CN107142253B

CN107142253B - Xylanase mutant and preparation method and application thereof

Info

Publication number: CN107142253B
Application number: CN201710176527.3A
Authority: CN
Inventors: 姚斌; 罗会颖; 游帅; 王苑; 涂涛; 黄火清; 苏小运; 柏映国; 王亚茹
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Animal Science of CAAS
Priority date: 2017-03-23
Filing date: 2017-03-23
Publication date: 2020-01-21
Anticipated expiration: 2037-03-23
Also published as: CN107142253A

Abstract

A xylanase mutant with high catalytic efficiency and high temperature resistance, a preparation method and application thereof relate to the field of genetic engineering and genetic engineering. The invention takes GH10 family xylanase XYL10C derived from filamentous fungus Bispora sp MEY-1 as a female parent, and adopts a molecular biology technology to carry out saturation mutation on the 175 th site E in the female parent, and the results show that the catalytic efficiency and the specific activity of eight mutants of the mutant XYL10C-CutN, E175H, E175N, E175D, E175V, E175L, E175Q and E175M are greatly improved compared with those of wild enzymes, and the important role of the site in the high specific activity and the high catalytic efficiency of XYL10C is discovered and researched through the saturation mutation, so that the method has important guiding significance for researching the high-efficiency catalytic mechanism of xylanase and other (alpha/beta) 8 barrel enzymes.

Description

Xylanase mutant and preparation method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a xylanase mutant with high catalytic efficiency and high temperature resistance, and a preparation method and application thereof.

Background

Cellulose, hemicellulose and lignin are the major components that make up the cell wall of plants, accounting for approximately 50% of the biomass in the world. Hemicellulose is mainly present on the surface of cell walls, and cellulose is wrapped in the hemicellulose, which in turn is covalently linked with lignin to form a network structure. Xylan is the main component of hemicellulose, is a renewable resource with the content second to cellulose in nature, and almost occupies one third of the organic carbon content of a sphere. Xylan has a complex structure, the main chain of the xylan is connected by xylopyranose through beta-D-1, 4-xyloside bonds, and the xylan has various substituents.

Due to the diversity and structural complexity of xylan, its complete hydrolysis requires the synergy of multiple enzymes to complete. Comprises xylanase, xylosidase, alpha-D-glucuronidase of hydrolytic side chain substituent group, acetyl xylan esterase, ferulic acid esterase, coumaric acid esterase and the like. By the synergistic action of these enzymes, xylan can be efficiently hydrolyzed. Among them, xylanase can degrade the beta-1, 4-glycosidic bond of xylan backbone, and is an enzyme playing the most important role in the degradation process of xylan. The xylanase has wide application value in the feed industry, the food industry, the paper industry, the energy industry, the textile industry, the medicine industry and other industries.

The molecular improvement of xylanase mainly focuses on the research and improvement of thermal stability mechanism. Ding et al (2013) investigated the thermolabile region of xylanase Sl-XlnA from Streptomyces lividans by molecular dynamics simulation to determine the factors affecting the thermostability of Sl-XlnA. Gallardo et al enhanced 20-fold the thermostability of xylanase Xyn10B from Paenibacius barcinonsis by forced evolution. Kamondi et al found that the optimum temperature was increased for 4 hybrids by random fragment substitution of two 10 families of xylanases by family shamilffing. However, the research on the mechanism and improvement of other properties of the xylanase of the 10 th family is very little, and particularly, the research on the efficient catalytic mechanism and improvement of the xylanase of the 10 th family is not reported.

Disclosure of Invention

An object of the present invention is to protect the amino acid sequences of xylanase obtained by cutting off the N-terminal sequence from XYL10C and xylanase mutants obtained by mutating the E175 site in XYL10C, and to investigate in detail the roles played by the N-terminal sequence and the E175 site in the high catalytic efficiency of XYL 10C.

It is still another object of the present invention to provide a recombinant vector comprising the E175 saturation mutant gene.

It is another object of the present invention to provide a recombinant strain comprising an E175 saturation mutant gene.

The full length of the wild type amino acid sequence is shown as SEQ ID NO. 1.

MSFHSLLISGLLASVAVAVPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDT YSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

The wild type mature amino acid sequence of the mutant is shown as SEQ ID NO. 2.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF QPDGPNTPLVKKAAYDGCLQALQHKAESP

The sequence of the mutant (wild type amino acid minus N end) claimed by the invention is shown in SEQ ID NO. 3.

WGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSELPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPST FAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175H is shown in SEQ ID NO. 4.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSHLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF QPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175N is shown in SEQ ID NO. 5.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADG KLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSNLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGIT VWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175D is shown in SEQ ID NO. 6.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSDLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF QPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175V is shown in SEQ ID NO. 7.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSVLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNM ADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF QPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175L is shown in SEQ ID NO. 8.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSLLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF QPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175Q is shown in SEQ ID NO. 9.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIW QSQLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYY ATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFFQPDGPNTPLVKKAAYDGCLQALQHKAESP

The amino acid sequence of the mutant E175M is shown in SEQ ID NO. 10.

VPKEAWGITVTETKTVSTTIIATVTELGTCSSTITSPTSDATTTTTSSATNTNPTTTLLATPQPSNWGLNNAARADGKLWFGTAADIPGLEQDDRYYMKEYNNTHDFGGTTPANIMKFMFTEPEQNVFNFTGAQEFLDIAFASHKLVRCHNLIWQSMLPTWVTNPTTNWTNETLSKVLQNHVYTLVSHFGDQCYSWDVVNEALSDDPAGSYQNNIWFDTIGPEYVAMAFEYAEKAVKDHKLNVKLYYNDYNIEYPGPKSTAAQNIVKELKARNIQIDGVGLESHFIAGETPSQATQITNMADFTSLDIDVAVTELDVRLYLPPNATSEAQQVADYYATVAACAATERCIGITVWDFDDTYSWVPSTFAGQGYADLFF QPDGPNTPLVKKAAYDGCLQALQHKAESP

The invention also provides a method for preparing the xylanase mutant, which comprises the following technical scheme:

1. designing mutation primers as shown in table 1, and obtaining complete gene segments of the mutants;

2. cloning the obtained mutant gene fragment to an expression vector pPIC9 to obtain a mutant recombinant plasmid;

3. and (3) converting the mutant recombinant plasmid into pichia pastoris GS115, and performing induced expression to obtain the xylanase with high catalytic efficiency.

The invention also provides a method for preparing xylanase, which comprises the following steps:

1. transforming yeast host cells by using the recombinant vector to obtain a recombinant strain;

2. culturing the recombinant strain, and inducing the expression of the recombinant xylanase;

3. recovering and purifying the expressed xylanase.

According to the invention, N-terminal truncation and E175 site saturation mutation are carried out on mature xylanase XYL10C to construct 21 mutants including E175 site deletion, a mutant recombinant vector is transformed into Pichia pastoris GS115, and tubule induction expression shows that the other 19 mutants have activity except that no activity is detected in tubule supernatants of three mutants of XYL10C-CutE and XYL 10C-E175W.

19 active mutants and the wild enzyme XYL10C were subjected to large flask induction, protein concentration and purification, and then basic enzymological properties were determined, as shown in Table 2, and as determined by beech xylan, 18 mutants among E175 site saturation mutations and mutant XYL10C-CutN had optimum temperatures of about 85 degrees, optimum pH of about 4.0-4.5, and pH ranges similar to each other.

Compared with the wild enzyme XYL10C, the specific activities of the mutant XYL10C-CutN, E175H, E175N, E175D, E175V, E175L, E175Q and E175M are respectively improved by 2.1 times, 2.4 times, 1.7 times, 1.9 times, 2.0 times, 1.4 times, 2.5 times and 4.3 times. Compared with the wild enzyme XYL10C, the catalytic efficiency of the mutant XYL10C-CutN, E175H, E175N, E175D, E175V, E175L, E175Q and E175M is respectively improved by 1.7 times, 2.3 times, 2.1 times, 1.4 times, 1.9 times, 1.7 times, 2.3 times and 4.9 times.

