Xylanase GH10 and application thereof

Abstract

The invention provides xylanase GH10 and application thereof, the xylanase shows the highest activity (Vmax = 0.6691 [mu] mol / min.mg) at 40 DEG C, and still retains gt at 4 DEG C; the method is suitable for preparing low-temperature fermented foods (such as steamed sponge cakes). Through a recombinant expression technology, the enzyme can be efficiently expressed in escherichia coli, and low-cost production is realized by optimizing a fermentation process. Application experiments prove that by adding the enzyme, the specific volume of the steamed sponge cake can be increased by 42%, and the bulkiness and flavor are remarkably improved. The invention provides a new scheme for low-temperature application of the lignocellulose degrading enzyme and upgrading of a traditional food process.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a xylanase GH10 and its application. Background Art

[0002] The origin of the steamed sponge cake can be traced back to ancient China and is closely related to the farming culture. The ancients used fermentation technology to make pastries. The steamed sponge cake got its name "fa" (which means "prosperity" in Chinese) because yeast or fermented glutinous rice wine was used in its fermentation, and it is often used as a lucky food during festivals or celebrations. Steamed sponge cakes in each region have their local characteristics. For example, in the Jiangnan region: Zhejiang (such as Quzhou and Longyou): Usually, rice flour is fermented with fermented glutinous rice wine, and the taste is soft and slightly sweet. Shanghai: Chongming cake is a large-sized steamed sponge cake, usually made by mixing glutinous rice and japonica rice. Guangdong: Called "Malagao", with brown sugar and coconut milk added, and it is dark brown after steaming, commonly seen in teahouses. Guangxi: Hakka steamed sponge cake is fermented with sticky rice flour and often decorated with red dates or wolfberries. Central China: Hunan and Hubei: Usually made with brown sugar or white sugar and steamed during festivals, meaning good luck. North China and Northwest China: In Shanxi, Shaanxi and other places, there are similar pasta to the steamed sponge cake, but more are mainly made of wheat flour and are called "date cake" or "yellow cake". Usually, to change the taste of the steamed sponge cake, the original formula of the steamed sponge cake is changed. For example, using a certain proportion of japonica rice flour and glutinous rice flour in combination, adding various auxiliary materials such as eggs, oils or nuts to enhance the aroma of the steamed sponge cake. However, since the raw material rice flour used in the preparation of the steamed sponge cake contains hemicellulose, which is mainly xylan, composed of β-1,4-xylose chains and may carry side chains such as arabinose and glucuronic acid, hemicellulose has strong water absorption, and excessive undigested hemicellulose will increase the viscosity of the flour and inhibit the gas retention ability. Adding xylanase during the fermentation process to decompose the excessive hemicellulose can form a gel network and improve the fluffiness of the steamed sponge cake. And the decomposition product xylose of hemicellulose generates furan and pyrazine flavor compounds in the Maillard reaction, giving the steamed sponge cake a caramel or sweet aroma. We take the steamed sponge cake in Central China as an example, using ordinary rice flour, yeast and white sugar.

[0003] Due to the insufficient degradation of hemicellulose (mainly xylan) in the rice flour of traditional steamed sponge cakes, the viscosity is too high, the gas retention ability is poor, and the fluffiness of the finished product is insufficient. Adding exogenous xylanase can decompose hemicellulose, but the existing enzyme preparations are inefficient in low-temperature fermentation (below 37°C), which restricts the process optimization.

[0004] Most of the currently known xylanases are derived from fungi and bacteria, but the optimal temperature of most enzymes is relatively high (50 - 60°C), and their activity decreases significantly at low temperatures (4 - 25°C), which limits their application in low-temperature fermented foods (such as traditional steamed sponge cakes). There are few reports on the GH10 family xylanases with both high activity at low temperature and acid resistance (pH 4 - 5) in existing research.

[0005] Therefore, it is necessary to develop a xylanase with good activity at low temperatures to solve the problem of insufficient activity of xylanase in low-temperature fermentation (such as making steamed sponge cake) in the prior art. Summary of the Invention

[0006] The object of the present invention is to provide a xylanase GH10 and its application. This xylanase GH10 has both high activity at low temperatures (retaining > 60% activity at 4°C) and acid resistance (pH 4.0), and can simultaneously improve the fluffiness and flavor of steamed sponge cake.

