Application of protein 0604 in degradation of solanine

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a protein 0604 capable of efficiently degrading solanine to degradation of solanine. The amino acid sequence of the protein is shown as SEQ ID NO: 1. The streptococcus lactis strain SKLAN202403XW is obtained through identification and separation in potato planting soil, biological protein is obtained from the strain, solanine can be efficiently decomposed, the content of solanine can be remarkably reduced, the toxicity of potato stems and leaves is reduced, and a foundation is provided for subsequent development and utilization.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to the application of protein 0604 in degrading solanine. Background Technology

[0002] Solanine is a toxic substance widely found in potatoes and their waste, posing a threat to food safety. Existing treatment methods are not only costly but also have negative environmental impacts.

[0003] Preliminary progress has been made in protein research on the degradation of solanine (α-solanine and α-chaconine). Studies have shown that some soil bacteria, such as... Arthrobacter genus and Glutamicibacter halophytocola S2 can hydrolyze solanine, and its degradation mechanism mainly relies on the synergistic action of three glycoside hydrolases: α-rhamnosidase (RhaA, belonging to the GH78 glycoside hydrolase family), β-glucosidase (GluA, GH3 family), and β-galactosidase (GalA, GH2 family), ultimately converting toxic solanine into non-toxic solanidine. These enzymes have been recombinantly expressed and their activity verified in microorganisms, showing good degradation efficiency. Some fungal proteins have also shown similar activities, but their specific structures and genetic information are still unclear. In addition, studies have found that insect gut bacteria also have degradation potential, providing a new direction for screening functional enzymes from biological proteins. Overall, although some key enzymes and their GH classifications and functions have been identified, their mechanisms of action, structural characteristics, and application expansion still need further exploration. Summary of the Invention

[0004] The purpose of this invention is to provide the application of protein 0604 in degrading solanine.

[0005] Another object of the present invention is to provide a method for degrading solanine.

[0006] The protein of the present invention capable of efficiently decomposing solanine has the amino acid sequence shown in SEQ ID NO:1.

[0007] SEQ ID NO:1 (gene0604) MQSRFSEKFLWGGATAANQLEGAYDRDGKGLSVADAMPGGKQRFAIIGSEEFDWTIDQEKYIYPNHRGIDHYDRFKEDLALFAEMGFKCYRFSIAWTRIFPNGDEGTPNEAGLEFYDQLI DECLKYDIEPVITISHYEMPLHLAKEYGGWKNRKLIDFYERFAQTVLERYSSKVKYWMTFNEINSAFHFPALSQGLVKSNGAGEYQNIFQAWHNQFVASSKAVKIGHELRSDIQIGCMII YATTYSIDANPVNQAATMIQNQEFNFFCTDVQVRGEYPAYTARTHKKYGVDPEKLEQTEKDFQLLKEYPVDYIGFSYYMSTAINETDPQAATSEGNLLGGVKNPFLEASEWGWQIDPEGL RIALNVLYNRYQKPLFIVENGLGAIDHMEEDGTIIDDYRIDYLRRHIEAMANAVADGVDLMGYTPWGCMDLVSASTGEMSKRYGFIYVDLDDEGNGTLNRSKKKSFDWYKEVIASNGVNL.

[0008] The nucleotide sequence of the gene encoding the protein capable of efficiently degrading solanine according to the present invention is shown in SEQ ID NO:2.

[0009] SEQ ID NO:2:

[0010] The beneficial effects of this invention are as follows: This invention identifies and isolates a strain of Streptococcus lactis from potato planting soil. Enterococcus lactis The strain SKLAN202403XW was used to obtain a biological protein, which belongs to the GH1 family. Blast analysis revealed that the most similar sequence was arylphospho-β-d-glucosidase from Bacillus, with a similarity of 60.54%. The functional characteristics of the currently discovered and reported GH2, GH3, and GH78 sequences are: removal of galactose residues (GH2); broad substrate range, capable of removing glucose residues (GH3); and removal of L-rhamnose residues (GH78). Gene0604 of the GH1 family discovered in this invention broadly includes β-glucosidase, β-xylosidase, and 6-phosphate-β-glucosidase; most are monosaccharide glycosidases, especially showing strong specificity for glucose and xylose residues; compared to GH3, GH1 enzymes have narrower substrate specificity but potentially higher catalytic efficiency.

[0011] The gene0604 of this invention can efficiently decompose solanine, significantly reducing its content and thus decreasing the toxicity of potato stems and leaves, providing a foundation for subsequent development and utilization. The gene0604 sequence of this invention possesses a highly specialized glucosidase function, showing greater potential for efficient SGA hydrolysis than GH3. The optimal pH for the gene0604 protease is between 4.5 and 6.5, exhibiting a weakly acidic environment; the optimal temperature is 40-55℃. GH1 enzyme has better temperature resistance and is suitable for industrial conditions. Attached Figure Description

[0012] Figure 1 Showing the results of colony PCR identification; Figure 2 The results of SDS-PAGE analysis of the recombinant protein are shown. Figure 3 The degradation results of α-solanine and α-carboline are shown; Figure 4 This indicates the optimal pH value for the gene0604 protein; Figure 5 This indicates the optimal temperature for gene0604 protein; Figure 6 Tests demonstrating the degradation of solanine by commercial enzymes; Figure 7 This experiment demonstrates the degradation of solanine by commercial Escherichia coli. Detailed Implementation Example 1: Cloning of the target gene

[0013] To verify the function of gene0604 in degrading solanine, it was cloned into the expression vector pUC57-Kan-tac-Clya-mFC. First, specific primers were designed with appropriate restriction enzyme sites (EcoRI and HindIII), and the gene0604 fragment, approximately 1600 bp in length, was amplified by PCR. The PCR product was detected by 1.0% agarose gel electrophoresis, purified, and ligated into the linearized pUC57-Kan-tac-Clya-mFC vector. The reaction was carried out overnight at 16°C using T4 DNA ligase. The recombinant plasmid was then transformed into [the target fragment] via heat shock. E. coli BL21(DE3) competent cells were cultured overnight on plates containing kanamycin (Kan, 50 μg / mL) resistance.

