TW201433637A - Xylanase having improved enzymatic activity - Google Patents

Abstract

A xylanase having improved enzymatic activity is disclosed. The amino acid sequence of the xylanase is a modified amino acid sequence of SEQ ID NO: 2, wherein Tryptophan at position 125 is substituted with Phenylalanine and Phenylalanine at position 163 is substituted with Tryptophan.

Description

Xylanase with enhanced enzyme activity

This case relates to a xylanase, especially a xylanase with enhanced enzyme activity.

Hemicellulose is one of the main components constituting the cell wall of plants. Among them, xylose (xylan) is the main component, and its number ranks second among the polysaccharides in nature. Therefore, the industrial application of polyxylose can be said to be very extensive and cannot be ignored. Polyxylose is a long-chain polysaccharide which is composed of xylose which is a five-carbon sugar and is bonded by a β-1,4-glycosidic bond. The xylose in nature has a complex and diverse structural composition, for example, it can be modified by a methyl group or a acetyl group, and even bonded to other sugar molecules to form a different Branched. Furthermore, hemicellulose such as polyxylose interacts with cellulose and lignin to form a tough plant cell wall. Many herbivores and microorganisms in the biological world, including bacteria and fungi, need to break down the polysaccharide polymer in the plant cell wall into monosaccharides that can be absorbed by the body as a source of survival energy. Therefore, many of these different microorganisms in nature contain commensal microorganisms in the gastrointestinal tract of herbivorous rumen animals, usually by producing different kinds of polysaccharide-degrading enzymes such as cellulase or xylanase. Decomposes the plant cell wall to obtain a monosaccharide that can be absorbed by the organism.

For the decomposition of the heterogeneous complex composition of polyxylose, it is necessary Different types of decomposing enzymes, such as: endo-β-D-xylanase, exo-xylosidase (β-1,4-xylosidase) or excision of its branches The acetylxylan esterase, the arabinose hydrolase (arabinase) and the glucuronidase (α-glucuronidase) are involved. Among them, endo-type xylanase is the most important. Endo-xylanase (EC 3.2.1.8) is a carbohydrate hydrolase that hydrolyzes the β-1,4-glycosidic bond on the polyxylose backbone, which in turn decomposes xylose and releases sugar. Oligrose can be further broken down into monosaccharides by exo-xylosidase (EC 3.2.1.37).

To date, xylanase has been widely used in industry, both in feed, paper, food and textile industries, and even in biomass energy. For different industrial applications, xylanases also need to meet their different applicable conditions and scope. For example, the feed industry requires acid-resistant enzyme proteins, whereas the paper industry prefers alkaline xylanases. Therefore, many studies are devoted to finding enzyme proteins that are more in line with the needs of different industries. In addition to the properties of enzyme proteins, their specific activity is also a major focus of improved industrial enzyme proteins. The higher the specific activity of the enzyme protein itself, the lower the cost of the industrial process. Therefore, industrial enzyme proteins which are compatible with industrial application conditions and high specific activity can be obtained, and are currently aimed at both academic and industrial circles.

In many related studies on obtaining better enzyme proteins, in addition to the direction of screening new genes from nature, the existing enzyme proteins have been modified. There are two major transformation strategies today: one is to randomly mutate genes, and then to screen out enzyme mutant proteins that are more suitable for their industrial conditions under specific conditions. The advantage of this strategy is that it does not require an in-depth analysis of the structure or mechanism of action of the enzyme protein, but directly finds better enzyme proteins under specific conditions, but the disadvantage is that it requires a lot of manpower and time to perform a large number of screenings, or There are efficient and accurate screening methods to match. Another strategy for transformation is by The mechanism of action and its mechanism of action to identify amino acids that are critical for the activity or properties of the enzyme protein, and site-directed mutagenesis and analysis of these specific amino acids, resulting in better enzyme muteins. The advantage is that it does not require a lot of time and manpower in a large number of mutation and screening steps, but it is necessary to understand the protein structure of the enzyme and its mechanism of action in order to find a specific amino acid with potential for transformation.

