Application of chitosanase mutant BAC / MH and its prepared chitosan oligosaccharide in abalone preservation

Abstract

This invention relates to a chitosanase mutant BAC / MH and the application of its prepared chitosan oligosaccharide in abalone preservation, belonging to the field of genetic engineering. The amino acid sequence of the chitosanase mutant BAC / MH is shown in SEQ ID NO:1. Compared with the wild-type chitosanase Csn-BAC, cysteine ​​at position 77 is mutated to methionine and threonine at position 236 is mutated to histidine. The chitosanase mutant BAC / MH of this invention can catalyze the production of chitosan oligosaccharide from chitosan, with a catalytic activity 6.13 times that of the wild-type chitosanase Csn-BAC. Furthermore, the chitosan oligosaccharide can improve the quality of abalone during cold storage preservation.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to the application of a chitosanase mutant BAC / MH and the chitosan oligosaccharide prepared therefrom in the preservation of abalone. Background Technology

[0002] Chitosan oligosaccharide (COS), as a degradation product of chitosan, has shown significant application value in food preservation due to its unique bioactivity and physicochemical properties. Compared with physical cleavage and chemical degradation methods, enzymatic hydrolysis for preparing COS has become the main technical route for COS preparation due to its advantages such as mild reaction conditions, high product specificity, high uniformity, and good environmental compatibility. Among them, chitosanase (EC 3.2.1.132), as a biocatalyst for specifically hydrolyzing the β-1,4-glycosidic bonds of chitosan, is widely found in bacteria, fungi, and plants. However, the low catalytic efficiency of chitosanases reported so far has further limited the progress of industrial application. Currently, the rational design of enzyme molecules through protein engineering technology has become an effective strategy to improve catalytic performance.

[0003] Abalone, a high-value marine shellfish, is widely favored by consumers for its rich nutrition and delicious taste. In 2024, my country's abalone farming output reached 245,000 tons. Fresh abalone is one of the main distribution and sales methods. However, abalone is prone to quality deterioration during refrigeration and preservation, mainly manifested in microbial proliferation leading to spoilage, lipid oxidation producing unpleasant flavors, and severe juice loss resulting in decreased water-holding capacity. Therefore, developing chitosan oligosaccharide natural preservatives is of great significance for improving the product quality of abalone during preservation. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a chitosanase mutant BAC / MH and the application of the prepared chitosan oligosaccharide in the preservation of abalone. The present invention improves the catalytic activity of chitosanase by performing site-directed mutagenesis, which is used for the efficient preparation of chitosan oligosaccharide, which can improve the preservation quality of fresh abalone.

[0005] This invention is achieved through the following technical solution: A chitosanase mutant BAC / MH, the amino acid sequence of which is shown in SEQ ID NO:1, involves site-directed mutations at amino acids 77 and 236 compared to wild-type chitosanase Csn-BAC (GenBank ID: ASB61783.1).

[0006] Furthermore, the wild-type chitosanase is derived from Bacillus sp. MD-5.

[0007] The present invention also provides a gene encoding the chitosanase mutant BAC / MH, the nucleotide sequence of which is shown in SEQ ID No:2.

[0008] The present invention also provides a recombinant plasmid containing the above-mentioned gene, wherein the vector of the recombinant plasmid is pET-28a.

[0009] The present invention also provides genetically engineered bacteria containing the above-mentioned recombinant plasmid.

[0010] Furthermore, the genetically engineered strain provided by the present invention is obtained by transforming the recombinant plasmid into Escherichia coli BL21(DE3).

[0011] The present invention also provides the application of the above-mentioned chitosanase mutant BAC / MH or the genetically engineered bacteria containing the above-mentioned chitosan to produce chitosan oligosaccharides, wherein the degree of polymerization of the chitosan oligosaccharides is 2 to 3, and the chitosan oligosaccharides can improve the preservation quality of abalone.

[0012] The present invention also provides an application of the prepared chitosan oligosaccharide in improving the preservation quality of abalone.

[0013] Furthermore, the application method involves soaking the pretreated fresh abalone in a solution containing the chitosan oligosaccharide for at least 30 minutes, then aseptically vacuum packaging it and storing it at a preservation temperature.

[0014] Furthermore, the concentration of the chitosan oligosaccharide solution is above 0.25 g / 100 mL, and the preservation temperature is 4℃.

