Multifunctional fiber-degrading enzyme and use thereof

Abstract

The application discloses a multifunctional fiber-degrading enzyme and application thereof, and belongs to the technical field of biology. The multifunctional fiber-degrading enzyme is named RuCelF, and the amino acid sequence is shown as SEQ ID NO. 1. Based on rumen microbial metagenomic resource mining technology, a new multifunctional fiber-degrading enzyme RuCelF is developed. The enzyme has double activities of cellulase and xylanase, can be used for degrading agricultural waste, reduces treatment cost, improves degradation efficiency, helps to improve the utilization rate and economic benefits of agricultural waste, and provides a new enzyme source and technical support for efficient resource utilization of agricultural waste.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a multifunctional cell-degrading enzyme and its applications. Background Technology

[0002] Agricultural waste is rich in polysaccharides such as cellulose and hemicellulose. If it can be efficiently degraded, it can be converted into high-value-added products such as biofuel, feed, and organic fertilizer, which have both economic and environmental value.

[0003] However, the degradation of these agricultural wastes currently faces a major challenge: low degradation efficiency. Existing technologies using single cellulases or xylanases can only target specific polysaccharide components. Crop straw, camellia seed cake, and tea seed cake have complex structures, typically containing cellulose, hemicellulose, and lignin. Distillers' grains, due to variations in raw materials and brewing processes, exhibit significant fluctuations in polysaccharide composition and content, which are intertwined with residual starch and protein. Single enzyme preparations are insufficient for efficient degradation; multiple enzymes are required for synergistic action. This not only increases degradation costs but also presents challenges such as difficulty in optimizing enzyme ratios and incompatible reaction conditions.

[0004] To address these issues, researchers in the field have begun focusing on the discovery and application of "multifunctional enzymes." Multifunctional enzymes (such as enzymes possessing both cellulase and xylanase activities) can degrade multiple polysaccharide components through a single enzyme molecule. This not only simplifies enzyme system composition and reduces application costs but also reduces spatial competition between different enzyme molecules, thereby improving degradation efficiency. Currently, the sources of multifunctional enzymes are mainly concentrated in soil microorganisms and saprophytic fungi. However, multifunctional enzymes from these sources generally suffer from narrow substrate specificity (e.g., they can only degrade cellulose from specific sources, showing poor degradation effects on complex polysaccharides in camellia seed cake and tea seed cake, as well as polysaccharides coexisting with starch and protein in distiller's grains), and difficulty adapting to the special environment of residual alcohol and organic acids that may be present in distiller's grains, making it difficult to meet practical application needs.

[0005] The rumen, as the core digestive organ of ruminants, is home to a large community of microorganisms (such as bacteria, archaea, and fungi) that primarily utilize cellulose and hemicellulose as carbon sources. Through long-term evolution, these microorganisms have developed highly efficient polysaccharide-degrading enzyme systems, including many multifunctional enzymes with broad-spectrum degradation activity. Compared to enzymes derived from soil microorganisms and saprophytic fungi, enzymes derived from rumen microorganisms have advantages such as strong substrate adaptability (able to adapt to the complex polysaccharide mixture within the rumen and function normally in environments containing proteins, organic acids, etc., exhibiting higher compatibility with the compositional environment of distillers' grains) and high catalytic efficiency (able to rapidly degrade cellulose and hemicellulose under mild conditions). These advantages make them an ideal resource for developing highly efficient multifunctional enzymes, especially in the degradation of complex agricultural wastes like distillers' grains, where they possess unique potential advantages. However, current research on multifunctional enzymes derived from rumen microorganisms is still significantly insufficient: existing studies mostly focus on the identification of single enzyme activities, with little exploration of multifunctional enzymes that simultaneously possess cellulase and xylanase activities, and there is a lack of systematic investigation into their degradation effects on crop straw, camellia seed cake, tea seed cake, and especially on special agricultural wastes such as distiller's grains, and a lack of verification of their degradation performance on complex component systems of distiller's grains. Summary of the Invention

[0006] The purpose of this invention is to provide a multifunctional cell-degrading enzyme and its applications to address the problems existing in the prior art. This invention develops a novel multifunctional cell-degrading enzyme, RuCelF, which has the ability to degrade agricultural waste. It can be used to degrade agricultural waste, improve degradation efficiency, and contribute to increasing the utilization rate and economic benefits of agricultural waste.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] In a first aspect, the present invention provides a multifunctional fiber-degrading enzyme RuCelF, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Secondly, the present invention also provides a gene encoding a multifunctional fiber-degrading enzyme RuCelF, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] Thirdly, the present invention also provides a recombinant vector containing the aforementioned gene.

