A multifunctional cellulase and its application in degrading agricultural byproducts

Abstract

This invention discloses a multifunctional cellulase and its application in degrading agricultural by-products, belonging to the field of biotechnology. The multifunctional cellulase is named RuCelI, and its amino acid sequence is shown in SEQ ID NO.1. Based on rumen microbial metagenomic resource mining technology, this invention has developed a novel multifunctional cellulase RuCelI. This enzyme possesses dual activities as both an endonuclease and a xylanase, enabling it to degrade agricultural by-products, reduce processing costs, improve degradation efficiency, and contribute to increasing the utilization rate and economic benefits of agricultural by-products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a multifunctional cellulase and its application in the degradation of agricultural by-products. Background Technology

[0002] Crops play a vital role in global food production, generating vast quantities of byproducts such as rice straw, corn stalks, wheat straw, soybean straw, rapeseed straw, and camellia seed meal. These byproducts are rich in lignocellulose and other components, representing a potential high-quality feed source for ruminants. However, current rumen digestibility of these byproduct fibers in ruminants is generally insufficient. The main obstacle lies in the dense cross-linked structure formed by cellulose, hemicellulose, and lignin, which severely hinders the utilization of substrates by rumen microorganisms and enzymes, significantly limiting fiber degradation efficiency and the improvement of animal production performance. Therefore, developing highly efficient degradation enzymes adapted to the rumen environment to break down the aforementioned cross-linked structure and improve fiber accessibility is key to solving the problem of efficient utilization of crop byproducts.

[0003] Enzymatic hydrolysis technology, as an important means to improve the degradation of agricultural by-products, has advantages such as mild operation, low cost, and low pollution. Among them, the synergistic effect of endoglucanase (the core component of the cellulase system) and xylanase (which can improve the accessibility of cellulose to enzymes), or bifunctional enzymes, has been proven to improve hydrolysis efficiency. However, existing enzyme preparations often suffer from unstable application effects and difficulty in fully exerting their effects due to the mismatch between their temperature and pH adaptation ranges and the rumen environment.

[0004] The rumen of ruminants is a veritable treasure trove of enzyme genes. Most enzymes produced by rumen microorganisms are adapted to the rumen environment. However, due to technological limitations, many rumen bacteria cannot be cultured in vitro, leaving a large number of potentially highly efficient enzyme genes untapped. The advent of metagenomic sequencing technology has made it possible to directly obtain enzyme genes from the rumen microbiota, overcoming the bottleneck of traditional microbial culture. Summary of the Invention

[0005] The purpose of this invention is to provide a multifunctional cellulase and its application in degrading agricultural by-products, thereby addressing the problems existing in the prior art. This invention develops a novel multifunctional cellulase, RuCelI, which can be used to degrade agricultural by-products, improving degradation efficiency and contributing to increased utilization and economic benefits of agricultural by-products. It provides a new enzyme source and technical support for the efficient resource utilization of agricultural by-products.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] In a first aspect, the present invention provides a multifunctional cellulase RuCelI, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Secondly, the present invention also provides a gene encoding a multifunctional cell-degrading enzyme RuCelI, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] Thirdly, the present invention also provides a recombinant vector containing the aforementioned gene.

[0010] Fourthly, the present invention also provides a recombinant bacterium containing the recombinant vector.

[0011] Fifthly, the present invention also provides the application of the aforementioned multifunctional cellolytic enzyme RuCelI in the degradation of cellulose and / or xylan.

[0012] In a sixth aspect, the present invention also provides the application of the aforementioned multifunctional cellolytic enzyme RuCelI in the degradation of agricultural by-products.

[0013] Preferably, the agricultural by-products include one or more of rice straw, corn straw, wheat straw, soybean straw, rapeseed straw, wheat bran, and camellia oil meal.

[0014] In a seventh aspect, the present invention also provides the use of the gene, the recombinant vector, or the recombinant bacteria in the preparation of the multifunctional cellulase RuCelI.

[0015] In an eighth aspect, the present invention also provides a method for degrading agricultural by-products, comprising the step of degrading agricultural by-products using the multifunctional cellulase RuCelI.