[0007] The present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a xylanase gene xyn-4670 derived from Stropharia rugosoannulata is provided, and the nucleotide sequence of the gene is as shown in SEQ ID NO: 1.

[0009]

[0010] In a second aspect of the present invention, there is provided a xylanase GH10, and the amino acid sequence of the xylanase GH10 is as shown in SEQ ID NO: 2.

[0011] GSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFQTAVSLLIDFPGYQSSGTINLRSTLASVSQAKGSTFHFGSTYDTYDADASFSQNVFSTFFNHIVAENGCKWDATEPTRGVPDLTECQAVQSYAATNGDTFRGHNTFWHSQTPTWLPGTVTASDLVDNVIPQHVQQTIQGMGTSVTSWDVVNEIVGDGVSNGMTALQCVKNKNDWPTQASDGSSTTLVTDLSFVHAAFSTALQYAGPNTRLAINDYNTGGNDAKTACMFTLLADINANAAVPYNRLAVGFQSHISPTSFVSKSALSATFSKLAALGANAMITELDIALPSGTSAYERLQAAIWGDYLDACLYASNCNEFINWDTRDDVSWLGTSEAGTLFDSNGNPKPAAFEVQARMQRFASGAPMLCATALGTSSCMASGPTGGSSTASSPSSSSTVSKPSSTSTAPASGATAAHWGQCGGIGWTGPTVCASPYTCQVSNAYYSQCL(SEQ ID NO: 2).

[0012] pI: 5.39 Molecular weight: 50704.83

[0013] In a third aspect of the present invention, there is provided a recombinant expression vector capable of expressing the xylanase GH10 as claimed in claim 2.

[0014] Further, the recombinant expression vector includes at least one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacterium expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

[0015] In a fourth aspect of the present invention, there is provided a recombinant bacterium or engineered cell line comprising the recombinant expression vector.

[0016] Furthermore, the host cell includes one of Escherichia coli host cell, yeast host cell, Bacillus subtilis host cell, lactic acid bacteria host cell, actinomycetes host cell, filamentous fungus host cell, and insect cell.

[0017] In the fifth aspect of the present invention, there is provided an application of the gene, the xylanase GH10, the recombinant expression vector, the recombinant bacterium or the engineered host cell line in the preparation of a product for fermenting Chinese rice cakes.

[0018] In the sixth aspect of the present invention, there is provided a method for preparing a low-temperature xylanase, the method comprising:

[0019] extracting total RNA from Stropharia rugosoannulata and reverse-transcribing it into cDNA;

[0020] using the cDNA as a template and performing PCR amplification with the primer pair shown in SEQ ID NO.3 - SEQ ID NO.4 to obtain the xyn-4670 gene;

[0021] inserting the xyn-4670 gene between the Nde1 and Not1 restriction enzyme cleavage sites of the expression vector pET-28a to construct an Escherichia coli recombinant expression vector pET-28a-xyn4670;

[0022] transforming the recombinant expression vector into a host cell and inducing expression at 18 - 25 °C, and purifying to obtain the low-temperature xylanase.

[0023] In the seventh aspect of the present invention, there is provided a method for preparing Chinese rice cakes, the method comprising: adding the xylanase to the raw rice flour of Chinese rice cakes for preparation.

[0024] Furthermore, the addition amount of the xylanase is 0.05 - 0.5% of the mass of the rice flour.

[0025] Furthermore, the enzyme can randomly cleave the β-1,4-glycosidic bond inside the xylan main chain to generate xylo-oligosaccharides (such as xylobiose, xylotriose).

[0026] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0027] 1. The xylanase GH10 provided by the present invention and its application. This xylanase GH10 has both low-temperature high activity (the optimal temperature is 40 °C, and it still retains >60% activity at 4 °C) and acid resistance (the optimal pH is 4.0); kinetic parameters: Km = 7.231 mg / mL, Vmax = 0.6691 μmol / min·mg. Stability: It retains 85% activity after being treated at 40 °C for 1 h and is stable within the pH range of 3 - 5.

[0028] 2. For the first time, the industrial application of this enzyme in traditional low-temperature fermented foods (steamed sponge cake) was realized, breaking through the bottleneck of viscosity control in low-temperature fermentation. Add purified enzyme solution (50 - 500 μL / g rice flour) to the rice flour and steam it after fermenting at 37°C for 1 h. Effect verification: When the enzyme dosage is 200 μL / g, the specific volume of the steamed sponge cake increases by 42%, and the texture indexes (chewiness, adhesiveness) are significantly optimized. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a PCR nucleic acid gel diagram and a protein SDS-page diagram.