As mentioned earlier, xylanase has been widely used in various industries for a long time, for example, in the paper industry or the feed industry. Good industrial xylanases, in addition to meeting their different working conditions, high protein yield and high catalytic efficiency are also the goal that has been pursued. Therefore, the specific activity of xylanase proteins is also a major focus of improved industrial enzyme proteins. The higher the activity of the enzyme protein, the lower the cost and the higher the profit. Therefore, this case intends to improve the enzyme protein activity by modifying the gene, and relatively reduce the process cost, thereby effectively improving the industrial value of xylanase application in industry.

The purpose of this case is to transform the existing xylanase, using structural analysis and site-directed mutagenesis technology to effectively enhance the activity of xylanase, thereby increasing the industrial application value of xylanase.

In order to achieve the above object, a preferred embodiment of the present invention provides a xylanase whose amino acid sequence is a mutation of the tryptophan acid at position 125 of SEQ ID NO: 2 to phenylalanine, and the 163th The position of the phenylalanine is mutated to the amino acid sequence of the tryptophan.

In order to achieve the above object, a preferred embodiment of the present invention provides a xylanase whose amino acid sequence is an amino acid sequence which mutates the tryptophan at position 125 of SEQ ID NO: 2 to amphetamine.

In order to achieve the above objectives, one of the preferred embodiments of the present invention provides a A xylanase whose amino acid sequence is an amino acid sequence which mutates the phenylalanine at position 163 of SEQ ID NO: 2 to tryptophan.

Figure 1 shows the gene and amino acid sequence of xylanase xynCDBFV.

Figure 2 shows the steric structure of the xylanase xynCDBFV protein and its complex structure with oligosaccharide binding.

Figure 3 shows the primer sequence used for site-directed mutagenesis.

Figure 4 shows the gene and amino acid sequence of the W125F mutant of the xylanase xynCDBFV.

Figure 5 shows the gene and amino acid sequence of the F163W mutant of the xylanase xynCDBFV.

Figure 6 shows the gene and amino acid sequence of the W125F/F163W mutant of the xylanase xynCDBFV.

Figure 7 shows the results of activity tests of xylanase xynCDBFV proprotein and mutant xylanase.

Some exemplary embodiments embodying the features and advantages of the present invention are described in detail in the following description. It should be understood that the present invention is capable of various modifications in the various aspects of the present invention, and the description and drawings are intended to be illustrative and not limiting.

The xylanase in this case is a multi-mutation gene isolated from the anaerobic fungus Neocallimastix patriciarum in the rumen and screened by directed mutation and site-directed mutagenesis techniques. It is called xynCDBFV (refer to Chen, YL, Tang, TY, and Cheng, KJ (2001) Can J Microbiol 47, 1088-1094). As shown in Fig. 1, the full length of the xynCDBFV gene is 678 bases (the DNA sequence is indicated by SEQ ID NO: 1) and 225 amino acids (the amino acid sequence is indicated by SEQ ID NO: 2). In order to increase the industrial application value of this xylanase, we have improved the specific activity and catalytic efficiency by structural analysis and site-directed mutagenesis, thereby reducing industrial costs. The method for modifying the xylanase in the present invention and the modified xylanase obtained therefrom will be described in detail below.

First, the xynCDBFV gene was constructed into the pPICZαA vector using Eco RI and Not I. After plastid DNA is linearized with Pme I, the DNA is then sent to Pichia by electroporation. The electrolyzed bacterial solution was then applied to a YPD dish containing 100 μg/ml of Zeocin antibiotic for two days at 30 °C. Colonies were selected and inoculated into 5 ml of YPD at 30 ° C until the next day, and again inoculated into 50 ml of BMGY and cultured at 30 ° C until the next day. Next, the medium was changed to 20 ml of BMMY containing 0.5% methanol to induce protein expression. Samples were taken and supplemented with 0.5% methanol every 24 hours. The strain with the best protein expression was selected to amplify the performance. After inoculating the strain into 5 ml of YPD, it was amplified into 500 ml of BMGY and cultured at 30 ° C until the next day. The original BMGY was then replaced with 250 ml of BMMY to induce protein expression. After 2 days of culture, the bacterial solution was centrifuged, and the supernatant was collected, followed by dialysis twice with 5 L of a buffer containing 25 mM Tris; pH 7.5. In order to obtain high-purity xynCDBFV enzyme protein, we used a fast protein liquid chromatography (FPLC) to separate xynCDBFV enzyme protein with protein purity above 95% by using DEAE anion exchange column, and 10 mg. The /ml concentration was stored at -80 °C under 25 mM Tris; 150 mM NaCl; pH 7.5.

