TWI609961B - Nucleic acid construct, recombinant expression vector and method thereof for producing a recombinant enzyme - Google Patents

Abstract

The present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a Teleogryllus emma cellulase, encoding a thermomyces lanuginosus endo-beta-1,4-D-xylanase nucleic acid sequence or Its combination. The present invention further provides a recombinant expression vector comprising the nucleic acid construct, and a recombinant host cell obtained by introducing the recombinant expression vector into a host cell. The invention still further provides a method for producing a target protein.

Description

Nucleic acid construct for producing recombinant enzyme, recombinant expression carrier and method thereof

This invention relates to a nucleic acid construct, and more particularly to a nucleic acid construct for the production of recombinant enzymes.

Cellulose is the most important source of biomass carbon on the earth. The annual dry weight of plants produced by photosynthesis is about 22 billion tons. The cell wall components (mainly including cellulose, hemicellulose and lignin) account for about the dry weight of plants. 90%. In recent years, research on biomass energy sources has shifted from sugar and starch crops to cellulosic energy crops, such as rice straw, corn stalks and agroforestry waste, which mainly use cellulose in plants. And hemicellulose is used as a carbon source for producing alcohol or butanol. At present, the cellulose conversion process still has no mass production experience. The technologies to be broken include: development of cellulose and hemicellulose decomposing enzymes, low-cost mass production of fibrinolytic enzymes, process integration and simplification, development of genetic recombinant fermentation strains, and simultaneous fermentation. The development of carbon sugar and hexasaccharide strains, fiber hydrolysis combined with five or six carbon sugar fermentation strains. Among them, fiber source treatment is one of the key points for the entire industrial process to be broken. To include research and development of cellulose and hemicellulolytic enzymes.

At present, the technology for degrading the fiber source by the enzyme method is mostly treated with various enzymes according to different ratios. This method is complicated and difficult to operate, and it is necessary to carry out the compounding of different proportions of enzymes according to different fiber sources, which is not conducive to industrial processes. Therefore, the role of the fibrinolytic enzyme and the treatment process are still to be improved.

The object of the present invention is to develop a single enzyme having two fibrinolytic enzymes (for example, cellulase and xylanase), and to prepare a process for separately producing two fibrinolytic enzymes, which is modified by the genetic recombination method of the present invention. And the modification of naturally occurring cellulases (eg, sputum cellulase) and xylanases (eg, thermophilic fungal xylanase), using recombinant DNA technology to effectively enhance recombinant cellulase and xylanase The yield is increased to increase the industrial value of the recombinant cellulase and xylanase, and the cost of producing the enzyme is greatly reduced.

The present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a cellulase, a nucleic acid sequence encoding a thermophilic fungal xylanase, or a combination thereof.

The present invention provides a recombinant expression vector comprising a nucleic acid construct according to the present invention, the nucleic acid construct comprising a nucleic acid sequence encoding a cellulase, a nucleic acid sequence encoding a thermophilic fungal xylanase, or a combination thereof .

The invention further provides a recombinant host cell for producing a target protein comprising a recombinant expression vector according to the invention.

The present invention provides a method for producing a target protein, which comprises The recombinant host cell according to the present invention is cultured in a culture solution; and the target protein is collected from the culture solution, wherein the target protein is a cellulase, a thermophilic fungal xylanase or the cellulose A fusion enzyme of the enzyme with the thermophilic fungal xylanase.

Fig. 1 shows the results of purification of Sephacryl S-100 HR gel filtration chromatography and its activity analysis by Telegryllus emma cellulase (hereinafter referred to as cellulase); The results of thermal stability analysis of the enzyme; Figure 3 shows the results of the best reaction temperature analysis of cellulase; Figure 4 shows the results of the best reaction pH analysis of cellulase in different buffer solutions; Thermomyces lanuginosus endo-beta-1,4-D-xylanase (hereinafter referred to as xylanase) was purified by Sephacryl S-100 HR colloidal filtration chromatography and its activity analysis results; The figure shows the results of thermal stability analysis of xylanase; the figure 7 shows the optimal reaction temperature analysis results of xylanase; the figure 8 shows the optimal reaction pH of xylanase in different buffer solutions. Value analysis results; Figure 9 shows the purification of the fusion enzyme by Sephacryl S-100 HR colloidal filtration chromatography and its activity analysis; Figure 10 shows the thermal stability analysis of cellulase and xylanase in the fusion enzyme result; Figure 11 shows the optimal reaction temperature analysis results of cellulase and xylanase in the fusion enzyme; and Figure 12 shows the optimal reaction of cellulase and xylanase in the different buffer solutions in the fusion enzyme. pH analysis results.

The embodiments of the present invention are described by way of specific examples, and those skilled in the art can readily understand the advantages and effects of the present invention from the disclosure. The present invention may be embodied or applied by other different embodiments, and the various details of the present invention may be variously modified and changed without departing from the spirit and scope of the invention.