[0011] Fourthly, the present invention also provides a recombinant bacterium containing the recombinant vector.

[0012] Fifthly, the present invention also provides the application of the aforementioned multifunctional cell-degrading enzyme RuCelF in the degradation of cellulose and / or xylan.

[0013] In a sixth aspect, the present invention also provides the application of the aforementioned multifunctional fiber-degrading enzyme RuCelF in the hydrolysis of agricultural waste.

[0014] Preferably, the agricultural waste includes one or more of the following: rice straw, corn stalks, wheat straw, soybean straw, rapeseed straw, wheat bran, distiller's grains, camellia seed meal, and / or tea seed meal.

[0015] In a seventh aspect, the present invention also provides the use of the said gene, the said recombinant vector, or the said recombinant bacteria in the preparation of the multifunctional cell-degrading enzyme RuCelF.

[0016] In an eighth aspect, the present invention also provides a method for hydrolyzing agricultural waste, comprising the step of hydrolyzing agricultural waste using the aforementioned multifunctional cell-degrading enzyme RuCelF.

[0017] Preferably, the hydrolysis temperature is 30-50℃ and the pH is 4.0-8.0.

[0018] The present invention discloses the following technical effects:

[0019] This invention develops a novel multifunctional cellulase, RuCelF, which possesses dual activities as both a cellulase and a xylanase. It exhibits broad pH tolerance, high temperature tolerance, and wide applicability. Verification has shown that this enzyme has the ability to degrade agricultural waste, improving degradation efficiency and contributing to increased utilization and economic benefits of agricultural waste. Attached Figure Description

[0020] 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.

[0021] Figure 1 The results of nucleic acid gel electrophoresis analysis of PCR products; where lane 1 is the RuCelF gene and M is the marker.

[0022] Figure 2 Double digestion verification of plasmid pET-RuCelF; lanes 1, 3, and 5 are double-digested pET-RuCelF plasmids, lanes 2, 4, and 6 are constructed pET-RuCelF plasmids, and M is the marker.

[0023] Figure 3 This is a graph showing the results of substrate-specific detection;

[0024] Figure 4This is a graph showing the pH-dependent detection results;

[0025] Figure 5 The results of the temperature dependence test are shown in the figure.

[0026] Figure 6 The graph shows the results of measuring the concentration of reducing sugars released when RuCelF hydrolyzes various agricultural wastes. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Example 1

[0033] 1. Production of RuCelF

[0034] 1.1 Cloning and Expression of RuCelF

[0035] (1) Using rumen microbial DNA (NCBI ID: PRJNA806344) as a template, the RuCelF gene was amplified by PCR using upstream primer F: 5'-CTTTAAGAAGGAGATATACGGATCCATGGAACTGGTACGACAAGCAC-3' (SEQ ID NO.3) and downstream primer R: 5'-AGTGGTGGTGGTGGTGGTGCTCGAGCCACGCGCTCTTCCCGTCCGC-3' (SEQ ID NO.4) to obtain the PCR product; the PCR amplification system is shown in Table 1 below.

[0036] Table 1 PCR amplification reaction system

[0037] 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

[0038] The PCR amplification conditions were: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 10 s, for 30 cycles; and 72℃ extension for 5 min.

[0039] (2) After the amplification reaction, the obtained PCR products were analyzed by nucleic acid gel electrophoresis to detect the size of the target band. The results are shown in the figure. Figure 1 The theoretical molecular weight of the RuCelF gene is 1142 bp (including homologous arms). Figure 1 It can be seen that the RuCelF gene has been successfully amplified, and the result is consistent with the theoretical molecular weight. The target fragment was recovered. The recovered target fragment was homologously recombinated using the HieffClone® Plus Multi One Step Cloning Kit (Yeasen, Shanghai) and inserted into the pET-28a expression vector to obtain the recombinant plasmid pET-RuCelF.