In order to solve the xynCDBFV egg by X-ray crystallography In the white structure, the crystal is first subjected to a sinking method at room temperature by a vapor diffusion method. Screening conditions were screened using different crystals, and the optimal conditions were determined by adjusting the corrections to 2 M Ammonium sulfate and 0.1 M Tris; pH 8.5. In addition, the steric structure of the protein of xynCDBFV was solved by molecular replacement (MR). In addition, the xynCDBFV protein crystal was further immersed in a solution containing 10 mM xylobiose or tetrasaccharide, and a diffraction pattern was obtained by X-ray, thereby obtaining a complex structure in which xynCDBFV binds to an oligosaccharide substance.

Fig. 2 shows the structure of the complex structure of xynCDBFV and its complex structure combined with oligosaccharides by X-ray crystallography. It was observed from the steric structure of the protein that the tryptophan (Trp125) at position 125 and the phenylalanine (Phe163) at position 163 were in the protease active region and could interact with oligosaccharides and have certain enzyme activities. The effect was chosen as the location for the site-directed mutagenesis.

In order to obtain a specific amino acid mutated enzyme protein, the site uses a site-directed mutagenesis technique and uses a xylanase xynCDBFV gene as a template for a polymerase chain reaction (PCR) step. Mutant primers are listed in Figure 3, where W125F refers to the mutation of tryptophan at position 125 to phenylalanine, and F163W refers to the mutation of phenylalanine at position 163 to tryptophan. Next, Dpn I was added to act at 37 ° C, the original template was removed, and the plastid was sent to E. coli competent cells, and sequencing was performed to confirm the successful mutant gene. Thereafter, the successfully mutated genes were each sent to Pichia pastoris according to the aforementioned method.

Figures 4 to 6 show the three mutant genes and amino acid sequences constructed in this case, including W125F, F163W and W125F/F163W. W125F means that the tryptophan at position 125 of xynCDBFV is mutated to phenylalanine, F163W is the 163th position of phenylalanine mutated to tryptophan, while W125F/F163W represents the mutation of the 125th position of tryptophan to phenylalanine and the 163th position of phenylalanine to tryptophan, in which W125F, The DNA sequences of F163W and W125F/F163W are designated by SEQ ID NOs: 3, 5, and 7, respectively, and the amino acid sequences of W125F, F163W, and W125F/F163W are designated by SEQ ID NO: 4, 6, and 8.

In order to verify the difference between the xylanase xynCDBFV original protein and the mutant xylanase, the enzyme activity was further measured in the present case. The xylanase activity test method is to mix 1% β-polyxylose with an appropriate concentration of enzyme protein (pH 0.05 M sodium acetate; pH 5.3) in a ratio of 9:1, and act at 55 ° C. minute. Next, 1 volume of 1% DNS was added to 100 ° C boiling water for 10 minutes to stop the reaction and color. Before measuring the absorbance, add 2.5 times the volume of water to the color tube and mix well. The absorbance was measured at the OD540 wavelength and converted to the enzyme activity unit. The standard curve for enzyme activity is determined by a xylose standard solution between 0 and 0.6 mg/ml. The 1 unit is defined as the amount of enzyme protein required to release 1 μmole of product per minute.

Figure 7 shows the results of the activity test of xylanase xynCDBFV original protein and mutant xylanase, in which different enzyme activities were compared under the benchmark of 100% of the original protein enzyme activity, and the statistical analysis was Using the two-tail analysis of T-test, a significant difference ( ^* ) was determined at P < 0.05, and the error bars were expressed as standard error of the mean (SEM). From the results shown in the figure, the specific activity of single mutation W125F and F163W is higher than that of the original protein, while the double mutation W125F/F163W has a more obvious specific activity, which is nearly 20%. In addition, in the Pichia pastoris expression system, the amount of mutant protein expression is comparable to that of the original protein. In terms of total activity, W125F/F163W also has the highest enzyme activity, which means that this mutant protein is indeed more valuable than the original protein.