[0015] The beneficial effects of this invention compared to the prior art are as follows: This invention addresses the problem of low catalytic activity in existing chitosanases. Based on chitosanase Csn-BAC, it employs multiple sequence alignment to select several potential amino acid sites that might affect its catalytic activity. For the first time, a double-mutant BAC / MH was obtained by mutating cysteine ​​(C) at position 77 and threonine (T) at position 236 to methionine (M) and histidine (H), respectively. The resulting mutant exhibits significantly increased enzyme activity, 6.13 times that of the wild type. Chitosan oligosaccharides prepared using the above chitosanase mutant can improve the quality of abalone during cold storage preservation. Attached Figure Description

[0016] Figure 1 Graph showing the product analysis of chitosan oligosaccharide preparation by chitosanase BAC / MH degradation of chitosan; Figure 2 Graph showing the changes in total bacterial count in abalone during storage at different concentrations of chitosan oligosaccharide treatment; Figure 3 Graph showing the change of TVB-N in abalone during storage with different concentrations of chitosan oligosaccharides; Figure 4 The graph shows the changes in TBARS in abalone during storage at different concentrations of chitosan oligosaccharides. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments. However, it should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are also within the protection scope of the present invention.

[0018] Example 1: Construction, induction of expression and purification of chitosanase mutant BAC / MH Using the plasmid of wild-type chitosanase Csn-BAC (GenBank ID: ASB61783.1, the wild-type chitosanase is derived from Bacillus sp. MD-5) as a template, a chitosanase mutant BAC / MH was constructed. Compared with wild-type chitosanase Csn-BAC, the mutant BAC / MH mutated cysteine ​​at position 77 to methionine and threonine at position 236 to histidine.

[0019] The recombinant bacteria containing the chitosanase mutant BAC / MH were inoculated into LB medium and cultured at 37°C until OD500. 600 After reaching a concentration of 0.6-0.8, IPTG at a final concentration of 0.1 mM was added to induce expression, and the cells were cultured at 20°C for 16 h. The cells were then collected by centrifugation, sonicated, and the protein was purified using a Ni-NTA affinity column. The target protein was eluted with an NPI-200, and then ultrafiltration was performed at 4°C for desalting and concentration.

[0020] Example 2: Activity detection and hydrolysis product analysis of chitosanase mutant BAC / MH The reaction system for detecting the chitosan hydrolysis activity of the chitosanase mutant BAC / MH consisted of colloidal chitosan (1%, w / v), sodium phosphate buffer (50 mM, pH 7), and an appropriate amount of the purified chitosanase mutant BAC / MH prepared in Example 1. The hydrolysis reaction was carried out at 30 °C for 10 min, then terminated by adding 375 μL of DNS reagent, followed by boiling for 10 min. The reducing sugar content in the mixture was measured at 520 nm. The enzyme activity of the mutant BAC / MH was found to be 255.56 U / mg, which is 6.13 times that of the wild-type chitosanase Csn-BAC (41.67 U / mg).

[0021] The hydrolysis products of the chitosanase mutant BAC / MH were analyzed using colloidal chitosan (1%, w / v) as a substrate. After the reaction, an equal volume of ethanol was added and the mixture was centrifuged. A suitable sample was spotted onto a silica gel plate, using propanol-ammonia-water (8:3:1, v / v / v) as the mobile phase and 0.1% ethanol-ninhydrin solution as the colorimetric reagent. Figure 1 As shown, the hydrolysis products of the chitosanase mutant BAC / MH are chitobiose and chitotriose. Compared with the wild-type chitosanase Csn-BAC, the degree of polymerization of the products of the mutant BAC / MH did not change.

[0022] Example 3: Preparation of chitosan oligosaccharide aqueous solution The method for preparing chitosan oligosaccharide aqueous solution is as follows: chitosan mutant BAC / MH is added to chitosan aqueous solution, wherein the mass-volume ratio of chitosan is 5% (5 g / 100 mL) and the mass-volume ratio of chitosan mutant BAC / MH is 2‰ (2 g / 1000 mL); enzymatic hydrolysis is performed at 30℃ for 24 h, followed by boiling, centrifugation, filtration, and the supernatant is the crude chitosan oligosaccharide aqueous solution containing chitobiose and chitotriose.