[0040] (3) The pET-RuCelF plasmid was extracted, verified by double digestion with BamHI and XhoI, and then amplified and sequenced by PCR. The results were obtained by... Figure 2 It can be seen that the double enzyme digestion results are consistent with the theoretical size. The 5000+ band is the linearized vector pET28a(+), and the other band is consistent with the size of the RuCelF gene, indicating that the expression plasmid pET-RuCelF was successfully constructed.

[0041] PCR amplification and sequencing results showed that the final amino acid sequence of RuCelF is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding RuCelF is shown in SEQ ID NO.2. Details are as follows:

[0042] SEQ ID NO.1:

[0043] MELVRQAPWIRGRNYMPASCANRVDQWQELGSAERIGEVERELALAEEIGFNAMRIIVEVQGFGVWLADHDGFMARFERYLTILDRHGMRACIVLGNDCMRPKEFWTMPTPGPQKFDWGYHGGRKMSQHGSFPDAIGYSPVDDPELAKPFYAMCEELMTKYRTDQRILMWNIWNEPGNSNRGDLTIPQMKKLFELAWRIDPEQPLAADLWDTYGEKTDGSAKAEHRRRVEALAGELSDVISYHSYGDYETQVKTIAFLKKRYRRPLFNTEWLHRIQRNTVETAYPLYYLEKVGCFCWGFVAGKYQTYEPWEGLWKLDEQGKADDLDFTRWQHDLIRPTMRPYDPKELRLIKRFTKLADGKSAW;

[0044] SEQ ID NO.2:

[0045]

[0046] 1.2 Preparation of RuCelF enzyme solution

[0047] (1) The pET-RuCelF plasmid was introduced into Escherichia coli BL21(DE3) by heat shock, and transformants were screened overnight at 37°C on LB agar plates containing kanamycin. Positive colonies were identified by PCR and specific primers. Positive colonies were picked into LB medium and then placed in a constant temperature shaking incubator for overnight culture. The bacterial culture was then aspirated and preserved.

[0048] (2) Inoculate 100 μL of bacterial culture into 10 mL of LB medium containing kanamycin and incubate overnight at 37°C and 120 rpm for recovery. After recovery, transfer the bacterial culture to 1 L of medium and incubate at 37°C and 120 rpm. When OD 600 When the expression level reached 0.6-0.8, 400 μL of 1M IPTG (final concentration 0.4 mM) was added, and the cells were incubated at 20℃ and 80 rpm for 20 h to induce expression. After incubation, the cells were collected by centrifugation at 4℃ and 8000 rpm for 5 minutes, resuspended in 0.01 M PBS, sonicated in ice water for 20 minutes, and then centrifuged at 4℃ and 8000 rpm for 20 minutes. The supernatant was used as the crude enzyme solution. The crude enzyme solution was purified using a Nuvia IMAC nickel affinity chromatography column (BioRad, USA) and a low-pressure protein purification system (LP; Bio-Rad, USA) to obtain RuCelF enzyme solution.

[0049] 2. Enzymatic Characterization of RuCelF

[0050] 2.1 Substrate specificity of RuCelF

[0051] To investigate the substrate specificity of the enzyme, sodium carboxymethyl cellulose, microcrystalline cellulose, wheat straw xylan, and chitosan were selected as substrates. The experimental system consisted of 1 mL of substrate at a concentration of 1% (m / v). The experimental group received 7.6 μg of RuCelF, while the control group received no enzyme. Incubation was performed at pH 6.0 and 40℃. Xylan was incubated for 30 minutes, while the other substrates were incubated for 2 hours. Enzyme activity was measured using the DNS method, and curves were plotted using glucose and xylose as standards. The reducing sugar content was calculated by reading the absorbance at 540 nm using a microplate reader. 1 U of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of xylose or glucose per minute.

[0052] like Figure 3As shown, RuCelF 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 RuCelF possesses significant endocellulase and xylanase activities, while also exhibiting relatively weak exocellulase activity, making it a multifunctional cell-degrading enzyme.