As used herein, the term "nucleic acid" or "nucleic acid sequence" means a macromolecular substance that exists in the nucleus in the form of a single or double strand, which can be divided into deoxyribonucleic acid (ie, DNA) and ribonucleic acid. (ie RNA). As used herein, the term "nucleic acid" or "nucleic acid sequence" can be used interchangeably with "gene", "DNA", or "DNA sequence."

As used herein, the term "fusion gene" means a DNA fragment in which two or more genes are fused in a single open reading frame for encoding two or more borrows. A protein that is linked by one or more peptide bonds.

As used herein, the term "codon usage" means that the frequency of codon usage in a nucleic acid sequence is not evenly distributed, some codons are used at higher frequencies, while others are less frequently present. This is now The sequence of the protein coding region is rendered to be appreciable statistically specific.

As used herein, the term "vector" means any recombinant expression system which, in vitro or in vivo, exhibits a modified cellulase sequence according to the invention, modified in any host cell. The xylanase sequence and the fusion enzyme sequence of the fusion cellulase and xylanase.

As used herein, the term "transformation" means the manner in which a nucleic acid sequence, recombinant DNA construct or recombinant vector is introduced into a selected host cell by a variety of techniques, including, but not limited to, Electroporation, microinjection, transfection of calcium phosphate or calcium chloride media, transfection of liposome vectors, transfection with bacteria or phage, transduction with retroviruses or other viruses, protoplasts Body fusion, Agrobacterium media transformation or other methods.

The present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a cellulase, a nucleic acid sequence encoding a thermophilic fungal xylanase, or a combination thereof.

According to a specific embodiment of the present invention, the nucleic acid sequence encoding the cellulase is a modified nucleic acid sequence encoding a cellulase, and the modified nucleic acid sequence encoding the cellulase is a yeast code. Sub-preferences are modified. According to a particular embodiment of the invention, the modified nucleic acid sequence encoding a cellulase has a sequence difference of up to 20% with the unmodified nucleic acid sequence encoding a cellulase, and the modified coding The nucleic acid sequence of 蟋蟀 cellulase is the nucleic acid sequence of SEQ ID NO: 2.

According to a specific embodiment of the invention, the thermophilic fungal xylan The nucleic acid sequence of the enzyme is a modified nucleic acid sequence encoding a thermophilic fungal xylanase, and the modified nucleic acid sequence encoding a thermophilic fungal xylanase is modified with the codon preference of the yeast. According to a particular embodiment of the invention, the modified nucleic acid sequence encoding a thermophilic fungal xylanase has a sequence difference of up to 25% with an unmodified nucleic acid sequence encoding a thermophilic fungal xylanase, And the modified nucleic acid sequence encoding a thermophilic fungal xylanase is the nucleic acid sequence of SEQ ID NO: 4.

The invention further provides a recombinant expression vector comprising a nucleic acid construct according to the invention. According to a particular embodiment of the invention, the recombinant expression vector may further comprise an inducible promoter sequence that controls the expression of the nucleic acid construct. According to another embodiment of the present invention, the recombinant expression vector may further comprise at least one selected from the group consisting of a marker gene sequence, a reporter gene sequence, an antibiotic resistance gene sequence, and a restriction enzyme cleavage position sequence. , a polyadenylation position sequence, a enhancer sequence, and a regulatory sequence.

The invention further provides a recombinant host cell for producing a target protein comprising a recombinant expression vector according to the invention. According to a particular embodiment of the invention, the host cell line is selected from at least one of the group consisting of a virus, a bacterial cell, a fungal cell, an algal cell, a plant cell, and an animal cell. In a preferred embodiment of the present invention, the recombinant host cell is a yeast cell, and the yeast strain is Yarrowia lipolytica .

The invention further provides a method for producing a target protein comprising the step of carrying out the recombinant host cell according to the present invention in a culture solution. And culturing the target protein from the culture solution, wherein the target protein is a cellulase, a thermophilic fungal xylanase or a fusion enzyme of the cellulase with the thermophilic fungal xylanase.

According to a specific embodiment of the present invention, a nucleic acid sequence encoding a modified cellulase (ie, SEQ ID NO: 2) is introduced into a recombinant expression vector, and the recombinant expression vector is introduced into a host cell by transformation. The recombinant cellulase is heat-resistant to 40 ° C (having a relative activity of 99%), the optimum reaction temperature is 50 ° C, and the optimum reaction pH is pH 3.5.

According to a specific embodiment of the present invention, a nucleic acid sequence encoding a modified thermophilic fungal xylanase (ie, SEQ ID NO: 4) is introduced into a recombinant expression vector and the recombinant expression vector is transformed by transformation. The introduced host cell expresses a recombinant xylanase which is heat resistant to 90 ° C (having a relative activity of 89%), an optimum reaction temperature of 65 ° C, and an optimum reaction pH of pH 6.5 to 7.5.