The invention proves that the key catalytic related site E175 site in the material XYL10C is confirmed, and the importance of the site on the high specific activity and the high catalytic efficiency of the enzyme is proved, namely the E175 site plays an important role in the high specific activity and the high catalytic efficiency of the enzyme. Provides important clues for researching the high-efficiency catalytic mechanism of the material XYL10C, and simultaneously provides a reliable reference site for improving the catalytic efficiency of other xylanases in the family, thereby proving the decisive role of the site in the catalytic efficiency of the family xylanases.

Drawings

FIG. 1: the crystal structure of the GH10 family xylanase XYL10C-CutN and the position of the E175 site in the three-dimensional structure.

FIG. 2: electrophoresis pictures of protein purification of the wild enzyme XYL10C, the mutant XYL10C-CutN and 18 active mutants.

FIG. 3: (FIGS. 3-1 and 3-2) pH optima for the wild-type enzyme XYL10C, the mutant XYL10C-CutN and the 18 active mutants.

FIG. 4: (FIGS. 4-1 and 4-2) temperature optima for the wild-type enzyme XYL10C, the mutant XYL10C-CutN, and the 18 active mutants.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

TABLE 1 primer construction of XYL10C-CutN and E175 saturation mutant with XYL10C as female parent

Test materials and reagents

1. Bacterial strain and carrier: the expression host Pichia pastoris GS115 and the expression plasmid vector pPIC9r are stored in the laboratory.

2. Enzymes and other biochemical reagents: the endonuclease was purchased from Fermentas, the recombinase from Quantum, and the zelkova from Sigma. The others are domestic analytical pure reagents (all can be purchased from common biochemical reagents).

3. Culture medium:

(1) LB culture medium: 0.5% yeast extract, 1% peptone, 1% NaCl, pH 7.0.

(2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose.

(3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin.

(4) BMGY medium: 1% yeast extract, 2% peptone, 1% glycerol (V/V), 1.34% YNB, 0.00004% Biotin.

(5) BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% Biotin, 0.5% methanol

(V/V)。

EXAMPLE 1 preparation of mutant XYL10C-CutN recombinant vector

GH10 family xylanase XYL10C-pPIC9R from Bispora sp & MEY-1 is used as a template, a primer XYL 10C-CutN-F is designed at a nucleic acid sequence corresponding to an amino acid sequence WGLNN and a primer XYL 10C-CutN-R is designed at a nucleic acid sequence corresponding to the end of the whole amino acid sequence, an amplification product is directly connected with a cut pPIC9R plasmid after being subjected to double digestion by EcoRI and Not I, and the competence of TransI-T1 is transformed.

Example 2 construction of E175 saturation mutant recombinant vector Using XYL10C as parent

Designing saturated mutation primers E175X-F and E175X-R at the E175 site of gene XYL10C, using XYL10C-pPIC9R as a template, using XYL10C-F and E175X-R and E175X-F and XYL10C-R as primers for PCR, amplifying upstream and downstream fragments, then using XYL10C-F and XYL10C-R as primers for overlap, performing double enzyme digestion on a product by EcoR I and Not I, directly connecting with a cut pPIC9R plasmid, and transforming TransI-T1 competence. Single-clone sequencing was picked and found that only mutant E175F E175M E175T E175W and CutE did not appear, so the remaining several mutants were re-primed as described above.

EXAMPLE 3 preparation of recombinant strains of xylanase mutants

The recombinant vector which is connected with the expression vector pPIC9r and has correct sequencing is linearized by an endonuclease BglII and transformed into pichia pastoris Gs115 to obtain a recombinant yeast strain GS115/XYL10C-CutN, GS115/XYL 10C-E175X.

Selecting a Gs115 strain containing the recombinant plasmid, inoculating the strain into a 10mL tubule of a 2mL BMGY culture medium, and performing shake culture at 30 ℃ and 220rpm for 48 h; after this time, the culture broth was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 1.5mL BMMY medium containing 0.5% methanol and induced again at 30 ℃ and 220rpm for 48 h. And finally taking the supernatant for enzyme activity detection.

