Cellulase and application thereof

Abstract

The invention discloses cellulase and application thereof, and belongs to the technical field of protein engineering. The invention develops a novel cellulase (named RuCel066) based on a rumen microbial metagenome resource mining technology, and the cellulase has the capability of effectively hydrolyzing agricultural by-products. Research and application of the enzyme are helpful for improving utilization rate and economic benefit of agricultural by-products.

Description

Technical Field

[0001] The present invention relates to the technical field of protein engineering, and particularly relates to a cellulase and its application. Background Art

[0002] As the most abundant renewable carbon source on earth, lignocellulosic biomass has become the core research object for replacing fossil fuels. This kind of biomass is composed of a composite network structure formed by cellulose, hemicellulose and lignin. Although it endows plants with mechanical strength, its dense crystal structure and the stubborn cross-linking of lignin-carbohydrates seriously reduce the enzymatic hydrolysis efficiency. Especially for agricultural by-products such as camellia seed meal, wheat bran, and corn straw, the complex matrix formed by their high content of cellulose and lignin leads to bottlenecks such as strong substrate specificity and low synergy efficiency in the traditional single-functional enzyme system, resulting in low sugar conversion rate and high industrial application cost.

[0003] Enzymatic hydrolysis technology has become a key path for biotransformation due to its environmental friendliness and high specificity. Among them, cellulase (including endoglucanase, exoglucanase, β-glucosidase, etc.) can improve the depolymerization efficiency of polysaccharides. Therefore, it is urgent to provide a variety of alternative cellulases. Summary of the Invention

[0004] The purpose of the present invention is to provide a cellulase and its application to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above purpose, the present invention provides the following solutions: One of the technical solutions of the present invention is a cellulase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0006] Another technical solution of the present invention is a DNA molecule encoding the cellulase, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] Another technical solution of the present invention is the application of the cellulase in hydrolyzing sodium carboxymethyl cellulose and xylan.

[0008] Another technical solution of the present invention is a method for hydrolyzing agricultural by-products to produce reducing sugars, using the cellulase to carry out hydrolysis reaction on the agricultural by-products.

[0009] Another technical solution of the present invention is a product for producing reducing sugars from agricultural by-products, including the cellulase.

[0010] Based on the above technical solutions, the present invention has the following technical effects: Based on the rumen microbial metagenomic resource mining technology, the present invention has developed a novel cellulase (named RuCel066), which has the ability to effectively hydrolyze agricultural by-products. The research and application of this enzyme will contribute to improving the utilization rate and economic benefits of agricultural by-products. Brief Description of the Drawings

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0012] Figure 1 Results of the pH dependence and tolerance of the RuCel066 enzyme.

[0013] Figure 2 Results of the temperature dependence of the RuCel066 enzyme.

[0014] Figure 3 Results of the temperature tolerance of the RuCel066 enzyme.

[0015] Figure 4 Effects of metal ions and common inhibitors on the activity of the RuCel066 enzyme.

[0016] Figure 5 Results of the sensitivity experiment of RuCel066 to SDS.

[0017] Figure 6 Results of the hydrolysis experiment of RuCel066 on agricultural by-products. Detailed Embodiments

[0018] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0019] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0021] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the description of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the description of this invention are obvious to those skilled in the art. The description and examples of this application are merely exemplary.

[0022] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0023] The technical solutions described in this invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0024] An embodiment of this invention provides a cellulase, the amino acid sequence of which is shown as SEQ ID NO.2.

[0025] An embodiment of this invention also provides a DNA molecule encoding the cellulase, the nucleotide sequence of which is shown as SEQ ID NO.1.

[0026] An embodiment of this invention also provides the application of the cellulase in hydrolyzing sodium carboxymethyl cellulose and xylan.

[0027] An embodiment of this invention also provides a method for hydrolyzing agricultural by-products to produce reducing sugars, using the cellulase to carry out a hydrolysis reaction on the agricultural by-products.