According to a specific embodiment of the present invention, the SEQ ID NO: 2, SEQ ID NO: 4 and the ligation sequence are joined to form a fusion nucleic acid sequence comprising a cellulase and a thermophilic fungal xylanase. According to another embodiment of the present invention, the nucleic acid sequence encoding the cellulase (SEQ ID NO: 2) and the nucleic acid sequence encoding the thermophilic fungal xylanase (SEQ ID NO: 4) are in three different lengths. The DNA fragment is ligated, and the six fusion enzyme sequences are obtained by arranging and combining the cellulase and xylanase DNA sequences before and after the three DNA sequences are ligated, wherein the fusion nucleic acid sequence is selected from SEQ ID NO : 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ The nucleic acid sequence of the group consisting of ID NO: 12 and SEQ ID NO: 13. According to still another embodiment of the present invention, the ligation sequence is selected from the group consisting of a short length DNA sequence (SEQ ID NO: 5), a medium length DNA sequence (SEQ ID NO: 6), and a long length DNA sequence (SEQ ID) NO: 7) At least one of the groups formed.

According to a specific embodiment of the present invention, the optimal reaction temperature of the cellulase and xylanase activities of the obtained fusion enzyme is 50 ° C and 70 ° C, respectively. Compared with the cellulase and xylanase alone, the optimal reaction temperature of the cellulase and xylanase of the fusion enzyme is slightly increased, and the optimal reaction temperature of the cellulase activity is unchanged (50 ° C). The optimum reaction temperature for the xylanase is increased to 70 °C. Considering the activity of two enzymes, the optimal reaction temperature of the fusion enzyme is 60 ° C. At this reaction temperature, the relative activity of cellulase is increased by 97% (81% when it is expressed alone), and the relative activity of xylanase is unchanged ( Maintained at 90%).

According to a specific embodiment of the present invention, a fusion enzyme nucleic acid sequence encoding a cellulase and a xylanase-encoding nucleic acid sequence (i.e., having SEQ ID NO: 9) is introduced into a yeast, and the transformation is carried out. The yeast (including SEQ ID NO: 9) was deposited on food on June 12, 2014 under the name: pY-CMD (Y. lipolytica Po1g) pY-CMD (in Yarrowia lipolytica Po1g) CPC-CMD. The Center for Biological Resources Conservation and Research of the Industrial Development Institute and the accession number BCRC 920088 given by the Center. This biomaterial has been tested and passed the viability test.

The invention will be further illustrated by the following examples, but it should be understood that these examples are for illustrative purposes only and should not be construed as limiting Implementation of the Ming.

Example Example 1 Selection and purification of cellulase ( Teleogryllus emma cellulase) and analysis of its enzyme activity and biochemical characteristics

In this example, a DNA construct having a cellulase activity of Teleogryllus emma cellulase (hereinafter referred to as cellulase) was constructed, and its enzyme activity and biochemical characteristics were tested.

(1) Selection and purification of cellulase: Step 1 - Synthesis of modified DNA sequence: DNA sequence derived from 蟋蟀 ( Teleogryllus emma ) cellulase (GenBank: EU126927.2), based on Yarrowia lipolytica The codon usage of ( Yarrowia lipolytica ), which modified its original DNA sequence (SEQ ID NO: 1) into a modified coding DNA sequence (SEQ ID NO: 2) (modified DNA sequence for chemical synthesis) The nucleotide fragment (Shanghai Spark Crystal Molecular Biotechnology Co., Ltd.) was synthesized, and the modified DNA sequence was 87% similar to the original DNA sequence.

Step 2 - introducing the modified DNA sequence into a commercially available vector

pYLSC1:

The modified DNA sequence and the expression vector pYLSC1 (YLEX expression kit, Yisheng Biotech Development Co., Ltd.) were treated with restriction enzymes SfiI and HindIII (New England Biolabs), respectively, at 37 ° C for 1 hour, and the product was subjected to Zymoclean ^TM Gel DNA. Recovery Kit (ZYMO RESEARCH) for DNA fragment purification. After obtaining the modified DNA sequence after restriction enzyme treatment and the expression vector pYLSC1 (YLEX expression kit, Yisheng Biotechnology Development Co., Ltd.), the above two were obtained by ligase (T4 DNA ligase, New England Biolabs). The fragment (the modified DNA sequence after restriction enzyme treatment and the purified expression vector pYLSC1) was subjected to a ligation reaction, and was allowed to act at 16 ° C for 1 hour to introduce the modified DNA sequence into pYLSC1 to form a recombinant plasmid.