[0053] 2.2 pH-dependent analysis

[0054] Based on the results of the substrate activity screening experiment, sodium carboxymethyl cellulose (NCC) with the highest RuCelF activity was selected as the characteristic substrate for pH-dependent enzyme analysis. The amount of RuCelF added was set at 7.6 μg. A blank control group without RuCelF 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), with 1% (w / v) NCC as the substrate, and the reaction was carried out at a constant temperature of 40℃ for 2 h. After the reaction, the enzyme activity was characterized by measuring the amount of reducing sugar released in the system. To visually represent the effect of pH on enzyme activity, enzyme activity was expressed as relative activity: the activity value at the pH corresponding to the highest enzyme 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 at the optimum pH.

[0055] The results are as follows Figure 4 As shown, the optimal reaction pH for RuCelF is 5.0. Within the pH range of 4.0-8.0, the enzyme activity retains more than 50% of its activity at the optimal pH (pH 5.0), and no significant drop in activity is observed within this range. This result confirms that RuCelF possesses a broad pH adaptation spectrum, providing experimental support for its application in the degradation of substrates with different pH environments, such as straw and distiller's grains.

[0056] 2.3 Temperature Dependence Analysis

[0057] In a 1 mL reaction system, 1% sodium carboxymethyl cellulose, followed by a pH 5.0 citrate-disodium hydrogen phosphate buffer solution and 7.6 μg RuCelF were added. A blank control group without RuCelF 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 30-70℃ for 2 h. 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.

[0058] The results are as follows Figure 5 The results show that the optimal reaction temperature of RuCelF is 40℃ (the relative activity at 50℃ is 100%). In the range of 30-50℃, it can maintain more than 90% of the maximum activity. However, when the temperature exceeds 60℃, its activity drops sharply to less than 20%, and it is almost completely lost.

[0059] Finally, under the above optimal conditions (pH 5.0, 40℃), the endonuclease activity of RuCelF was determined to be 43.3 U / mg and the xylanase activity was 36.5 U / mg.

[0060] 3. RuCelF's degradation of various agricultural wastes

[0061] In a 1 mL reaction system, 1% of agricultural waste (wheat straw, rice straw, soybean straw, corn stalks, rapeseed straw, wheat bran, distiller's grains, camellia seed meal, and tea seed meal), a pH 5.0 citrate-disodium hydrogen phosphate buffer solution, and 10.0 μg of RuCelF were added respectively. The mixture was incubated at 40°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.

[0062] Depend on Figure 6 It was found that RuCelF exhibited significant degradation capabilities for various agricultural wastes, with particularly outstanding effects on distillers' grains, camellia seed meal, and tea seed meal. Specifically, under the same reaction conditions, after 6 hours of treatment, RuCelF released 912.0 μg / mL, 745.0 μg / mL, and 466.2 μg / mL of reducing sugars from distillers' grains, camellia seed meal, and tea seed meal, respectively, far exceeding the sugar production yields from other tested agricultural wastes. This result indicates that RuCelF not only possesses dual cellulase-xylanase activity and a broad pH spectrum, but also can efficiently adapt to complex agricultural waste substrates such as distillers' grains, camellia seed meal, and tea seed meal, providing crucial experimental evidence for its industrial application in the resource utilization of these agricultural wastes (such as conversion into bio-feed or fermentation substrates).

[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 RuCelF, characterized by, The amino acid sequence of the multifunctional fiber-degrading enzyme RuCelF is shown as SEQ ID NO.

1.

2. A gene encoding a multifunctional cellulolytic enzyme RuCelF, c h a r a c t e r i z e d 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 fiber-degrading enzyme RuCelF of claim 1 in degrading cellulose and / or xylan.

6. Use of the multifunctional fiber-degrading enzyme RuCelF of claim 1 in hydrolyzing agricultural waste.

7. Use according to claim 6, characterized in that, The agricultural waste comprises one or more of rice straw, corn straw, wheat straw, bean straw, rape straw, wheat bran, vinasse, oil tea cake and / or tea leaf cake.

8. Use of the gene of claim 2, the recombinant vector of claim 3 or the recombinant bacteria of claim 4 in preparing the multifunctional fiber-degrading enzyme RuCelF of claim 1.

9. A method of hydrolyzing agricultural waste, characterized by, The method comprises the step of hydrolyzing agricultural waste by using the multifunctional fiber-degrading enzyme RuCelF of claim 1.

10. The method of claim 9, wherein, The temperature of the hydrolysis is 30-50℃, and the pH is 4.0-8.0.

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