Cellulose excision enzyme gene

Abstract

The invention provides a cellulose excision enzyme gene, and belongs to the technical field of biological genetic engineering. The cellulose excision enzyme gene provided by the invention at least contains a DNA fragment of one of the following nucleotide sequences; 1) a nucleotide sequence as shown in SEQ ID NO.1 in a sequence table; and 2) a nucleotide sequence which at least has more than 90% of homology with the nucleotide sequence as shown in SEQ ID NO.1 and is used for coding proteins with the same biological functions. Cellulose excision enzyme coded by the cellulose excision enzyme gene is high in activity.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a cellulase gene. Background Technology

[0002] Cellulose is a linear polysaccharide composed of thousands of D-glucose units linked by β-1,4-glycosidic bonds, forming unbranched cellulose chains. Lignocellulosic biomass is primarily composed of three components: cellulose (40–60%), hemicellulose (20–40%), and lignin (10–20%). Cellulose degradation is crucial for the global carbon cycle, representing a major carbon flow from the carbon fixation pool to the atmosphere. Cellulases have become key biocatalysts in various industrial applications, first used in the feed industry in the early 1980s. Cellulases are complex enzyme systems that hydrolyze the β-1,4-glucosidic bonds of cellulose, converting it into cellobiose and glucose. Cellulases are not single enzymes but complexes of multiple enzymes, including endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21), and are mainly produced by bacteria and fungi.

[0003] Cellulases are classified into various GH families, such as GH1, GH3, GH5, GH6, GH8, GH9, GH12, GH45, GH48, GH51, GH61, and GH74. Cellulose glycosidic bonds are randomly hydrolyzed by endoglucanases to produce oligosaccharides. These oligosaccharides are then cleaved at both the reducing and non-reducing ends by exoglucanases to produce cellobiose, which is further hydrolyzed by β-glucosidases to release sugar molecules such as glucose. The bioconversion of cellulose is influenced by the microcrystalline structure of cellulose-rich materials. Although numerous traditional physical and chemical methods have been used to pretreat these materials with some success, secondary pollution has also occurred, negatively impacting their utilization. Therefore, microbial enzymes are a good alternative for improving the availability of cellulose bioresources. Cellulases are mainly obtained from microbial sources, and significant differences in enzyme activity and stability exist among cellulases from different microbial sources. Many bacteria, protozoa, fungi, animals, and plants can produce cellulases. With increasing demand, the need to explore different ecological habitats to find new microbial isolates has become crucial.

[0004] Various microorganisms can produce cellulases, including fungi, bacteria, and actinomycetes. Fungi are the preferred source and major contributor of cellulases in industrial applications, accounting for 80% of cellulose decomposition in nature. Fungi have an advantage over other microorganisms because they can secrete large amounts of biodegrading enzymes when growing on inexpensive substrates. Fungal cellulases are produced in the form of secretory proteins, facilitating enzyme isolation and extraction. In contrast, bacterial cellulases have low yields, low activity, and a limited enzyme system (mainly Cx enzymes), and cannot be secreted extracellularly. Furthermore, the need for cell disruption in practical applications increases the cost of enzyme use. Currently, the industrial cultivation / growth of cellulases is generally challenging. Therefore, the search for new microorganisms to degrade cellulose and the isolation of cellulases with higher specific activity remain a hot research topic for the future. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a cellulase gene, wherein the cellulase encoded by the cellulase gene of the present invention has high activity.

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

[0007] This invention provides a cellulase gene, wherein the gene contains at least one of the following nucleotide sequences in a DNA fragment;

[0008] 1) The nucleotide sequence of SEQ ID NO.1 in the sequence listing;

[0009] 2) A nucleotide sequence that has at least 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes a protein with the same biological function.

[0010] The present invention also provides a cellulase exonuclease, which is a cellulase exonuclease encoded by the above-mentioned cellulase exonuclease gene.

[0011] The present invention also provides a cellulase-producing Bjerkandera adusta, which is classified as Bjerkandera adusta, with accession number CGMCC NO.40380, accession date of January 6, 2023, and deposited at the China General Microbiological Culture Collection Center; the cellulase includes the cellulase described above.

[0012] Preferably, the activity of the cellulase is not less than 89.06 IU / mL.

[0013] This invention also provides the application of the above-mentioned cellulase in the preparation of cellulose-degrading products.

[0014] This invention also provides the application of *Bacillus citrinum* producing cellulase in the preparation of cellulose-degrading products.

[0015] The present invention utilizes a cellulose exonuclease encoded by a cellulose exonuclease gene, which has an activity of 98.60±9.54 IU / mL. Attached Figure Description

[0016] Figure 1 Images of the strains screened in this invention;

[0017] Figure 2 This diagram shows the proportion of carbohydrate-active enzymes in the *Cymbidium* strain that produces cellulase exonuclease according to the present invention.

[0018] Figure 3 The sequence of the cellulase gene obtained from transcriptome sequencing;

[0019] Figure 4 Homology of the cellulose exonuclease gene sequence of this invention in the NCBI database. Detailed Implementation

[0020] This invention provides a cellulase gene, wherein the gene contains at least one of the following nucleotide sequences in a DNA fragment;

[0021] 1) The nucleotide sequence of SEQ ID NO.1 in the sequence listing;

[0022] 2) A nucleotide sequence that has at least 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes a protein with the same biological function.

[0023]

[0024] This invention provides a cellulase exonuclease, which is a cellulase exonuclease encoded by the above-mentioned cellulase exonuclease gene.

[0025] This invention provides a cellulase-producing Bjerkandera adusta, which is classified as Bjerkandera adusta, with accession number CGMCC NO.40380, accession date of January 6, 2023, and deposited at the China General Microbiological Culture Collection Center; the cellulase includes the cellulase described above.