[0023] Example 4: Application of chitosan oligosaccharide prepared using the chitosanase mutant of Example 3 in improving the quality of abalone under low-temperature refrigeration, specifically including the following steps: (1) Abalone sample preparation: Live abalone were covered with crushed ice for 10 minutes, then the shells and viscera were removed and cleaned. The abalone were then rinsed with pure water and drained.

[0024] (2) The prepared abalone was immersed in chitosan oligosaccharide solutions with concentrations of 0.25 g / 100 mL, 0.5 g / 100 mL, and 1.0 g / 100 mL, prepared by adding the crude chitosan oligosaccharide aqueous solution prepared in Example 3, for 30 min. The surface water was drained and recorded as K-0.25, K-0.5, and K-1.0, respectively. At the same time, a control group was set up, in which the prepared abalone was not subjected to any immersion treatment and was recorded as RS. The 0.25 g / 100 mL means that 100 mL of solution contains 0.25 g of chitobiose and chitotriose.

[0025] (3) After processing, the abalone is placed in a UV-sterilized polyethylene preservation bag, vacuum-packed, and then stored in a refrigerator at 4°C.

[0026] For each experimental group that underwent the above treatment, samples were taken on days 0, 2, 4, 6, 8, 10, and 12 to measure the following indicators: Determination of cooking loss rate: Take refrigerated abalone, accurately weigh it and record it as M1, place it in a cooking bag, seal it, put it in an 85℃ water bath for 30 min, remove it, cool it, blot the surface moisture with filter paper, and weigh it again and record it as M2. Each group of samples was measured in triplicate, and the average value was taken. The loss rate was calculated according to the following formula: ; Centrifugation loss rate determination: Take refrigerated abalone and weigh it, recording the weight as M1. Cut filter paper to a suitable size, wrap the sample in the filter paper, and place it in a centrifuge tube. Centrifuge at 5000 r / min for 20 min at 4℃. Weigh the sample after centrifugation and record the weight as M2. Perform three parallel measurements for each sample group, take the average value, and calculate the loss rate using the following formula: ; The results showed that the centrifugation loss rate in the RS group increased from 19.08% (0 d) to 32.58% (12 d), while the K-0.5 group had a loss rate of 28.67% at 12 d, significantly lower than the RS group (p < 0.05). Regarding the cooking loss rate, the RS group increased from 36.54% (0 d) to 47.08% (12 d), while the K-0.5 group only increased to 39.96% (12 d), a decrease of 7.12% compared to the RS group. These results demonstrate that the chitosan oligosaccharide can effectively maintain the water-holding capacity of muscle proteins and reduce juice loss.

[0027] Total bacterial count determination: Performed according to GB 4789.2—2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Bacterial Count". Accurately weigh 5.0 g of muscle into a sterile homogenizing bag, add 45 mL of sterile physiological saline, and agitate for 30 seconds to prepare a 1:10 dilution. Then perform 10-fold serial dilutions, selecting 2–3 suitable dilutions. Spread 100 μL of each solution onto plate counting agar medium and incubate at 30°C for 48 h before counting.

[0028] like Figure 2 As shown, the total bacterial count in the RS group increased rapidly from 2.31 log CFU / g (0 d) to 7.79 log CFU / g (12 d); the growth of the total bacterial count in the chitosan oligosaccharide treatment group slowed down significantly. At 12 d, the K-0.5 and K-1.0 groups were 6.70 and 6.62 log CFU / g, respectively, which were 1.09 and 1.17 CFU / g lower than the RS group (p < 0.05), showing a good antibacterial effect.

[0029] TVB-N value determination: Performed according to GB 5009.228-2016 "National Food Safety Standard - Determination of Volatile Basic Nitrogen in Food". A Kjeltec 8400 automatic Kjeldahl nitrogen analyzer (FOSS, Denmark) was used for determination, and results are expressed in mg / 100 g.

[0030] TBARS determination: Mix 5 g of sample with 45 mL of pre-cooled 7.5% trichloroacetic acid for 2 min, filter, take an equal volume of filtrate and mix with TBARS solution (0.02 mol / L), boil for 40 min, cool and measure absorbance at 532 nm. The result is expressed as mg MDA / kg.