In summary, in order to enhance the activity of xylanase, the three-dimensional structure of xylanase xynCDBFV was analyzed in this case, and the mutants were obtained by site-directed mutagenesis of Trp125 and Phe163 located in the active region of the steric structure of the protein. The enzyme proteins W125F, F163W and W125F/F163W all have higher enzymatic activities than the original protein, so the production cost can be reduced, and the industrial application potential is more practical.

Furthermore, xylanase is widely used in industry. For example, in the fruit juice production of the food industry, it is necessary to decompose a large amount of pectin in fruits by pectinase. However, pectin will be combined with hemicellulose and the like. The coexistence of substances, so in the production process of juice, the addition of xylanase to the juice to be clarified can help the pectinase, thereby improving the clarity of the juice. In addition, in the production of beer, the addition of xylanase during the saccharification process can effectively help break down the main raw materials such as barley or wheat, which can reduce the viscosity and increase the extraction yield and filtration effect. In the feed industry, liquid xylanase is sprayed on a carrier such as bran and then mixed with the feed to help monogastric animals such as pigs and chickens to decompose these hemicellulose-containing materials. The feed further promotes the digestion and absorption of plant material by the intestinal tract of the animal. In the textile industry, for example, xylanase is used for retting of plant material to obtain hemp or flax fibers. In addition, xylanase is particularly important in the manufacturing process of the paper industry. The most critical part of the pulp making process is the pulp bleaching step, which breaks down the hemicellulose tightly bound to lignin and removes it. With brown residual lignin and its derivatives, which effectively achieve the result of bleaching, xylanase can replace the traditional bleaching method using chlorine-containing chemicals to reduce the production of toxic by-products. Finally, in terms of biomass energy, the addition of xylanase during the saccharification process can help break down plant material into monosaccharides that can be used by microorganisms, allowing specific microorganisms to ferment by monosaccharides to produce a large number of Bio-alcohol. thus It can be seen that xylanase can be applied in many different industrial fields and has certain economic value. In this case, genetic engineering technology is used to transform proteins, which greatly increases the activity of xylanase and further reduces production costs. Increase the value of its industrial application. Therefore, the xylanase proposed in this case is of great industrial value and is submitted in accordance with the law.

This case has been modified by people who are familiar with the technology, but it is not intended to be protected by the scope of the patent application.

<110> Ji Ye Biotechnology Co., Ltd.

<120> Xylanase with enhanced enzyme activity

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Claims (10)

A xylanase having an amino acid sequence is a mutation of a tryptophan acid at position 125 of SEQ ID NO: 2 to phenylalanine, and a phenylalanine at position 163 to an amino acid sequence of tryptophan. The xylanase according to claim 1, wherein the gene encoding the SEQ ID NO: 2 is a xynCDBFV gene isolated and mutated from the rumen fungus Neocallimastix patriciarum . The xylanase according to claim 1, wherein the xylanase amino acid sequence is the amino acid sequence of SEQ ID NO: 8. A xylanase whose amino acid sequence is an amino acid sequence which mutates the tryptophan at position 125 of SEQ ID NO: 2 to amphetamine. The xylanase according to claim 4, wherein the gene encoding the SEQ ID NO: 2 is a xynCDBFV gene isolated and mutated from the rumen fungus Neocallimastix patriciarum . The xylanase according to claim 4, wherein the xylanase amino acid sequence is the amino acid sequence of SEQ ID NO: 4. A xylanase whose amino acid sequence is an amino acid sequence which mutates phenylalanine at position 163 of SEQ ID NO: 2 to tryptophan. The xylanase according to claim 7, wherein the gene encoding the SEQ ID NO: 2 is a xynCDBFV gene isolated and mutated from the rumen fungus Neocallimastix patriciarum . The xylanase according to claim 7, wherein the xylanase amino acid sequence is the amino acid sequence of SEQ ID NO: 6. An industrial use of a xylanase according to any one of claims 1-9, wherein the industry is selected from the group consisting of the food industry, the feed industry, the textile industry, the paper industry, and the biomass energy industry.