Step 3 - Expression of cellulase using Yarrowia lipolytica : The recombinant plastid obtained in the above step 2 is transformed into Yarrowia lipolytica by transformation, and the transformation condition is First, the recombinant plastid was linearized using the restriction enzyme NotI, and then YLOS buffer, DTT solution, and vector DNA (YLEX-Yarrowia Expression Set User Manual) were added to the linearized plastid to prepare a YLOS mixture, which was YLOS. After the mixed solution was mixed with Yarrowia lipolytica, the transformation was completed by incubating at 39 ° C for 1 hour. Next, the above-mentioned transformed yeast is cultured in a 4 liter fermentation tank to express the cellulase of the recombinant plastid, which is a yeast extract containing recombinant plastids at 0.25 liter of yeast extract-peptone- Glucose (Yeast Extract-Peptone-Dextrose, hereinafter referred to as YPD) was cultured at 22 ° C for 72 hours, and then 2 liters of YPD culture solution was added, and cultured at 28 ° C for 3 to 4 days in a 4 liter fermentation tank to express recombinant cellulase.

Step 4 - Purification of recombinant cellulase: The recombinant cellulase supernatant obtained in the above step 3 is concentrated, and then Sephacryl S-100 HR colloidal filtration chromatography (gel filtration) Chromatography) Purification of recombinant cellulase.

(2) Determination of activity and biochemical characteristics of purified recombinant cellulase: assay of activity of recombinant cellulase: The purified cellulase enzyme purified by this method is carboxymethyl cellulose (hereinafter referred to as CMC). For the substrate to measure the activity, 10 μL of recombinant cellulase solution was added to 990 μL of substrate solution (1% CMC in 50 mM sodium acetate, pH 5.0), and allowed to act at 50 ° C for 10 minutes, and 1 mL of DNS coloring solution (10 g / L 3 ) was added. , 5-dinitrosalicylic acid, 300g / L sodium potassium tartrate tetrahydrate, 16g / L sodium hydroxide), at 100 ° C for 20 minutes, then add 1mL deionized water and cooled in an ice bath, after mixing evenly The absorbance value is measured at a wavelength of 595 nm, and the absorbance value is substituted into the glucose standard calibration curve to obtain the enzyme activity data. As shown in Fig. 1, the highest activity measured by the purified recombinant cellulase was 31 units/ml (U/mL).

Biochemical characteristics analysis of cellulase: The biochemical analysis of the thermal stability, the optimal reaction temperature and the optimum reaction pH was carried out by taking the dividing tube with the highest cellulase activity in the above steps.

1. Thermal stability analysis: After the recombinant cellulase was treated at different temperatures (including 30 to 80 ° C) for 30 minutes, the enzyme activity was measured.

2. Optimal reaction temperature analysis: 10 μL of recombinant cellulase solution was added to 990 μL of substrate solution. (1% CMC in 50 mM sodium acetate, pH 5.0), 10 min at different temperatures (4 to 80 ° C), add 1 mL of DNS coloring solution (10 g / L of 3,5-dinitrosalicylide) Acid, 300g / L of sodium potassium tartrate tetrahydrate, 16g / L of sodium hydroxide), at 100 ° C for 20 minutes, then add 1mL of deionized water and cooled in an ice bath, mixed and measured at 595nm wavelength absorbance The enzyme activity data can be obtained by substituting the absorbance value into the glucose standard calibration curve.

Optimal reaction pH analysis: 10 μL of recombinant cellulase solution was added to the substrate solution of different pH (1% CMC in 50 mM sodium acetate, pH 3.0 to 6.0, or 50 mM sodium citrate, pH 3.0 to 7.0, Or 50 mM sodium phosphate, pH 6.0 to 9.0, or 50 mM Tris-HCl, pH 7.0 to 11.0), at 50 ° C for 10 minutes, add 1 mL of DNS coloring solution (10 g / L of 3,5-dinitrogen Salicylic acid, 300g / L of sodium potassium tartrate tetrahydrate, 16g / L of sodium hydroxide), at 100 ° C for 20 minutes, then add 1mL of deionized water and cooled in an ice bath, mixed evenly at 595nm wavelength The enzyme activity data can be obtained by measuring the absorbance value and substituting the absorbance value into the glucose standard test curve.

As shown in Fig. 2, the cellulase derived from hydrazine was heat resistant to 40 ° C (the relative activity was 99%), and when the treatment temperature reached 45 ° C, its relative activity was reduced to 74%. As shown in Figures 3 and 4, the optimum reaction temperature for cellulase is 50 ° C and the optimum pH is pH 3.5.

Example 2 Selection and purification of thermomyces lanuginosus endo-beta-1,4-D-xylanase and analysis of its enzyme activity and biochemical characteristics

In this example, a DNA construct having the activity of Thermomyces lanuginosus endo-beta-1,4-D-xylanase (hereinafter referred to as xylanase) was constructed, and its enzyme activity and biochemical characteristics were tested.