[0031] like Figure 3 , Figure 4 As shown, the TBARS value in the RS group increased sharply from 0.16 mg MDA / kg (0 d) to 4.58 mg MDA / kg (12 d), and the TVB-N value increased from 2.26 mg / 100 g (0 d) to 21.76 mg / 100 g (12 d). In contrast, the increase in the above indicators in the chitosan oligosaccharide treatment group was significantly reduced. At 12 d, the TBARS value and TVB-N value in the K-1.0 group were 1.02 mg MDA / kg and 6.08 mg / 100 g lower than those in the RS group, respectively. Moreover, the differences between each treatment group and the control group were significant (p < 0.05), indicating that chitosan oligosaccharide has strong antioxidant capacity and can effectively inhibit protein degradation caused by microorganisms and endogenous enzymes.

[0032] In summary, the chitosan oligosaccharide (degree of polymerization 2-3) prepared by the chitosanase mutant BAC / MH in this invention comprehensively improves the quality of abalone during low-temperature refrigeration through multiple pathways, including inhibiting microbial proliferation, slowing down protein degradation and lipid oxidation, and improving water-holding capacity. The concentration of 0.5 g / 100 mL achieves the best balance between treatment effect and cost, demonstrating significant value for industrial application.

[0033] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0034] The amino acid sequence of the chitosanase mutant BAC / MH (SEQ ID NO.1): MNISLKKKAGFWKKAAISLLVFTMFFTLMMSETVFAAGLNKDQKRRAEQLTSIFENGTTEIQYGYVEPLGDGRGYTMGRAGFTTATGDALEVVEEYTKAVPNNKLKKYLPELRRLAKEESDDISNLKGFASAWKSLANDKEFRAAQDKVNDRLYYQPAMKRSDNAGLKTALARAVMYDTVIQHGDGDDPDSFYALIKRTNKKAGGSPKDGIDEKKWLNKFLDVRYDDLMNPANHDHRDEWRESVARVDVLRSIAKENNYNLNGPIQVHSKEYGHFVIK;

[0035] Nucleotide sequence of the chitosanase mutant BAC / MH gene (SEQ ID NO.2): ATGAATATCAGTTTGAAGAAAAAAGCGGGCTTCTGGAAAAAGGCAGCAATCTCATTACTTGTTTTCACCATGTTTTTTACCCTGATGATGAGCGAAACGGTTTTTGCGGCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTCGAAAACGGCACGACGGAGATCCAATATGGATATGTCGAGCCATTGGGTGACGGGCGAGGCTATACAATGGGACGGGCAGGTTTTACAACGGCTACAGGGGATGCATTGGAAGTGGTGGAAGAATACACAAAGGCAGTTCCGAATAACAAACTGAAAAAGTACCTGCCTGAATTGCGCCGTCTGGCCAAGGAAGAAAGCGATGACATAAGCAATCTTAAGGGCTTCGCTTCTGCCTGGAAGTCGCTTGCAAATGATAAGGAATTTCGCGCTGCCCAAGACAAAGTAAATGACCGTTTGTATTATCAGCCTGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTAATGTACGATACGGTTATTCAGCATGGCGATGGTGATGATCCTGACTCCTTTTATGCCTTGATTAAACGTACGAACAAAAAAGCGGGCGGGTCACCAAAAGACGGAATAGACGAGAAGAAGTGGTTAAATAAATTCTTGGACGTACGCTATGACGATCTGATGAATCCGGCAAATCATGACCACCGTGACGAGTGGAGAGAGTCGGTTGCCCGTGTGGACGTGCTCCGCTCTATCGCCAAGGAGAACAACTACAATCTAAACGGACCAATTCAGGTTCATTCAAAGGAATACGGTCATTTCGTAATCAAG。

Claims

1. A chitosanase mutant BAC / MH, characterized in that, The amino acid sequence of the chitosanase mutant BAC / MH is shown in SEQ ID NO.

1. Compared with the wild-type chitosanase Csn-BAC, cysteine ​​at position 77 is mutated to methionine and threonine at position 236 is mutated to histidine.

2. The gene encoding the chitosanase mutant BAC / MH of claim 1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A recombinant plasmid containing the gene of claim 2, characterized in that, The vector for the recombinant plasmid is pET-28a.

4. Genetically engineered bacteria containing the recombinant plasmid as described in claim 3.

5. The application of the chitosanase mutant BAC / MH of claim 1 or the gene of claim 2 in catalyzing the production of chitosan oligosaccharides, wherein the degree of polymerization of the chitosan oligosaccharides is 2-3, and the chitosan oligosaccharides can improve the preservation quality of abalone.

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