[0016] Preferably, the degradation temperature is 30-40℃ and the pH is 5.0-8.0.

[0017] The present invention discloses the following technical effects:

[0018] This invention develops a novel multifunctional cellulase, RuCelI, which possesses dual activities as both an endonuclease and xylanase. It exhibits broad pH tolerance, high temperature tolerance, and wide applicability. Verification has shown that this enzyme has the ability to degrade agricultural by-products, improving degradation efficiency and thus contributing to increased utilization and economic benefits. This provides a new enzyme source and technological support for the efficient resource utilization of agricultural by-products. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The substrate specificity of RuCelI;

[0021] Figure 2 Temperature dependence of RuCelI;

[0022] Figure 3 The pH dependence of RuCelI;

[0023] Figure 4 The effect of metal ions on the activity of RuCelI;

[0024] Figure 5 The effects of inhibitors and detergents on RuCelI activity;

[0025] Figure 6 RuCelI is used to degrade agricultural byproducts. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now 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, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Example 1

[0032] 1. Production of RuCelI

[0033] 1.1 Cloning of RuCelI and Construction of Recombinant Expression Vectors

[0034] (1) Using rumen microbial DNA (NCBI ID: PRJNA806344) as a template, primers -F: 5′-CTTTAAGAAGGAGATATACGGATCCATGCAACTCAAGAATTTCTATC-3′ (SEQ ID NO.3) and R: 5′-CAAGGCTCTCGGCGTCGGAAAACTCGAGCACCACCACCACCACCACT-3′ (SEQ ID NO.4) were designed, and the RuCelI gene was cloned by PCR. The PCR amplification system is shown in Table 1 below.

[0035] Table 1 PCR amplification reaction system

[0036] reagents Dosage (μL) 2×Canace Plus PCR buffer 15 DNA template 0.5 Primer F 1.2 Primer R 1.2 Hieff Canace Plus High-Fidelity DNA Polymerase 0.6 <![CDATA[ddH2O]]> Supplement to 30

[0037] PCR amplification conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 20 s, for 30 cycles; and 72℃ extension for 5 min. HieffClone was used. ® The Plus One-Step Cloning Kit (catalog number: 10911ES20; Shanghai Yisheng Biotechnology Co., Ltd., China) ligates PCR amplification products into the pET-28a vector, which has been double-digested with restriction endonucleases BamHI and XhoI. The constructed plasmid is named pET-RuCelI and transformed into *E. coli* DH5α competent cells. After overnight incubation at 37°C, PCR verification is performed, and the plasmid is extracted and sequenced.

[0038] PCR amplification and sequencing results showed that the final amino acid sequence of RuCelI is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding RuCelI is shown in SEQ ID NO.2. Details are as follows:

[0039] SEQ ID NO.1:

[0040] MQLKNFYPKMSILGIATVMALTACSDENPTLPQVTDPNGGIYNPTPASSSDMNPTSSGAVIDPTSSSAVIDPTSSSAVVVDPSTLPAEGPITQPAGLGILVDDFEDGDNLSKINDYWYTYNDNDNGGASIITTPLNEEENIIPGRVNNGSNYALQVNYTLDRGDYEFDPYVGWGVQVAPDEANGRFGGLTYWYKGGAHEVHVEITDVEDYDVHLAKFPASRTWKQAVVRFKDLVQGGWGKEVPFDAKHIMAISFQAKGSKSKVMTDSLFIDNIYLQDSSEVEKDQPDMEIMDPVIPTVEFTEAEITVTNPLQEKAMKYLNKGVNFTNWLENADGKFKSFELGEKDVQILAENGFKSLRLPIDLDLYATNRDAFIAGTDTELKFDDDTLFLVLDSFVEWTAKYNMSLVIDYHEYDNSYNATSAKDANYIKMMAEVWKHVAAHYAESPREDLFFELLNEPDMSDGKVTAATWTTAAQAMIDAIRSVNTKHTILFGDAQWYSITLLAKRTPFTDDNIIYVIHTYEPFAFTHQGGSWTDYATIHDIPFPYDPAKWSTVSGDFGVNKSTKSYVRTAIKNYYKTGSKEAILEQILKAKKWAATNNVPVIINEFGALNLRSTAESRINYLTAMREICDTLQIPWTHWGYTGNFSVIENGKLIEGLDKALGVGK;

