Multifunctional cellulolytic enzyme and application thereof in degradation of agricultural by-products

Abstract

The invention discloses a multifunctional cellulolytic enzyme and application thereof in degradation of agricultural by-products, and belongs to the technical field of biology. The multifunctional cellulolytic enzyme is named RuCelI, and the amino acid sequence of the multifunctional cellulolytic enzyme is as shown in SEQ ID NO. 1. Based on a rumen microbial metagenome resource mining technology, a novel multifunctional cellulose decomposition enzyme RuCelI is developed, and the enzyme has double activities of endo-cellulase and xylanase, can be used for degrading agricultural by-products, reducing the treatment cost and improving the degradation efficiency, and is beneficial to improving the utilization rate and the economic benefit of the agricultural by-products.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a multifunctional fiber-degrading enzyme and its application in degrading agricultural by-products. BACKGROUND

[0002] Crops play an important role in global food production, and the various by-products generated by crops, such as rice straw, corn stalks, wheat straw, soybean stalks, rapeseed stalks, and oil tea dregs, are in large quantities. These by-products are rich in lignocellulose and other components, and are potential high-quality feed sources for ruminants. However, the current rumen of ruminants has a generally insufficient fiber digestion rate for these by-products. The main obstacle is that cellulose, hemicellulose, and lignin form a dense cross-linked structure, which seriously hinders the utilization of substrates by rumen microorganisms and enzymes, and greatly limits the efficiency of fiber degradation and the improvement of animal production performance. Therefore, developing efficient degrading enzymes that adapt to the rumen environment, breaking the above cross-linked structure, and improving fiber accessibility are the key to solving the problem of efficient utilization of crop by-products.

[0003] Enzymatic hydrolysis technology is an important means to improve the degradation of crop by-products, and has the advantages of mild operation, low cost, and small pollution. The synergistic effect of endoglucanase (a core component of cellulase system) and xylanase (which can improve the accessibility of cellulose to enzymes), or enzymes with dual functions, has been proven to improve the hydrolysis efficiency. However, existing enzyme preparations often do not match the rumen environment in terms of temperature and pH adaptation range, resulting in unstable application effects and difficulty in fully playing a role.

[0004] The rumen of ruminants is a high-quality enzyme gene library. The enzymes produced by rumen microorganisms can generally adapt to the rumen environment, but due to technical limitations, most rumen bacteria cannot be cultured in vitro, resulting in a large number of potential efficient enzyme genes not being developed and utilized. The emergence of metagenomic sequencing technology provides the possibility of directly obtaining enzyme genes from rumen microbial communities, breaking through the bottleneck of traditional microbial culture. SUMMARY

[0005] The present application aims to provide a multifunctional fiber-degrading enzyme and its application in degrading agricultural by-products to solve the problems existing in the prior art. The present application develops a new multifunctional fiber-degrading enzyme RuCelI, which can be used to degrade crop by-products and improve the degradation efficiency, thereby helping to improve the utilization rate and economic benefits of crop by-products, and providing new enzyme sources and technical support for efficient resource utilization of crop by-products.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions:

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

[0008] In a second aspect, the present application also provides a gene encoding the multifunctional cellulolytic enzyme RuCelI, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0009] In a third aspect, the present application also provides a recombinant vector comprising the gene.

[0010] In a fourth aspect, the present application also provides a recombinant bacterium comprising the recombinant vector.

[0011] In a fifth aspect, the present application also provides the multifunctional cellulolytic enzyme RuCelI for use in degrading cellulose and / or xylan.

[0012] In a sixth aspect, the present application also provides the multifunctional cellulolytic enzyme RuCelI for use in degrading agricultural by-products.

[0013] Preferably, 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.

[0014] In a seventh aspect, the present application also provides the gene, the recombinant vector or the recombinant bacterium for use in preparing the multifunctional cellulolytic enzyme RuCelI.

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

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

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

[0018] The present application develops a new multifunctional cellulolytic enzyme RuCelI, which has both endo-cellulase and xylanase activities, is resistant to a wide pH range, has high temperature resistance, and has a wide range of applications. It has been verified that the enzyme has the ability to degrade crop by-products, can be used to degrade crop by-products, improve degradation efficiency, help to improve the utilization rate and economic benefits of crop by-products, and provides a new enzyme source and technical support for efficient resource utilization of crop by-products. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to illustrate the embodiments of the present application or prior art technical solutions more clearly, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0020] Figure 1 Substrate specificity of RuCell;

[0021] Figure 2 Temperature dependence of RuCell;

[0022] Figure 3 pH dependence of RuCell;

[0023] Figure 4 Effect of metal ions on RuCell activity;

[0024] Figure 5 Effect of inhibitors and detergents on RuCell activity;

[0025] Figure 6 Degradation of agricultural by-products by RuCell. DETAILED DESCRIPTION

[0026] The detailed description set forth below will illustrate the various exemplary embodiments of the present application, and is not intended to limit the present application, but to explain certain aspects, features and embodiments of the present application.