(1) Selection and purification of thermophilic fungal xylanase: Step 1 - Synthesis of modified DNA sequence: DNA sequence derived from Thermomyces lanuginosus xylanase (GenBank: U35436.1), based on The codon preference of Yarrowia lipolytica , the original DNA sequence (SEQ ID NO: 3) was modified to a modified DNA sequence (SEQ ID NO: 4) (modified DNA sequence for chemical synthesis) The nucleotide fragment (Shanghai Spark Crystal Molecular Biotechnology Co., Ltd.) was synthesized by the method, and the modified DNA sequence has an affinity of 82% with the original DNA sequence.

Step 2: Introducing the modified DNA sequence into the commercial vector pYLSC1: treating the modified DNA sequence and the expression vector pYLSC1 (YLEX performance kit, Yisheng Biotechnology Development Co., Ltd.) with restriction enzymes SfiI and HindIII (New England Biolabs), respectively 1 hour at 37 ℃ role product was purified fragment DNA was recovered DNA colloidal Zymoclean ^TM kit (ZYMO RESEARCH). After the restriction enzyme treatment and the purified modified DNA sequence and the expression vector pYLSC1 (YLEX expression kit, Yisheng Biotech Development Co., Ltd.), the above two fragments were restricted by ligase (T4 DNA ligase, New England Biolabs). After the enzyme treatment, the purified DNA sequence and the expression vector pYLSC1) were subjected to a ligation reaction, and the reaction was carried out at 16 ° C for 1 hour, and the modified DNA sequence was introduced into pYLSC1 to form a recombinant plasmid.

Step 3 - Expression of xylanase by Yarrowia lipolytica: The recombinant plastid obtained in the above step 2 is transformed into Yarrowia lipolytica by transformation, and the transformation condition is: first use The restriction enzyme NotI linearizes the recombinant plastid, and then YLOS buffer, DTT solution, and vector DNA (YLEX-Yarrowia Expression Set User Manual) are added to the linearized plastid to prepare a YLOS mixture, and the YLOS mixture is combined with After mixing Yarrowia lipolytica, the transformation was completed by incubating at 39 ° C for 1 hour. Next, the above-mentioned transformed yeast was cultured in a 4 liter fermentation tank to express the cellulase of the recombinant plastid, which was a yeast containing the recombinant plastid at 0.25 liter YPD (Yeast Extract-Peptone- Dextrose) was cultured at 22 ° C for 72 hours, and then 2 liters of YPD culture solution was added, and cultured at 28 ° C for 3 to 4 days in a 4 liter fermentation tank to express recombinant xylanase.

Step 4 - Purification of xylanase: After the recombinant xylanase supernatant obtained in the above step 3 was concentrated, the recombinant xylanase was purified by Sephacryl S-100 HR colloidal filtration chromatography.

(2) Determination of activity and biochemical characteristics of purified recombinant xylanase: assay for activity of recombinant xylanase: The purified xylanase system was purified by using xylan as a substrate. For the activity assay, add 10 μL of recombinant xylanase solution to 990 μL. The substrate solution (1% xylan in 50 mM sodium acetate, pH 5.0) was applied at 70 ° C for 10 minutes, and 1 mL of DNS coloring solution (10 g/L 3,5-dinitrosalicylic acid, 300 g/L IV) was added. Hydration of sodium potassium tartrate, 16g / L sodium hydroxide, at 100 ° C for 20 minutes, then add 1mL of deionized water and cooled in an ice bath, mix well, measure the absorbance at 595nm wavelength, and then substitute the absorbance into xylose Enzyme activity data can be obtained from the standard calibration curve. As shown in Figure 5, the highest activity measured by the purified recombinant xylanase was 166 U/mL.

Analysis of Biochemical Characteristics of Recombinant Xylanase: The biochemical properties of the heat-stabilized, optimal reaction temperature and optimum pH value of the stalk tube with the highest xylanase activity in the above steps were taken out.

1. Thermal stability analysis: After the recombinant xylanase was treated at different temperatures (30 to 100 ° C) for 30 minutes, the enzyme activity was measured.

2. Optimal reaction temperature analysis: 10 μL of recombinant xylanase solution was added to 990 μL of substrate solution (1% xylan in 50 mM sodium acetate, pH 5.0) and applied at different temperatures (20 to 80 ° C). Minutes, add 1mL of DNS coloring solution (10g / L of 3,5-dinitrosalicylic acid, 300g / L of sodium potassium tartrate tetrahydrate, 16g / L of sodium hydroxide), at 100 ° C, 20 Minutes, then add 1mL of deionized water and cool in an ice bath. After mixing, the absorbance is measured at 595nm wavelength, and then the absorbance is substituted into the xylose standard calibration line to obtain the enzyme activity data.