[0031] Figure 2 It is an SDS-page diagram of the purified protein, and the size of the target protein is 50 kDa.

[0032] Figure 3 It is the optimum temperature measured through orthogonal experiments.

[0033] Figure 4 It is the optimum pH measured through orthogonal experiments.

[0034] Figure 5 By changing the substrate concentration, the xylose content generated in the reaction was calculated according to the standard curve of the reaction between xylose and DNS at a wavelength of OD540, so as to obtain the Lineweaver - Burk diagram of xylanase xyn - 4670.

[0035] Figure 6 It is five groups of pictures of steamed sponge cakes, two in each group. Groups 1, 2, 3, and 4 are experimental groups, and the enzyme addition amounts are 500 μL, 200 μL, 100 μL, and 50 μL respectively. The 5th group is the control group without adding enzyme.

[0036] Figure 7 It is the change of adhesiveness of the steamed sponge cake measured by a texture analyzer under the condition of adding enzyme solutions with different volumes.

[0037] Figure 8 It is the change of chewiness of the steamed sponge cake measured by a texture analyzer under the condition of adding enzyme solutions with different volumes. <S

[0038] Figure 9 It is the change of hardness of the steamed sponge cake measured by a texture analyzer under the condition of adding enzyme solutions with different volumes. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0040] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0041] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or by existing methods.

[0042] A xylanase GH10 of the present application and its application will be described in detail below in conjunction with examples and experimental data. The pET28a(SUMO) vector of the present application is a commercial vector and can be purchased from a biological company, such as addgene, with the catalog number MLCC1025; Escherichia coli Rosetta 2(DE3) can be obtained commercially. In the present application, there are no special requirements for the type of expression vector and the strain of Escherichia coli.

[0043] Example 1: Cloning of the xylanase xyn-4670 gene

[0044] Extract the RNA genome of Stropharia rugosoannulata from the mycelium of Stropharia rugosoannulata, and obtain the cDNA group of Stropharia rugosoannulata through reverse transcriptase. Design primers:

[0045] Forward primer (degenerate primer): 5’-GCTSVHTSHTBBTHC-3’ (SEQ ID NO.3);

[0046] Reverse primer: 5’-TTTTTTTTTTTT-3’ (SEQ ID NO.4).

[0047] Use the cDNA as a template to clone the target gene xyn-4670 through PCR technology. The PCR program is set with the denaturation temperature at 94°C for 5 minutes per cycle, the annealing temperature at 52°C for 30 seconds per cycle, the extension temperature at 72°C for 2 minutes per cycle, and the number of cycles is 34 cycles. The PCR results are as Figure 1 .

[0048] The amplified target gene xyn-4670 was ligated to the expression vector pET-28a to construct the recombinant expression vector pET-28a-xyn4670 of Escherichia coli. It was fermented at 37 °C for 4 h to an OD600 of between 0.6 and 0.8 using the ZYM-5052 lactose induction medium, and then transferred to 18 °C for fermentation for 20 h. After harvesting the bacteria by centrifugation, a crude enzyme solution was obtained by ultrasonic disruption, and a purified enzyme solution was obtained by nickel column purification. The size of the protein was verified by SDS-page diagram, as Figure 2 , and the size of the target protein was 50 kDa.

[0049] Example 2: Determination of the Optimal Temperature and Optimal pH of the Enzyme

[0050] 0.1 g of arabinoxylan was dissolved in 10 mL of water as the substrate for the determination of xylanase activity. Through orthogonal experiments, the activities of xylanase were measured at pH 3, 4, 5, 6, 7, 8 and temperatures of 4 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, respectively. The results were as Figure 3 , 4.

[0051] From Figure 3 and Figure 4 of the results, it can be seen that the optimal temperature is 40 °C and the optimal pH is pH = 4.

[0052] Example 3: Determination of Enzyme Kinetics

[0053] The standard curve of xylose was calculated by reacting known different concentrations of xylose with DNS and measuring the OD value under the condition of OD540. Reaction systems were prepared with different concentrations of arabinoxylan, and after measuring the OD values, the xylose production was calculated according to the standard curve, and the Lineweaver-Burk diagram was drawn, as Figure 5 .

