or more of the amino acids at position 81, 263, 284, 432, 439, 583, 585, 586, 611, or 615 of maltooligosaccharyl trehalose synthase derived from Sulfolobus acidocaldarius. These mutants have an improved enzymatic activity as compared with the maltooligosyl trehalose synthase of its parent.

In one embodiment of the invention, the sulfolobus acidocaldarius (s.acidocaldarius).

In one embodiment of the invention, the amino acid sequence of the maltooligosaccharyl trehalose synthase from sulfolobus acidocaldarius is shown as SEQ ID No. 1.

In one embodiment of the present invention, the 81 th glycine (Gly) is changed to serine (Ser), and the mutant is named G81S.

In one embodiment of the present invention, the mutant is obtained by converting glutamic acid (Glu) at position 263 to glycine (Gly) and is named as E263G.

In one embodiment of the invention, the mutant is a mutant wherein the phenylalanine (Phe) at position 284 is changed to valine (Val), and the mutant is designated F284V.

In one embodiment of the present invention, the mutant is obtained by changing glycine (Gly) at position 432 into aspartic acid (Asp), and is named G432D.

In one embodiment of the present invention, the mutant is a mutant in which threonine (Thr) at position 439 is changed to alanine (Ala), and the mutant is named T439A.

In one embodiment of the invention, the mutant is a mutant wherein phenylalanine (Phe) at position 583 is changed to leucine (Leu) and the mutant is designated F583L.

In one embodiment of the present invention, the mutant is a mutant in which glutamine (Gln) at position 585 is changed to arginine (Arg), and the mutant is named Q585R.

In one embodiment of the invention, the mutant is obtained by changing glycine (Gly) at position 586 to aspartic acid (Asp) and is designated G586D.

In one embodiment of the present invention, the mutant is a mutant in which isoleucine (Ile) at position 611 is changed to threonine (Thr), and the mutant is named as I611T.

In one embodiment of the present invention, the mutant is obtained by converting serine (Ser) at position 615 into glycine (Gly), and is named as S615G.

In one embodiment of the invention, the mutant is obtained by changing phenylalanine (Phe) at position 284 to valine (Val) and threonine (Thr) at position 439 to alanine (Ala), resulting in F284V/T439A.

In one embodiment of the invention, the mutant is obtained by changing phenylalanine (Phe) at position 284 to valine (Val) and glycine (Gly) at position 586 to aspartic acid (Asp), resulting in F284V/G586D.

In one embodiment of the invention, the mutant is a T439A/G586D obtained by changing threonine (Thr) at position 439 to alanine (Ala) and glycine (Gly) at position 586 to aspartic acid (Asp).

In one embodiment of the invention, the mutant is a mutant wherein threonine (Thr) at position 439 is changed to alanine (Ala) and glutamine (Gln) at position 585 is changed to arginine (Arg), resulting in T439A/Q585R.

In one embodiment of the invention, the mutant is obtained by converting glycine (Gly) at position 432 into aspartic acid (Asp) and converting glycine (Gly) at position 586 into aspartic acid (Asp), resulting in G432D/G586D.

In one embodiment of the present invention, the mutant is G81S/F284V/S615G obtained by changing glycine (Gly) at position 81 to serine (Ser), phenylalanine (Phe) at position 284 to valine (Val), and serine (Ser) at position 615 to glycine (Gly).

In one embodiment of the present invention, the mutant is obtained by changing phenylalanine (Phe) at position 284 to valine (Val), threonine (Thr) at position 439 to alanine (Ala), glycine (Gly) at position 586 to aspartic acid (Asp), resulting in F284V/T439A/G586D.

In one embodiment of the present invention, the mutant is a mutant wherein glycine (Gly) at position 81 is changed to serine (Ser), phenylalanine (Phe) at position 284 is changed to valine (Val), threonine (Thr) at position 439 is changed to alanine (Ala), and serine (Ser) at position 615 is changed to glycine (Gly), resulting in G81S/F284V/T439A/S615G.

In one embodiment of the present invention, the mutation is obtained by changing glycine (Gly) at position 81 to serine (Ser), phenylalanine (Phe) at position 284 to valine (Val), or glycine (Gly) at position 586 to aspartic acid (Asp), and changing serine (Ser) at position 615 to glycine (Gly) G81S/F284V/G586D/S615G.

In one embodiment of the present invention, the mutant is a mutant wherein glutamic acid (Glu) at position 263 is changed to glycine (Gly), phenylalanine (Phe) at position 284 is changed to valine (Val), phenylalanine (Phe) at position 583 is changed to leucine (Leu), isoleucine (Ile) at position 611 is changed to threonine (Thr), and serine (Ser) at position 615 is changed to glycine (Gly), giving E263G/F284V/F58 583L/I611T/S615G.

Another technical problem to be solved by the invention is to provide a preparation method of the maltooligosyl trehalose synthase 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 synthase gene as a template; plasmid vectors containing the genes encoding the mutants were constructed.

(2) The mutant plasmid is transformed into a host cell.

(3) Selecting positive clones, carrying out fermentation culture, centrifuging and collecting cells, wherein the cell wall-broken supernatant is the crude enzyme solution of the maltooligosyl trehalose synthase mutant.

The plasmid vector, in one embodiment of the present invention, is any one of the pET series, pUC series, or pGEX series.

The host cell is a bacterial or fungal cell.

The bacteria are gram-negative bacteria or gram-positive bacteria.

The invention provides a series of maltooligosyl trehalose synthetase mutants with improved enzyme activity in host bacteria. Under proper culture conditions, the activity of mutants G81S, E263G, F284V, G432D, T439A, F583L, Q585R, G586D, I611T, S615G, F284V/T439A, F284V/G586D, G432D/G586D, T439A/Q585R, T439A/G586D, G81S/F284V/S615 72, F284V/T439V/G586V, G81V/F284V/T439V/S615V, G81V/F284V/G586 72/S615V, E263V/F284/F583/I611V/S615V, E263V/F583/I611V/S V, the activity of the trehalose synthase is 1.2 times, 1.2.1.1.4 times, 1.2.1.4 times, 1.2.4 times, 1.4 times, 1.2 times, 1.4 times, 1.2 times, 1 times, 1.4 times, 1 times, 2 times, 1.4 times, 2 times of the activity of the.

Detailed Description