[0028] In some specific embodiments, the agricultural by-products include wheat bran, wheat, rice straw, wheat straw, corn straw, soybean straw, rape straw, and camellia oleifera meal.

[0029] In some specific embodiments, the addition amount of the cellulase is 4.16 U; the conditions of the hydrolysis reaction are: pH is 5.0 - 6.0, and the temperature is 20 - 40 °C.

[0030] In some specific embodiments, the conditions of the hydrolysis reaction are: adding 1 - 5 mM of Mn 2+ .

[0031] An embodiment of the present invention also provides a product for producing reducing sugar by using agricultural by-products, including the cellulase.

[0032] Example 1

[0033] Production steps of RuCel066 1.1 Cloning and expression of RuCel066 (1) Using rumen microbial DNA as a template, the RuCel066 gene was PCR amplified with the primer pair (SEQ ID NO.3: 5'-CTTTAAGAAGGAGATATACGGATCCATGAAGAAATTATCAGTAGTT-3' and SEQ ID NO.4: 5'-AGTGGTGGTGGTGGTGGTGCTCGAGTTTGCGACTTGACTCCGGCGC-3') to obtain a PCR product; the PCR amplification system is shown in Table 1 below, and the PCR amplification program is shown in Table 2 below.

[0034] Table 1 PCR amplification system (20 μL) , Table 2 PCR amplification program , (2) The pcr product was inserted into the pET-28a expression vector by homologous recombination using the Hieff Clone® Plus Multi One Step cloning kit (Yeasen, Shanghai) and transformed into Escherichia coli Top10 cells by heat shock method.

[0035] (3) The pET-RuCel066 plasmid was extracted, verified by PCR amplification, and sequenced. The nucleotide sequence is shown in SEQ ID NO.1:

[0036] 1.2 Preparation of RuCel066 enzyme solution.

[0037] (1) Introduce the pET-RuCel066 plasmid into Escherichia coli BL21 (DE3) by heat shock and screen for transformants overnight at 37 °C on an LB agar plate containing kanamycin; (2) Identify positive colonies by PCR and specific primers; (3) Culture the bacterial solution in an LB liquid medium containing kanamycin and culture at 37 °C until the OD600 reaches 0.7; (4) Add isopropyl-β-thiogalactoside (IPTG) at a final concentration of 0.2 mM to induce the expression of RuCel066; (5) Then transfer the culture to 20 °C and continue culturing for more than 18 hours; (6) After the culture is completed, centrifuge the mixture at 8000 rpm for 10 minutes at 4 °C to collect the bacteria; (7) Resuspend the bacterial pellet in PBS and disrupt it with ultrasound; (8) Lyse the cells using the ultrasonic disruption method, then separate the supernatant from the pellet by centrifugation, and aspirate the supernatant as the crude enzyme solution; (9) Purify the crude RuCel066 enzyme solution by affinity chromatography using a 5 mL BioScale Mini Nuvia IMAC nickel-charged column (BioRad, Hercules, CA, USA) connected to a low-pressure chromatography system (Bio LP; Bio-Rad, Hercules, CA, USA); Purify to obtain the RuCel066 enzyme solution.

[0038] The sequence of the RuCel066 enzyme is shown in SEQ ID NO.2: SEQ ID NO.2: MKKLSVVIVALLAVVSACVAQEKKVRPMDNEAIQFAMSMGIGWNLGNQMDAHYNGCSYEEGWGNKAATQQTFNGLAKAGFKSVRIPVTWMGHIGNAPTYTIEKGWMDRVAELVDMAHKAGLIVIINIHHDGFGAADTPSKGYHWLDLPGAAANEEVNQRIKQQLTMVWLQIGKRFANYDEWLVFETLNECQDGQWGGGANRTDNGAQYRVLNEWNQVCVNAIRAAGGKNETRYIGIPGYVCNPDLTVENLIIPEDIVENRIMVAVHSYDPWDFAGSAEKNEWGHTGIDVVPGSDENAYVGMLNRLFNMYVRRGIPVYFGEFGAVRRTNPADEPFRLYYFRYVCKAMRDRRIPALYWDNGNSKAGNDGFGVINHATGRYIGNGEQAVRAMIDSWENNDPNYTLQAVYDSAPESSRK*。