[0027] It should be understood that the terms used in the present application merely describe particular embodiments and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, and any other stated value or intermediate value within the stated range is also encompassed within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise defined, 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference in this specification is not intended as an admission that the reference is prior art, but rather that the reference is part of the state of the art.

[0029] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from this specification, which is to be construed as broadly as the permissible under the applicable law. Other embodiments will be apparent from the specification to persons of ordinary skill in the art. The specification and examples are illustrative only.

[0030] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that are intended to be synonymous, and are generally used to permit for the inclusion of additional

[0031] Example 1

[0032] 1. Production of RuCell

[0033] 1.1 Cloning of RuCell and construction of recombinant expression vector

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

[0035] Table 1 PCR amplification reaction system

[0036] Reagents Amount used (μL) 2x 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 ddH2O q.s. to 30

[0037] The PCR amplification conditions were 98°C pre-denaturation for 3 min; 98°C denaturation for 10 s, 60°C annealing for 20 s, 72°C extension for 20 s, 30 cycles; and 72°C extension for 5 min. The HieffClone ® Plus One-Step Cloning Kit (Item No.: 10911ES20; Shanghai Yingjie Biological Technology Co., Ltd.) was used to connect the PCR amplification product to the pET-28a vector double-digested by restriction enzymes BamHI and XhoI. The constructed plasmid was named pET-RuCell, and was transformed into E. coli DH5a competent cells. After overnight culture at 37°C, PCR verification was performed, the plasmid was extracted, and sequencing was performed.

[0038] The PCR amplification sequencing results showed that the amino acid sequence of RuCell was as shown in SEQ ID NO. 1, and the nucleotide sequence of the gene encoding RuCell was as shown in SEQ ID NO. 2. The 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 realize the expression of RuCelI, the recombinant plasmid pET-RuCelI was transformed into E. coli BL21 (DE3) competent cells by heat shock method, and then coated on Luria-Bertani (LB) agar medium containing kanamycin and cultured at 37°C overnight. The transformants verified positive by PCR were picked and inoculated into 2 mL LB liquid medium containing kanamycin; the overnight culture was added to 400 mL LB medium at an inoculation amount of 1%, and cultured at 37°C, 180 rpm, while the untransformed E. coli BL21 (DE3) was cultured 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 the induction was continued at 20°C, 80 rpm for 24 h. After the induction culture was completed, the culture was centrifuged (8000 rpm, 10 min); the E. coli precipitate was resuspended and lysed with phosphate buffer (PBS), and then ultrasonicated in an ice water bath for 20 min, centrifuged again (8000 rpm, 20 min), and 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 chromatography column (purchased from Bio-Rad, USA), and the RuCelI enzyme solution was obtained.

[0045] 2. Enzymatic property analysis of RuCelI

[0046] 2.1 Substrate specificity of RuCelI

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

[0048] As shown in Figure 1 , RuCelI showed activity on all four tested substrates, and the activity differed: the activity on carboxymethyl cellulose sodium was the highest, the activity on xylan was second, the activity on microcrystalline cellulose was weaker, and the activity on chitosan was the weakest. Combined with the substrate property analysis (carboxymethyl cellulose sodium is the characteristic substrate of endo-cellulase, xylan is the characteristic substrate of xylanase, and microcrystalline cellulose is the characteristic substrate of exo-cellulase), it was shown that RuCelI had significant endo-cellulase and xylanase activity, and also had relatively weak exo-cellulase activity, and was a multifunctional enzyme with fiber degradation activity.

[0049] 2.2 Temperature dependence of RuCelI

[0050] In a 1 mL reaction system, 1% of carboxymethyl cellulose sodium (CMC-Na) or wheat straw xylan (Xylan) was added, and then pH 6.0 citric acid-sodium hydrogen phosphate buffer solution and 19.0 micrograms of RuCelI were added. A blank control group without the addition of RuCelI was set up to exclude the interference of substrate self-degradation on the results. The above reaction system was incubated in a constant temperature water bath shaker at a gradient temperature of 20-90°C for 30 min (wheat straw xylan as the substrate) and 2 h (carboxymethyl cellulose sodium as the substrate). After the reaction was completed, the enzyme activity was characterized by determining the released reducing sugar content in the system. The enzyme activity was calculated as the relative activity: the activity value at the temperature corresponding to the highest activity of the enzyme (i.e., the optimum temperature) was set as 100%, and the enzyme activity at the other temperature conditions was calculated as the percentage relative to the optimum temperature activity.

[0051] Figure 2The results show that the optimum temperature of the enzyme is 30°C when CMC-Na is used as the substrate, and even if the temperature is raised to 90°C, it can still maintain more than 50% of the maximum enzyme activity; while when xylan is used as the substrate, the optimum temperature is 40°C, and when the temperature is raised to the interval of 50-70°C, the enzyme activity decreases significantly. The above shows that the endo-cellulase function of RuCelI has good high-temperature adaptability.