[0054] From Figure 5 it can be seen that the kinetic parameters of xylanase were calculated as Km = 7.231 mg / mL and Vmax = 0.6691 μmol / min.

[0055] Example 4: Determination of Enzyme Activity at Low Temperature

[0056] 0.1 g of arabinoxylan was dissolved in 10 ml of water to prepare 10 mg / mL arabinoxylan as the substrate. 100 μL of the substrate and 190 μL of pH 4 buffer were taken, and then 10 μL of the purified enzyme solution (protein concentration 0.05 mg / mL) was added and reacted at different temperatures (4 - 70 °C) for 10 min, and the activity was determined by the DNS method.

[0057] Result: The highest activity OD was 2.28 (OD540) at 40 °C, 1.68 (OD540) at 20 °C, and 1.39 (OD540) at 4 °C.

[0058] Example 5. Application of xylanase xyn-4670 in steamed sponge cake

[0059] Prepare five beakers, add 45 g of organic rice flour to each beaker, add 45 g of water in a 1:1 ratio, stir well, add yeast powder at 3% by weight of the rice flour and 5 g of white sugar, and then add 0 μL, 50 μL, 100 μL, 200 μL, and 500 μL of purified enzyme solution to the beakers in sequence.

[0060] After the yeast ferments fully for 1 hour at 37 °C, pour the experimental groups in each beaker into three molds respectively, and then ferment for 10 minutes at 37 °C. The specific situation is as Figure 6 shown.

[0061] Put the fermented steamed sponge cakes into a steamer and steam for 20 - 30 minutes. After cooling, cut out two pieces of each steamed sponge cake, about 1 cm long and 8 mm thick, for testing.

[0062] There are two parallel groups for each experimental group, and two pieces of steamed sponge cakes to be tested are cut out from each group. There are a total of four steamed sponge cakes to be tested for each experimental group. Each group to be tested is detected three times under a texture analyzer, for a total of 12 times. Take the average value as shown in Figure 7 、 8 、9.

[0063] It can be seen from Figures 7 - 9 that increasing the dosage of xylanase can significantly improve the taste of the steamed sponge cake.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

[0065] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

[0066] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic inventive concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0067] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A xylanase gene xyn-4670 derived from Stropharia rugosoannulata, characterized in that, The nucleotide sequence of the said gene is as shown in SEQ ID NO:

1.

2. A xylanase GH10, characterized in that, The amino acid sequence of the said xylanase GH10 is as shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, The said recombinant expression vector can express the xylanase GH10 as claimed in claim 2.

4. The recombinant expression vector according to claim 3, wherein The said recombinant expression vector includes at least one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

5. A recombinant bacterium or engineered host cell line comprising the recombinant expression vector as claimed in any one of claims 3-4.

6. The recombinant bacterium or engineered host cell line according to claim 5, wherein The said host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, an actinomycete host cell, a filamentous fungus host cell, and an insect cell.

7. Use of the gene as claimed in claim 1, the xylanase GH10 as claimed in claim 2, the recombinant expression vector as claimed in any one of claims 3-4, and the recombinant bacterium or engineered host cell line as claimed in any one of claims 5-6 in the preparation of a product for fermenting rice cakes.

8. A preparation method of a low-temperature xylanase, characterized in that, The said method includes: Extracting total RNA from Stropharia rugosoannulata and reverse-transcribing it into cDNA; Using the said cDNA as a template and performing PCR amplification with the primer pair shown in SEQ ID NO.3-SEQ ID NO.4 to obtain the xyn-4670 gene; Inserting the said xyn-4670 gene between the Nde1 and Not1 restriction enzyme cleavage sites of the expression vector pET-28a to construct the Escherichia coli recombinant expression vector pET-28a-xyn4670; Transforming the said recombinant expression vector into a host cell and inducing expression at 18-25 °C, and obtaining a low-temperature xylanase through purification.

9. A preparation method of sponge cake, characterized in that, The said method includes: preparing by adding the xylanase as claimed in claim 2 to the raw material rice flour of the rice cake.

10. The preparation method of a rice cake according to claim 9, characterized in that, The addition amount of the said xylanase is 0.05-0.5% of the mass of the rice flour.

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