[0039] Enzymatic Characterization of 2 RuCel066 (1) Substrate Specificity The substrate specificity of RuCel066 was investigated using a panel of five substrates: sodium carboxymethyl cellulose, microcrystalline cellulose, xylan, chitosan, and Poria cocos polysaccharide. Purified 2.8 U RuCel066 was added to 50 mM citrate-disodium hydrogen phosphate buffer (pH = 6) containing 1% (w / v) substrate and incubated in a shaking water bath at 40 °C for 30 minutes. The reducing sugar concentration of the reaction solution was measured using alkaline 3,5-dinitrosalicylic acid (DNS), and the amount of reducing sugar was calculated using a standard curve. One unit (U) of enzyme activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar in 1 min under the assay conditions.

[0040] As shown in Table 3, the enzyme exhibited varying degrees of activity towards the five substrates, with RuCel066 showing significantly higher activity towards sodium carboxymethyl cellulose and xylan than towards the other substrates.

[0041] Table 3 Substrate Specificity of RuCel143 , (2) pH Dependence and Tolerance Based on the above experiments, sodium carboxymethyl cellulose was selected for property analysis, and the addition amount of RuCel066 was 1.4 U. The pH-dependent activity of RuCel066 was evaluated using 50 mM disodium hydrogen phosphate-citrate buffer in the pH range (3.0 - 8.0). Using 1% (w / v) CMC-Na as the substrate, the reaction was carried out at 40 °C for 30 minutes, and the released reducing sugar was measured after the reaction. To determine the pH tolerance, RuCel066 was pre-incubated in the same buffer system without substrate at 30 °C for 30 minutes. The residual enzyme activity was measured under the optimal reaction conditions. The pH stability of RuCel066 was expressed as the percentage of the residual activity relative to the untreated enzyme sample, which was set as 100%.

[0042] As Figure 1 shown, for sodium carboxymethyl cellulose, the activity of RuCel066 reached its peak at pH 5.0 and maintained more than 80% of its activity in the pH range of 5.0 - 6.0.

[0043] (3)Temperature dependence and tolerance Based on the above experiments, sodium carboxymethyl cellulose was selected for property analysis, and the addition amount of RuCel066 was 1.4 U. The optimal temperature for RuCel066 activity was determined by reacting in 50 mM disodium hydrogen phosphate-citrate buffer (pH 5.0) and 1% (w / v) CMC-Na at temperatures ranging from 20 °C to 80 °C for 30 minutes. The thermal stability was evaluated by incubating the enzyme at 50 °C, 60 °C, 70 °C, and 80 °C for different durations (5, 10, 20, and 30 minutes) at pH 5.0 and then measuring the residual activity under the optimal conditions. The thermal stability was evaluated by comparing the remaining activity with the activity of the untreated enzyme sample (set as 100%).

[0044] As Figure 2 shown, RuCel066 exhibited the highest activity at 30 °C and maintained more than 90% of its activity in the range of 20 - 40 °C. At 50 °C, the activity decreased to 49.7%, and at 60 °C, only 1.19%. For the temperature tolerance results as Figure 3 shown, at 50 °C, the activity decreased over time and remained at 17.4% after 30 minutes. However, the detectable activity at 60 °C, 70 °C, and 80 °C was quite weak.