[0052] 2.3 pH-dependent analysis

[0053] Based on the above results, the pH-dependent analysis of the enzyme was carried out with CMC-Na or xylan as the characteristic substrate, and the amount of RuCelI added in the test was set to 19.0 μg. A blank control group without RuCelI was set to exclude the interference of substrate self-degradation on the results. A 50 mM citric acid-disodium hydrogen phosphate buffer solution (pH range 3.0-8.0) was used to construct different pH reaction systems, and when 1% (w / v) CMC-Na was used as the substrate, the reaction was carried out at 30°C constant temperature water bath for 2 h; when 1% (w / v) xylan was used as the substrate, the reaction was carried out at 40°C constant temperature water bath for 30 min; after the reaction was completed, the enzyme activity was characterized by measuring the content of reducing sugar released in the system. In order to intuitively reflect the influence of pH on enzyme activity, the enzyme activity was calculated as relative activity: the activity value corresponding to the pH at which the enzyme showed the highest activity (i.e. the optimum pH) was set to 100%, and the enzyme activity under other pH conditions was calculated as a percentage relative to the optimum pH activity.

[0054] Figure 3 The results show that whether CMC-Na or xylan is used as the substrate, the optimum pH of RuCelI is 6.0; when the pH is lower than 4.0, the enzyme activity decreases significantly, but when the pH is 5.0-8.0, RuCelI can maintain more than 58% of the maximum activity.

[0055] Finally, under the above optimum conditions (30°C, pH 6.0), the endo-cellulase activity of RuCelI is 221.5 U / mg, and the xylanase activity is 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, and when configuring, 1% (w / v) CMC-Na or 1% (w / v) xylan is first added as the substrate, and then 1 mM or 5 mM metal ions / chemical reagents (including Na + , Mg2 + , Cu 2+ , K+ , Ca2 + , Ni 2+ , Zn 2+ , Mn 2+ , sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), β-mercaptoethanol), or one of the 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; the reaction conditions were adjusted according to the substrate, and when sodium carboxymethylcellulose was used as the substrate, the reaction was carried out at pH 6.0 and 30°C for 2 h, and when wheat straw xylan was used as the substrate, the reaction was carried out at pH 6.0 and 40°C for 30 min; a reaction system without any of the above additives was set up as a blank control, and the enzyme activity was set to 100%, in order to study the tolerance of RuCelI to the above substances.

[0058] By Figure 4 and Figure 5 It can be seen that the effects of different metal ions and chemical reagents on the endo-cellulase activity (using sodium carboxymethylcellulose as the substrate) and xylanase activity (using wheat straw xylan as the substrate) of RuCelI are significantly different: the former activity can be activated by Na + , K + , Cu 2+ , Mn 2+ , and DTT, among which Cu 2+ has the most prominent activation effect (the activity is increased by 122.4% at 1 mM, and still increased by 34.2% at 5 mM), followed by Na + (30.4% and 41.1% at 1 mM and 5 mM, respectively), Mn 2+ (62.6% and 50.8% at 1 mM and 5 mM, respectively), and EDTA, SDS, and β-mercaptoethanol show inhibitory effects, and the activity is only 5% of the initial activity after treatment with 5 mM SDS; the latter activity can be activated by Mg 2+ , Ni 2+ , Ca 2+ , DTT, and Tween 20, and unlike the effect on endo-cellulase, EDTA and SDS can also significantly activate the enzyme activity, and the activity is increased by 1.6-fold at 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 above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A multifunctional cellulase RuCelI, characterized in that, The amino acid sequence of the multifunctional cellulase RuCelI is shown in SEQ ID NO.

1.

2. A gene encoding a multifunctional cell-degrading enzyme RuCelI, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 2.

4. A recombinant bacterium, characterized in that, The recombinant bacteria contain the recombinant vector as described in claim 3.

5. The use of the multifunctional cellolytic enzyme RuCelI according to claim 1 in the degradation of cellulose and / or xylan.

6. The application of the multifunctional cellolytic enzyme RuCelI according to claim 1 in the degradation of agricultural by-products.

7. The application according to claim 6, characterized in that, The agricultural by-products include one or more of the following: rice straw, corn straw, wheat straw, bean straw, rapeseed straw, wheat bran, and camellia oil meal.

8. The use of the gene of claim 2, the recombinant vector of claim 3, or the recombinant bacteria of claim 4 in the preparation of the multifunctional cellulase RuCelI of claim 1.

9. A method for degrading agricultural by-products, characterized in that, The method includes the step of degrading agricultural byproducts using the multifunctional cellolytic enzyme RuCelI as described in claim 1.

10. The method according to claim 9, characterized in that, The degradation temperature is 30-40℃, and the pH is 5.0-8.0.

Images