3. Optimal reaction pH analysis: 10 μL of recombinant xylanase solution was added to the substrate solution of different pH (1% xylan in 50 mM sodium acetate, pH 3.0 to 6.0, or 50 mM sodium citrate, pH 3.0 to 7.0, or 50 mM sodium phosphate, pH 6.0 to 9.0, or 50 mM Tris-HCl, pH 7.0 to 11.0), at 70 ° C for 10 minutes, add 1 mL of DNS color solution (10 g / L of 3 , 5-dinitrosalicylic acid, 300g / L of sodium potassium tartrate tetrahydrate, 16g / L of sodium hydroxide), at 100 ° C for 20 minutes, then add 1mL of deionized water and cooled in an ice bath, mixing After homogenization, the absorbance value is measured at a wavelength of 595 nm, and the absorbance value is substituted into the xylose standard product calibration line to obtain the enzyme activity data.

As shown in Fig. 6, the purified xylanase can be heat resistant to 90 ° C (the relative activity is 89%), and the relative activity is 97 when the treatment temperature reaches 80, 85, 90 and 100 ° C, respectively. 92, 89 and 78%. As shown in Figures 7 and 8, the optimum temperature for the purified xylanase is 65 ° C and the optimum pH is pH 6.5 to 7.5.

Example 3 Construction, Purification, Enzyme Activity Analysis and Biochemical Characterization of a Fusion Enzyme DNA Construct with Cellulase and Xylanase Activity

In this example, a fusion DNA construct having a cellulase and xylanase activity fusion enzyme is constructed, and the activity and biochemical characteristics of the fusion enzyme are determined.

Step 1 - ligation of cellulase and xylanase gene sequences: the modified cellulase DNA fragment (SEQ ID NO: 2) and the modified xylanase DNA fragment (SEQ ID NO: 4) are 3 different lengths DNA fragments (shown in Table 1) were ligated using the In-Fusion® HD selection kit (Clontech). Six kinds of fusion enzyme sequences (SEQ ID NOS: 8 to 13) were obtained by aligning and combining the cellulase and xylanase DNA sequences with the three DNA sequences of Table 1.

Step 2: Obtaining the fusion gene sequence on the commercial vector pYLSC1: designing the corresponding primers of the six fusion enzyme sequences obtained in the above step 1 (see Table 2) for polymerase chain reaction (polymerase chain reaction) , hereinafter referred to as PCR), the reaction conditions: 98 ° C reaction for 40 seconds, then the reaction 98 ° C / 20 seconds → 62 ° C / 20 seconds → 72 ° C / 30 seconds for 35 cycles, and finally at 72 ° C for 10 minutes, PCR products DNA fragments were purified performed by Zymoclean ^TM colloidal DNA recovery kit (ZYMO RESEARCH). The expression vector pYLSC1 (YLEX performance kit, Yisheng Biotech Development Co., Ltd.) was treated with restriction enzymes SfiI and HindIII (New England Biolabs), and was treated at 37 ° C for 1 hour. The product was subjected to Zymoclean ^TM colloidal DNA recovery kit (ZYMO). RESEARCH) Purification of DNA fragments. Each of the reaction products was subjected to DNA recombination (i.e., In-Fusion cloning reaction) using the fusion HD enzyme (In-Fusion® HD selection kit, Clontech) to limit the above PCR products. The enzyme SfiI and HindIII (New England Biolabs) treated expression vector pYLSC1 and the ligated DNA fragment were fused and transfected, and the ligation reaction was carried out at 50 ° C for 15 minutes to obtain 6 cellulase and xylanase DNA sequences. The DNA construct of the fusion enzyme.

* The primer sequence comprises: a partial fragment representing a plastid (pYLSC1), a restriction enzyme sequence, a partial fragment of a cellulase or xylanase DNA sequence, and a ligated DNA sequence.

Step 3 - Expression of the fusion enzyme by Yarrowia lipolytica : The fusion DNA constructs of the six fusion enzymes obtained in the above step 2 were respectively transformed into Yarrowia lipolytica. The recombinant plastid was first linearized using the restriction enzyme NotI, and then YLOS buffer, DTT solution, and vector DNA (YLEX-Yarrowia Expression Set User Manual) were added to the linearized plastid to prepare a YLOS mixture, which was mixed. After the solution was mixed with Yarrowia lipolytica, the transformation was completed by incubating at 39 ° C for 1 hour. Subsequently, each of the inoculum containing the recombinant plastid of the fusion enzyme was cultured at 28 ° C for 3 to 4 days in a 15 ml culture tube (containing 6 ml of YPD) to express the recombinant enzyme. The enzyme activity of each fusion enzyme was determined according to the cellulase activity assay of Example 1 above and the xylanase activity assay of Example 2 (SEQ ID NO: 9. Cellulase activity: 1 U/mL; Carbohydrase activity: 6.6 U/mL). Thereafter, the one having the highest enzyme activity (SEQ ID NO: 9) was taken out, and the recombinant fusion enzyme was expressed in a 4 liter fermentation tank.