[0045] (4)Effects of metal ions and common inhibitors The effects of K⁺, Ca²⁺, Na⁺, Mg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Ni²⁺, EDTA, SDS, β-ME, and DTT on enzyme activity were examined at final concentrations of 1 mM and 5 mM. Additionally, the effects of Tween-20 and Triton X-100 on enzyme activity were evaluated at final concentrations of 1% (v / v) and 5% (v / v). All reactions were carried out under the optimal conditions (30 °C, pH = 5) of 50 mM sodium hydrogen phosphate-citrate buffer with an addition amount of 1.4 U of RuCel066. The reaction mixture without any additives served as the positive control, and its activity was defined as 100%.

[0046] The results are as Figure 4 shown: Mn 2+ significantly increased the cellulase activity of RuCel066, and at concentrations of 1 mM and 5 mM, the cellulase activities of RuCel066 were increased by 70.2% and 103.6%, respectively. In contrast, Cu 2+ significantly inhibited the activity of RuCel066, reducing the activity to 2.3% and 0.86% at 1 mM and 5 mM, respectively. Other metal ions, such as K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ and Ni 2+ , had less effect on the activity of RuCel066.

[0047] In addition, RuCel066 was significantly sensitive to SDS, as Figure 5 shown. The activity of RuCel066 was reduced to almost zero by 1 mM and 5 mM of SDS, while high concentrations of EDTA and DTT reduced the activity to 76.6% and 71.4%, respectively. The inhibitory effect of β-mercaptoethanol was also obvious, and the enzyme activity was reduced to 87.9% and 81.0% at 1 mM and 5 mM. In contrast, Tween-20 and Triton X-100 had less effect on the activity of RuCel066.

[0048] 3 Hydrolysis of agricultural by-products by RuCel066 To evaluate the hydrolysis ability of RuCel066 on agricultural by-products, experiments were conducted on wheat bran, wheat, rice straw, wheat straw, corn straw, soybean straw, rape straw and camellia oleifera seed cake at a final concentration of 1% (w / v, 0.01 g of substrate added to 1 ml of liquid). The reaction was carried out in 50 mM phosphate-citrate buffer (pH 5.0) at 30 °C, and the enzyme addition amount was 4.16 U. A control reaction without enzyme was carried out in parallel to establish the baseline hydrolysis of each substrate. The hydrolysis efficiency was evaluated by comparing the reducing sugars released from the enzyme-treated samples and the control samples.

[0049] The results are as Figure 6 shown. The average concentrations of reducing sugars released by the hydrolysis of RuCel066 on these substrates are: wheat bran (122.99 µg / mL), wheat (127.10 µg / mL), rice straw (99.06 µg / mL), wheat straw (37.35 µg / mL), corn straw (226.97 µg / mL), soybean straw (97.35 µg / mL), rape straw (102.54 µg / mL), camellia oleifera seed cake (59.97 µg / mL).

[0050] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A cellulase, characterized in that, Its amino acid sequence is as shown in SEQ ID NO.

2.

2. A DNA molecule encoding the cellulase according to claim 1, characterized in that, Its nucleotide sequence is as shown in SEQ ID NO.

1.

3. Use of the cellulase according to claim 1 in hydrolyzing sodium carboxymethyl cellulose and xylan.

4. A method for hydrolyzing agricultural by-products to produce reducing sugars, characterized in that, Use the cellulase according to claim 1 to carry out a hydrolysis reaction on the agricultural by-products.

5. The method according to claim 4, characterized in that, The agricultural by-products include wheat bran, wheat, rice straw, wheat straw, corn straw, soybean straw, rape straw and camellia oleifera cake.

6. The method according to claim 4, characterized in that, The addition amount of the cellulase is 4.16 U; the conditions for the hydrolysis reaction are: pH is 5.0 - 6.0, and the temperature is 20 - 40 °C.

7. The method according to claim 4, characterized in that, The conditions for the hydrolysis reaction are as follows: add 1 - 5 mM of Mn 2 + .

8. A product for producing reducing sugars using agricultural by-products, characterized in that, It includes the cellulase according to claim 1.

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