[0026] In this invention, the activity of the cellulase is preferably not less than 89.06 IU / mL.

[0027] This invention also provides the application of the above-mentioned cellulase in the preparation of cellulose-degrading products.

[0028] This invention also provides the application of *Bacillus citrinum* producing cellulase in the preparation of cellulose-degrading products.

[0029] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] Cellulase-producing strains were initially screened from the soil. The colony diameter was determined using the streak plate method, and the results are shown in Table 1. Among the 10 strains, the ratio of the clear zone to the colony diameter, from largest to smallest, was: F71 > L51 > L62 = G72 > F73 > L74 > L73 > L71 > L61 > G73. The top three strains with the largest clear zone diameters were then used for subsequent enzyme activity assays.

[0032] Table 1. Ratio of transparent zone to colony diameter for different strains

[0033]

[0034] The cellulase activities of three strains (F71, L51, and L62) were determined, and the results are shown in Table 2. Measured by carboxymethyl cellulase (CMC) activity, strain F71 exhibited the highest activity at 214.26 ± 2.37 IU / mL, followed by strain L51 (208.09 ± 9.79 IU / mL), while strain L62 showed relatively lower activity at 196.51 ± 3.84 IU / mL. Regarding filter paper enzyme (FPA) activity, strain F71 showed the best performance, reaching 40.50 ± 1.05 IU / mL, significantly higher than strains L51 (38.16 ± 0.98 IU / mL) and L62 (36.65 ± 0.62 IU / mL). The results of exonuclease-β-1,4-glucanase (CBH) activity assays showed that the enzyme activity of strain F71 was 98.60±9.54 IU / mL, significantly higher than that of strains L51 (61.48±2.95 IU / mL) and L62 (67.23±3.29 IU / mL). There were certain differences in the activities of different cellulase components among different strains, with strain F71 showing a relative advantage in all three enzyme activity indicators. These results indicate that F71 had the highest cellulase activity, and F71 was subsequently selected for cellulase gene screening.

[0035] Table 2. Cellulase activity determination of each strain

[0036]

[0037] The selected F71 strain was subjected to genomic DNA extraction using the Ezup column-based fungal genomic DNA extraction kit. After successful DNA extraction, the DNA was identified and its purity was determined by 1% agarose gel electrophoresis and stored at -20℃ for later use. PCR amplification was performed using the universal primers for fungal identification, ITS1 / ITS4 (ITS1(5′TCCGTAGGTGAACCTGCGG3′), ITS4(5′TCCTCCGCTTATTGATA TGC 3′). The PCR products were sent to Sangon Biotech Co., Ltd. for sequencing. The sequenced sequences were entered into NCBI, and BLAST alignment was used for sequence homology analysis to molecularly identify the fungal species. The strain was identified as a novel strain within the fungal species.

[0038] The strain was sent to Megi and Novogene for whole genome sequencing (Megi) and transcriptome sequencing (Beijing Novogene Technology Co., Ltd.) to perform gene function analysis and identify its β-1,4-glucanase gene, cellulase gene, and cellulase gene.

[0039] The strain attributes obtained from whole-genome sequencing are shown in Table 3.

[0040] Table 3. Classification of bacterial strains by attributes

[0041]

[0042]

[0043] See the corresponding filtered images. Figure 1 Carbohydrate enzyme analysis in whole-genome sequencing showed ( Figure 2 This strain contains 39.02% glycoside hydrolases, 28.18% coenzymes, 14.09% carbohydrate lipases, 4.34% carbohydrate-binding modules, and 1.63% polysaccharide lyases. This indicates that this strain has good carbohydrate degradation activity.

[0044] RNA was extracted from strain F71, and cDNA was obtained by reverse transcription. Cellulase genes were screened based on the whole genome and transcriptome, and then... Figure 3 Primers were designed using Primer5 to obtain the gene sequence, and Phusion was used. TM High-fidelity DNA polymerase was used to run the amplified PCR product on a nucleic acid gel. If the product band was single, the PCR product was purified. The purified product was ligated into the pBM23 vector, transformed, plated, streaked, cultured, and plasmid extracted. Plasmid identification PCR was performed, followed by sequencing. After sequencing, the sequence was compared with the target gene, and the overlap rate was 100%, indicating successful sequencing. The CDS of the cellulase exonuclease gene sequence was obtained. After homology comparison with the NCBI gene bank, the highest homology between this gene and similar genes in the gene bank was found to be 79.42%. This confirmed it as a new gene sequence. Homology is shown in [see attached image]. Figure 4 .

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cellulase gene, characterized in that, The gene contains at least one of the following DNA segments with nucleotide sequences; 1) The nucleotide sequence of SEQ ID NO.1 in the sequence listing; 2) A nucleotide sequence that has at least 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and encodes a protein with the same biological function.

2. A cellulase exonuclease, characterized in that, Cellulose exonuclease encoded by the cellulose exonuclease gene as described in claim 1.

3. A type of *Cinnamomum casuarinae* producing cellulase, characterized in that, The *B. jerkandera adusta* is classified and named, with accession number CGMCC NO.40380, accession date January 6, 2023, and deposited at the China General Microbiological Culture Collection Center; the cellulase includes the cellulase described in claim 2.

4. The *Cinnamomum casuarinae* strain producing cellulase according to claim 3, characterized in that, The activity of the cellulase is not less than 89.06 IU / mL.

5. The application of the cellulase described in claim 2 in the preparation of cellulose-degrading products.

6. The application of the cellulase-producing *Cymbidium* strain according to claim 3 in the preparation of cellulose-degrading products.

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