Step 4 - Purification of the fusion enzyme: The fusion enzyme supernatant obtained in the above step 3 was concentrated, and then purified by a Sephacryl S-100 HR colloidal filtration chromatography.

Step 5 - Activity measurement of fusion enzyme: The activity of cellulase and xylanase was measured by the fusion enzyme purified in the above step 4.

Step 6 - Analysis of Biochemical Characteristics of Fusion Enzyme: The biochemical characteristics of the thermodynamic stability, the optimum reaction temperature and the optimum reaction pH were determined by taking the highest activity measured in the above step 5.

1. Thermal stability analysis: After the fusion enzyme was treated at various temperatures (30 to 100 ° C) for 30 minutes, the activity of the cellulase and xylanase of the fusion enzyme was analyzed.

2. Optimal reaction temperature analysis: the fusion enzyme was applied at various temperatures (20 to 80 ° C) for 10 minutes. Thereafter, the activity of the cellulase and xylanase of the fusion enzyme was analyzed according to the cellulase activity assay of Example 1 and the xylanase activity assay of Example 2.

3. Optimal reaction pH analysis: Adding the fusion enzyme to a substrate solution of different pH (1% CMC or xylan in 50 mM sodium acetate, pH 3.0 to 6.0, or 50 mM sodium citrate, pH 3.0 to 7.0, or 50 mM sodium phosphate, pH 6.0 to 9.0, or 50 mM Tris-HCl, pH 7.0 to 11.0), at 50 ° C for 10 minutes, according to the cellulase activity assay of Example 1 and the xylanase activity assay of Example 2 for fusion Activity analysis of cellulase and xylanase of enzymes.

As shown in Figure 9, the purified enzymes obtained by the purification can measure the highest activity of cellulase and xylanase at 45 U/mL and 180 U/mL, respectively, which is slightly higher than the highest enzyme activity of the two enzymes alone (respectively 31 U/mL and 166 U/mL), wherein the cellulase activity of the fusion enzyme is increased to 145% of the recombinant cellulase alone, and the xylanase activity is increased to 108% of the recombinant xylanase alone. .

As shown in Figure 10, the cellulase and xylanase of the fusion enzyme are respectively heat resistant to 45 ° C (relative activity of 95%) and 85 ° C (relative activity of 84%), when the treatment temperature reaches 50 ° C The cellulase relative activity was 66%; when the treatment temperature reached 90 ° C, the xylanase relative activity was 57%. Compared with the cellulase and xylanase alone, the thermal stability of the cellulase of the fusion enzyme increased slightly to 45 ° C (relative activity of 95%), while the cellulase alone showed 45 ° C relative Activity is only 74%; xylanase activity The thermal stability decreased slightly to 85 ° C (relative activity of 84%), while the xylanase alone showed a relative activity of 92% at 85 ° C.

As shown in Fig. 11, the optimal reaction temperature of the fusion enzyme showed that the optimal reaction temperature of the cellulase and xylanase activities of the fusion enzyme was 50 ° C and 70 ° C, respectively. Compared with the cellulase and xylanase alone, the optimal reaction temperature of the cellulase and xylanase of the fusion enzyme is slightly increased, and the optimal reaction temperature of the cellulase activity is unchanged (50 ° C). The optimum reaction temperature for the xylanase is increased to 70 °C. Considering the activity of two enzymes, the optimal reaction temperature of the fusion enzyme is 60 ° C. At this reaction temperature, the relative activity of cellulase is increased by 97% (81% when it is expressed alone), and the relative activity of xylanase is unchanged ( Maintained at 90%).

As shown in Fig. 12, the optimum pH value of the fusion enzyme showed that the optimum reaction pH values of the cellulase and xylanase of the fusion enzyme were pH 3.5 and pH 7, respectively. Compared with the cellulase and xylanase alone, the optimal reaction pH of the cellulase and xylanase activities of the fusion enzyme is unchanged. Considering the cellulase and xylanase activities, the optimum pH of the fusion enzyme is pH 5.5.

Taken together, the fusion enzymes linked to cellulase and thermophilic fungal xylanase have better enzyme activity and biochemical characteristics than those of each enzyme.

The above-described embodiments are merely illustrative of the principles of the invention and its effects, and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, those who have this expertise in the technical field are All equivalent modifications or variations that are made without departing from the spirit and scope of the inventions disclosed herein are still covered by the appended claims.

<110> Taiwan Zhongyou Co., Ltd.