[0041] SEQ ID NO.2:

[0042]

[0043] 1.2 Preparation of RuCelI enzyme solution

[0044] To achieve RuCelI expression, the recombinant plasmid pET-RuCelI was transformed into *E. coli* BL21(DE3) competent cells using a heat shock method. The transformed cells were then plated on Luria-Bertani (LB) agar containing kanamycin and cultured overnight at 37°C. PCR-positive transformants were picked and inoculated into 2 mL of LB liquid medium containing kanamycin. This overnight culture was then added at a 1% inoculum to 400 mL of LB medium and cultured with shaking at 37°C and 180 rpm. Untransformed *E. coli* BL21(DE3) cells were cultured simultaneously as a control. When the optical density (OD value) of the culture reached 0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to induce expression, and induction was continued at 20°C and 80 rpm for 24 h. After induction culture, the culture was centrifuged (8000 rpm, 10 min); the E. coli precipitate was collected, resuspended and lysed with phosphate-buffered saline (PBS), then sonicated in an ice-water bath for 20 min, and centrifuged again (8000 rpm, 20 min). The supernatant was collected as the crude enzyme solution. The RuCelI protein in the crude enzyme solution was purified using a 5 mL Bio-Scale Mini Nuvia IMAC Ni-Charged column (purchased from Bio-Rad, USA) to obtain the RuCelI enzyme solution.

[0045] 2. Enzymatic Characterization of RuCelI

[0046] 2.1 Substrate specificity of RuCelI

[0047] To clarify the substrate preference of RuCelI, its substrate specificity was first determined. The substrates used included sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose, wheat straw xylan, and chitosan. The total reaction volume was 1 mL, with a substrate concentration of 1% (m / v) and a 0.05 M citrate-disodium hydrogen phosphate buffer (pH 6.0). 19.0 μg of RuCelI was added to the system, and the reaction was carried out at pH 6.0 and 40 °C: 30 minutes with xylan as the substrate and 2 hours with other substrates. Enzyme activity was determined using the DNS (3,5-dinitrosalicylic acid) method, and the amount of reducing sugar released was calculated based on the absorbance at 540 nm and a standard curve. Specifically, when sodium carboxymethyl cellulose was used as the substrate, glucose was used as the standard to calculate the amount of reducing sugar released; when xylan was used as the substrate, xylose was used as the standard. Enzyme activity 1 U is defined as the amount of enzyme required to produce 1 μmol of xylose or glucose per minute.

[0048] like Figure 1 As shown, RuCelI exhibited activity against all four tested substrates, with varying degrees of activity: highest activity against sodium carboxymethyl cellulose, followed by xylan, weaker activity against microcrystalline cellulose, and weakest activity against chitosan. Combined with substrate characteristic analysis (sodium carboxymethyl cellulose is a characteristic substrate for endocellulase, xylan is a characteristic substrate for xylanase, and microcrystalline cellulose is a characteristic substrate for exocellulase), this indicates that RuCelI possesses significant endocellulase and xylanase activities, while also exhibiting relatively weak exocellulase activity, making it a multifunctional cell-degrading enzyme.

[0049] 2.2 Temperature dependence of RuCelI

[0050] In a 1 mL reaction system, 1% sodium carboxymethyl cellulose (CMC-Na) or wheat straw xylan was added, followed by a pH 6.0 citrate-disodium hydrogen phosphate buffer solution and 19.0 μg RuCelI. A blank control group without RuCelI was set up to eliminate the interference of substrate degradation on the results. The above reaction system was incubated in a constant temperature water bath shaker with a temperature gradient of 20-90℃ for 30 min (wheat straw xylan as substrate) and 2 h (sodium carboxymethyl cellulose as substrate). After the reaction, the enzyme activity was characterized by measuring the amount of reducing sugar released in the system. Enzyme activity was expressed as relative activity: the activity value corresponding to the temperature at which the enzyme exhibited the highest activity (i.e., the optimum temperature) was set as 100%, and the enzyme activity under other temperature conditions was calculated as a percentage of the activity at the optimum temperature.