Selecting a Gs115 strain with the highest enzyme activity of a supernatant, inoculating the strain in 30mL YPD medium for culturing for 48h for seed amplification culture, then inoculating the strain in 1L triangular flask of 300mL BMGY medium according to 1% of inoculation volume, and placing the strain in 30 ℃ and 220rpm for shake cultivation for 48 h; then, the culture solution was centrifuged at 3000g for 5min, the supernatant was discarded, and the pellet was resuspended in 200mL of BMMY medium containing 0.5% methanol, and then cultured again at 30 ℃ under induction at 220 rpm. And (3) supplementing 1mL of methanol every 12h to keep the methanol concentration in the bacterial liquid at 0.5%, and simultaneously taking the supernatant for enzyme activity detection.

Example 4 Activity analysis of recombinant xylanase mutants and wild type

The DNS method comprises the following steps: the specific method comprises the following steps: under the given conditions of pH and temperature, 1mL of reaction system comprises 100 μ L of appropriate diluted enzyme solution and 900 μ L of substrate, the reaction is carried out for 10min, 1.5mL of DNS is added to stop the reaction, and the reaction is boiled in boiling water for 5 min. After cooling, the OD was measured at 540 nm. 1 enzyme activity unit (U) is defined as the amount of enzyme required to generate 1. mu. mol reducing end per minute of xylan under the given conditions.

Example 5 determination of the Properties of the recombinant xylanase mutants and wild type

1. The optimum pH determination method of the recombinant xylanase mutant and the wild type comprises the following steps:

the recombinant xylanase mutant purified in example 3 and the wild type were subjected to enzymatic reactions at different pH to determine their optimum pH. The substrate zelkova was assayed for xylanase activity in 0.2mol/L citric acid-disodium hydrogen phosphate buffer at 80 ℃ at different pH. The results (FIG. 3) show that the optimum reaction pH of the recombinant xylanase mutant and the wild type is approximately 4.0-4.5, and the similar relative enzyme activity is obtained in the pH range of 3.0-6.0.

2. The method for measuring the optimal temperature of the recombinant xylanase mutant and the wild type comprises the following steps:

the optimal temperature of the recombinant xylanase mutant and the wild type is determined by performing enzymatic reactions in respective optimal pH buffer systems of 0.2mol/L citric acid-disodium hydrogen phosphate buffer and at different temperatures. The results of the enzyme reaction optimum temperature measurement (FIG. 4) show that the optimum temperature of the recombinant xylanase mutant and the wild type is about 80-95 ℃.

3. The method for determining the kinetic parameters of the mutant and the wild type of the recombinant xylanase comprises the following steps:

the first-order reaction time of the reaction was determined with reference to the method of wangkun in this laboratory (wangkun, 2013). The reaction time for measuring Km and Vmax was determined to be 5 min. Using different concentrations of xylan (10,8,5,2.5,2,1,0.75,0.5mg/mL) as a substrate, the enzyme activity was measured under the same conditions (80 ℃ C., pH4.0), the corresponding reaction rate was calculated, and Km value and Vmax were calculated using GraFit7 software.

The calculation results are shown in table 2.

TABLE 2 summary of the basic enzymological Properties of XYL10C and all mutants

Claims

1. A xylanase mutant is characterized in that the full length of an amino acid sequence is shown in a sequence table SEQ ID NO: 1, and the full length of the amino acid sequence of the xylanase mutant is shown as a sequence table SEQ ID NO: 3, respectively.

2. A method for preparing a xylanase mutant according to claim 1, comprising the steps of:

(1) the full length of the amino acid sequence is shown as the sequence table SEQ ID NO: 1, inserting the xylanase gene into an expression plasmid pPIC9 to obtain a recombinant plasmid XYL10C-pPIC 9;

(2) designing a primer according to a target fragment, adding a restriction enzyme cutting site at the joint of an expression plasmid pPIC9 at the 5' end of a primer pair, and amplifying by taking the recombinant plasmid in the step (1) as a template to obtain a coding gene of the xylanase with an N-terminal sequence of which 84 amino acids are removed;

(3) connecting the obtained coding gene enzyme digestion with the cut pPIC9 plasmid to obtain a mutant recombinant plasmid;

(4) transforming the mutant recombinant plasmid into pichia pastoris GS115, and carrying out induced expression to obtain the xylanase mutant with an N-terminal sequence of which 84 amino acids are removed.