<120> Nucleic acid construct for producing recombinant enzyme, recombinant expression plastid and method thereof

<160> 13

<210> 1

<211> 1362

<212> DNA

<213> 蟋蟀 ( Teleogryllus emma )

<223> Cellulase DNA

<400> 1

<210> 2

<211> 1362

<212> DNA

<213> Artificial sequence

<223> Modified cellulase DNA

<400> 2

<210> 3

<211> 678

<212> DNA

<213> Thermophilic fungus ( Thermomyces lanuginosus )

<223> Xylanase DNA of thermophilic fungi

<400> 3

<210> 4

<211> 678

<212> DNA

<213> Artificial sequence

<223> Modified xylanase DNA of thermophilic fungi

<400> 4

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<223> ligating the shorter DNA fragment of SEQ ID NO: 2 and SEQ ID NO:

<400> 5

<210> 6

<211> 39

<212> DNA

<213> Artificial sequence

<223> ligating DNA fragments of equal length in SEQ ID NO: 2 and SEQ ID NO:

<400> 6

<210> 7

<211> 75

<212> DNA

<213> Artificial sequence

<223> ligating the longer DNA fragment of SEQ ID NO: 2 and SEQ ID NO:

<400> 7

<210> 8

<211> 2064

<212> DNA

<213> Artificial sequence

<223> First fusion enzyme sequence

<400> 8

<210> 9

<211> 2076

<212> DNA

<213> Artificial sequence

<223> Second fusion enzyme sequence

<400> 9

<210> 10

<211> 2112

<212> DNA

<213> Artificial sequence

<223> Third fusion enzyme sequence

<400> 10

<210> 11

<211> 2064

<212> DNA

<213> Artificial sequence

<223> Fourth fusion enzyme sequence

<400> 11

<210> 12

<211> 2076

<212> DNA

<213> Artificial sequence

<223> Fifth fusion enzyme sequence

<400> 12

<210> 13

<211> 2112

<212> DNA

<213> Artificial sequence

<223> Sixth fusion enzyme sequence

<400> 13

Claims (17)

A nucleic acid construct comprising a nucleic acid sequence encoding a cellulase and a nucleic acid sequence encoding a thermophilic fungal xylanase, wherein the nucleic acid sequence encoding the cellulase is modified or not The nucleic acid sequence of SEQ ID NO: 1 wherein the modified nucleic acid sequence of SEQ ID NO: 1 has up to 20% sequence difference with the unmodified SEQ ID NO: 1 nucleic acid sequence and is encoded a nucleic acid sequence of SEQ ID NO: 3, wherein the nucleic acid sequence encoding the thermophilic fungal xylanase is modified or unmodified, wherein the modified nucleic acid of SEQ ID NO: 3 The sequence and the unmodified SEQ ID NO: 3 nucleic acid sequence have up to 25% sequence difference and encode the same thermophilic fungal xylanase. The nucleic acid construct of claim 1, wherein the modified nucleic acid sequence of SEQ ID NO: 1 is modified with a codon preference of the yeast. The nucleic acid construct of claim 1, wherein the modified nucleic acid sequence of SEQ ID NO: 1 is the nucleic acid sequence of SEQ ID NO: 2. The nucleic acid construct of claim 1, wherein the modified nucleic acid sequence of SEQ ID NO: 3 is modified with the codon preference of the yeast. The nucleic acid construct of claim 1, wherein the nucleic acid construct The nucleic acid sequence of SEQ ID NO: 3, which is modified, is the nucleic acid sequence of SEQ ID NO: 4. The nucleic acid construct of claim 2, wherein the yeast is Yarrowia lipolytica . The nucleic acid construct of claim 1, further comprising a ligation sequence that ligates the nucleic acid sequence encoding the cellulase enzyme to the nucleic acid sequence encoding the thermophilic fungal xylanase to form a fusion nucleic acid sequence. The nucleic acid construct of claim 7, wherein the linker sequence is selected from at least one of the group consisting of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7. The nucleic acid construct of claim 7, wherein the fusion nucleic acid sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO At least one of the group consisting of: 12 and SEQ ID NO: 13. A recombinant expression vector comprising the nucleic acid construct of any one of claims 1 to 9. The recombinant expression vector of claim 10, further comprising an inducible promoter sequence that controls expression of the nucleic acid construct. The recombinant expression vector of claim 10, further comprising at least one selected from the group consisting of: a marker gene sequence, a reporter gene sequence, an antibiotic resistance gene sequence, a restriction enzyme cleavage position sequence, a poly Adenylation position sequence, enhancer sequence And regulatory subsequences. A recombinant host cell for producing a target protein, which comprises the recombinant expression vector of any one of claims 10 to 12. The recombinant host cell according to claim 13 is at least one selected from the group consisting of a virus, a bacterial cell, a fungal cell, an algal cell, a plant cell, and an animal cell. The recombinant host cell of claim 14, wherein the fungal cell line is a yeast. The recombinant host cell of claim 15, wherein the yeast is Yarrowia lipolytica . A method for producing a target protein, comprising: culturing a recombinant host cell according to claim 13 in a culture medium; and collecting the target protein from the culture solution, wherein the target protein is a ruthenium fiber A zymase, a thermophilic fungal xylanase or a fusion enzyme of the sputum cellulase with the thermophilic fungal xylanase.