[0051] Figure 2The results showed that when sodium carboxymethyl cellulose (CMC-Na) was used as a substrate, the optimal temperature for this enzyme was 30℃, and even when the temperature was increased to 90℃, it could still maintain more than 50% of its maximum enzyme activity. However, when wheat straw xylan was used as a substrate, the optimal temperature was 40℃, and the enzyme activity decreased significantly when the temperature rose to the 50-70℃ range. These results indicate that the endonuclease function of RuCelI exhibits excellent high-temperature adaptability.

[0052] 2.3 pH-dependent analysis

[0053] Based on the above results, sodium carboxymethyl cellulose or wheat straw xylan were used as characteristic substrates to conduct enzyme pH-dependent analysis. The addition amount of RuCelI was set at 19.0 μg. A blank control group without RuCelI was set up to eliminate the interference of substrate degradation on the results. Different pH reaction systems were constructed using 50 mM citrate-disodium hydrogen phosphate buffer solution (pH range 3.0-8.0). When using 1% (w / v) sodium carboxymethyl cellulose as the substrate, the reaction was carried out at a constant temperature water bath of 30℃ for 2 h; when using 1% (w / v) wheat straw xylan as the substrate, the reaction was carried out at a constant temperature water bath of 40℃ for 30 min. After the reaction, the enzyme activity was characterized by measuring the content of reducing sugars released in the system. To visually demonstrate the effect of pH on enzyme activity, enzyme activity was expressed as relative activity: the activity value corresponding to the pH at which the enzyme exhibited the highest activity (i.e., the optimum pH) was set as 100%, and the enzyme activity under other pH conditions was calculated as a percentage of the activity relative to the optimum pH.

[0054] Figure 3 The results showed that the optimal pH for RuCelI was 6.0, regardless of whether sodium carboxymethyl cellulose or wheat straw xylan was used as the substrate. When the pH was below 4.0, the enzyme activity decreased significantly, but when the pH was between 5.0 and 8.0, RuCelI could maintain more than 58% of its maximum activity.

[0055] Finally, under the above optimal conditions (30℃, pH 6.0), the endonuclease activity of RuCelI was determined to be 221.5 U / mg and the xylanase activity was 140.3 U / mg.

[0056] 2.4 Resistance of RuCelI to metal ions and other additives

[0057] The total volume of the reaction system is 1 mL. During preparation, 1% (w / v) sodium carboxymethyl cellulose or 1% (w / v) wheat straw xylan is added first as a substrate, followed by the addition of 1 mM or 5 mM metal ions / chemical reagents (including Na+). + Mg2 + Cu 2+ K+ Ca2 + Ni 2+ Zn 2+ Mn 2+ The following were added: one of sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), and β-mercaptoethanol; or one of the following surfactants (Tween 20 or Triton X-100) at a concentration of 0.05% (v / v) or 0.25% (v / v); and finally, 19.0 μg of RuCelI was added. The reaction conditions were adjusted according to the substrate: when sodium carboxymethyl cellulose was used as the substrate, the reaction was carried out at pH 6.0 and 30℃ for 2 h; when wheat straw xylan was used as the substrate, the reaction was carried out at pH 6.0 and 40℃ for 30 min. At the same time, a reaction system without any of the above additives was set as a blank control and its enzyme activity was set to 100% to study the tolerance of RuCelI to the above substances.

[0058] Depend on Figure 4 and Figure 5 The results showed that different metal ions and chemical reagents had significant effects on the endonuclease activity (using sodium carboxymethyl cellulose as a substrate) and xylanase activity (using wheat straw xylan as a substrate) of RuCelI: the former activity could be affected by Na+. + K + Cu 2+ Mn 2+ and DTT activation, in which Cu 2+ The activation effect was most prominent (activity increased by 122.4% at 1 mM and still by 34.2% at 5 mM), Na + (Increased by 30.4% and 41.1% at 1 mM and 5 mM respectively), Mn 2+ (Increased by 62.6% and 50.8% at 1 mM and 5 mM, respectively) EDTA, SDS, and β-mercaptoethanol showed inhibitory effects, especially after treatment with 5 mM SDS, where only 5% of the initial activity remained; the latter's activity could be increased by Mg... 2+ Ni 2+ Ca 2+ DTT and Tween 20 activate the enzyme, and unlike its effect on endonuclease, EDTA and SDS also significantly activate the enzyme activity. 5 mM Mg 2+ Ca 2+The levels were increased by 26.8% and 28.1% respectively, while 5 mM EDTA and SDS were increased by 41.3% and 57.0% respectively. This characteristic provides an advantage for practical applications. RuCelI can be used in combination with EDTA to chelate inhibitory metal ions in feed ingredients and solve their interference with enzyme activity. It can also be used with SDS in the washing industry to help remove polysaccharide dirt.