[0014] The following day, single colonies were picked from the plate for colony PCR identification, and the amplification products were detected by agarose gel electrophoresis. Figure 1 Positive clones showed a band size consistent with the target fragment (approximately 1.2 kb). For example... Figure 1 As shown, lanes 1–12 represent PCR results of different clones, with most showing clear amplification bands, indicating successful insertion. Finally, the positive clone strain corresponding to lane 1 was selected for shake-flask enrichment culture for subsequent expression and functional verification experiments. Example 2: Heterologous expression and functional verification of genes

[0015] Protein expression was induced for 16 hours at 16°C by adding 0.5 mM IPTG. The expressed recombinant protein carried an N-terminal His tag, facilitating subsequent purification. SDS-PAGE analysis showed that the recombinant protein was successfully and efficiently expressed, and its molecular weight was consistent with the predicted values: gene0604 was approximately 48 kDa. Figure 2 The protein was purified by nickel ion affinity chromatography, followed by adsorption using a magnetic rack. Gel density analysis showed that the purity of the eluted fraction exceeded 85%. The total concentration of the purified protein, determined by the BCA method, was 0.86 mg / mL for gene0604.

[0016] To assess its enzyme activity, purified gene0604 protein was incubated with 100 μg / mL α-solanine and α-carboxane in phosphate buffer at 37°C and pH 7.0 for 24 hours, respectively. The reaction products were then analyzed by high-performance liquid chromatography (HPLC). The results showed that... Figure 3 The content of glycoalkaloids in the treatment group decreased significantly, while the control group showed no significant change.

[0017] To evaluate the effectiveness of the protein in practical applications, fresh potato stems and leaves were homogenized and then incubated with purified gene0604 protein at a concentration of 0.86 mg / mL for 24 hours. The reaction products were then analyzed by high-performance liquid chromatography (HPLC). The results showed that the purified gene0604 protein significantly degraded two solanines, and its degradation effect was superior to that of wild-type fungi.

[0018] To determine the optimal activity conditions for the protein, the purified protein was compared at different temperatures and pH values, and its activity was detected by ELISA. The results showed that the optimal pH for the protein was 5.5, indicating a weakly acidic environment; and that the protein activity was higher at 42℃ than at other temperature conditions. Figure 4 , Figure 5 ). Example 3: Degradation test of solanine using existing commercial enzymes and strains.

[0019] Commercially available β-D-glucosidase (Weifang Kangdian), along with α-rhamnosidase and galactosidase (Youlian Hebang), were cultured in solanine at 37°C for 12 hours, followed by detection using a solanine ELISA. The blank control group contained only the same concentration of solanine sample without any additional enzymes; the control group contained only the culture medium. Results showed that the three enzymes had no significant effect on the degradation of solanine, and the commonly available commercially available enzymes could not effectively break down solanine. Figure 6 ).

[0020] Meanwhile, the degradation experiment of solanine in *E. coli* containing GH1 family genes was also conducted. The experimental conditions were 37℃, 180 rpm for 12 hours. The blank control group contained only the same concentration of solanine sample, and the control group contained only the culture medium. This *E. coli* contains four GH1 family gene segments: gene0877, gene1085, gene2241, and gene4469, theoretically possessing a stronger degradation ability than the *Enterococcus lactis* strain SKLAN202403XW, which only contains one gene (gene0604, GH1 family). The results showed no significant difference in solanine concentration compared to the blank control group, indicating that this *E. coli* could not effectively degrade solanine. Figure 7 ).

[0021] Translational alignment of gene 0604 using BLASTp revealed that the protein encoded by gene 0604 is similar to arylphospho-β-D-glucosidases from multiple species, with the highest similarity being 60.54%, indicating structural differences from known proteins.

[0022] Table 1. Results of BLASTp similarity analysis of gene 0604 protein gene0604 - GH1 family protein highest score Total Score Coverage E value Similarity Multiple species: Arylphospho-β-D-glucosidase [Bacillus] 581 581 100% 0.0 60.54% Glycoside hydrolase family 1 protein [Clostridium difficile] 546 546 98% 0.0 56.64% 6-Phosphoproteosidelanase [Streptococcus pneumoniae] 509 509 99% 2e-177 55.99% 6-Phosphoproteosidelanase AscB [Escherichia coli] 504 504 100% 2e-175 51.57% Multiple species: Glycoside hydrolase family 1 protein [Streptococcus] 500 500 99% 8e-174 51.88% .

[0023] Although the GH1 family could potentially encode corresponding β-D-glucosidases to degrade solanine, results show that firstly, similar commercial enzymes cannot degrade solanine; secondly, bacteria containing GH1 family genes cannot degrade solanine. Similarity analysis revealed that this may be a significant reason why the GH1 family genes cannot encode enzymes capable of degrading solanine in practice due to significant differences in enzyme structure. In contrast, protein 0604 of this application can degrade solanine, demonstrating the uniqueness of this enzyme within the GH1 family. Furthermore, current studies have only reported genes in the GH2, GH3, GH42, and GH78 families as being associated with solanine degradation; no studies have yet linked the GH1 family to solanine degradation.

[0024] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. Application of protein-degraded solanine with amino acid sequence as shown in SEQ ID NO:

1.

2. A method for degrading solanine, characterized in that, The method includes the step of acting a protein with an amino acid sequence such as SEQ ID NO:1 on solanine.

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