[0059] 3. Degradation of agricultural by-products by RuCelI

[0060] In a 1 mL reaction system, 1% of agricultural by-products (wheat straw, rice straw, soybean straw, corn stalks, rapeseed straw, wheat bran, and camellia seed meal), a pH 6.0 citrate-disodium hydrogen phosphate buffer solution, and 38.0 μg of RuCelI were added respectively. The mixture was incubated at 30°C for 6 hours. No enzyme was added to the control group. After the reaction, the amount of reducing sugar released was determined using DNS.

[0061] like Figure 6 As shown, RuCelI exhibited degradation activity against a variety of tested agricultural byproducts, but the degradation efficiencies varied. Specifically, the enzyme showed the best degradation efficiency against rice straw, with a reducing sugar production of 151.0 μg / mL; the degradation effect on wheat straw was second best, with a reducing sugar production of 145.8 μg / mL; the reducing sugar concentration released after enzymatic hydrolysis of camellia seed cake was 132.7 μg / mL, which, although lower than the first two straw substrates, still maintained a relatively high degradation level; in contrast, the degradation efficiency of soybean straw was the lowest, with a reducing sugar production of only 64.2 μg / mL. The above results indicate that RuCelI has strong substrate compatibility with straw-based agricultural byproducts (especially rice straw and wheat straw) and specialty processing byproducts (camellia oil meal). This is closely related to its dual cellulase and xylanase activity. The synergistic presence of cellulose and hemicellulose in rice straw and wheat straw provides a target for the enzyme's dual catalytic function. The hemicellulose component abundant in camellia oil meal also forms a good match with the enzyme's xylanase activity.

[0062] In summary, RuCelI demonstrates clear application potential in the resource utilization of agricultural by-products, particularly in the bioconversion of bulk straws such as rice straw and wheat straw, as well as camellia oil meal, where it possesses technological advantages. Further optimization of enzymatic hydrolysis process parameters (such as substrate pretreatment methods, enzyme dosage, and reaction time) can further enhance its degradation efficiency for agricultural by-products and improve its application value in the comprehensive management of agricultural waste.

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A multifunctional fiber-degrading enzyme RuCell, characterized by, The amino acid sequence of the multifunctional cellulolytic enzyme RuCelI is shown as SEQ ID NO.

1.

2. A gene encoding a multifunctional cellulolytic enzyme, RuCell, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector comprises the gene of claim 2.

4. A recombinant bacterium, characterized in that, The recombinant bacteria comprise the recombinant vector of claim 3.

5. Use of the multifunctional cellulolytic enzyme RuCelI of claim 1 in degrading cellulose and / or xylan.

6. Use of the multifunctional cellulolytic enzyme RuCelI of claim 1 in degrading agricultural by-products, which include one or more of rice straw, corn straw, wheat straw, bean straw, rape straw, wheat bran and oil tea cake.

7. Use of the gene of claim 2, the recombinant vector of claim 3 or the recombinant bacteria of claim 4 in preparing the multifunctional cellulolytic enzyme RuCelI of claim 1.

8. A method of degrading an agricultural by-product, characterized by, comprising the step of degrading agricultural by-products by using the multifunctional cellulolytic enzyme RuCelI of claim 1; the agricultural by-products include one or more of rice straw, corn straw, wheat straw, bean straw, rape straw, wheat bran and oil tea cake.

9. The method of claim 8, wherein, the temperature for the degradation is 30-40℃, and the pH is 5.0-8.0.

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