Pichia pastoris engineering bacterium capable of efficiently expressing chitinase and application of pichia pastoris engineering bacterium in shrimp shell high-valued treatment

Abstract

The invention discloses pichia pastoris engineering bacteria capable of efficiently expressing chitinase and application of the pichia pastoris engineering bacteria in shrimp shell high-value processing, and belongs to the field of biological engineering. The pichia pastoris engineering bacterium takes pichia pastoris GS115 as a host, the high-purity CtChi70 enzyme preparation can be prepared by signal peptide optimization, high-copy strain screening and cofactor overexpression in combination with high-density fermentation in a fermentation tank, ultrafiltration concentration in a hollow fiber column and freeze-drying flow, and CtChi70 accounts for more than or equal to 90% of total protein in fermentation liquor. The lyophilized enzyme preparation is used for a chitin substrate extracted from shrimp shells after mild treatment, and a product mainly comprising chitobiose can be efficiently generated. According to the invention, high yield of CtChi70 and simple preparation of the preparation are realized, and an economical, environment-friendly and high-yield feasible path is provided for high-value utilization of waste shrimp shell resources.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a genetically engineered Pichia pastoris strain, which can efficiently express CtChi70 chitinase, and the application of the strain in large-scale production of CtChi70 chitinase and in conversion of chitin from shrimp shells into chitobiose. BACKGROUND

[0002] Chitin is the second most abundant renewable polysaccharide in nature (next to cellulose), which widely exists in the shell of crustaceans (such as shrimps and crabs). The shrimp shell waste generated by global aquatic processing industry is roughly in the order of millions of tons per year, which will bring huge pressure to the environment if not properly disposed. Chitobiose, which is composed of two D-glucosamine units connected by a beta-1,4 glycosidic bond, is an important substrate for synthesizing high-value products, human milk oligosaccharides (HMOs).

[0003] At present, microbially derived chitinases are widely used for chitin hydrolysis, but the enzyme yield of conventional expression systems is usually low, which is difficult to meet the needs of industrialization. Pichia pastoris is a commonly used protein expression host, which has the advantages of efficient secretion expression and easy genetic engineering. CtChi70 is a highly specific chitinase that can convert chitin into high-purity chitobiose, but its efficient expression in Pichia pastoris still faces challenges.

[0004] Therefore, it is of great significance to construct an engineered strain that can efficiently secrete and express CtChi70, and to establish a conversion system that matches the chitin extraction process, in order to realize the high-value utilization of waste biomass and reduce the cost of industrial production. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present application is to provide a Pichia pastoris engineered strain that can efficiently express chitinase CtChi70 and a construction method thereof. The Pichia pastoris engineered strain uses Pichia pastoris GS115 as the host, and is constructed by signal peptide optimization, high-copy strain screening, and overexpression of auxiliary factors, combined with high-density fermentation in a fermenter, hollow fiber column ultrafiltration concentration, and freeze-drying process, to prepare high-purity CtChi70 enzyme preparation, with CtChi70 accounting for ≥90% of the total protein in the fermentation broth. The freeze-dried enzyme preparation can be used to efficiently generate chitobiose-based products from chitin extracted from shrimp shells after mild treatment. The present application not only realizes the high production and simple preparation of CtChi70, but also provides an economic, environmentally friendly and high-yield feasible path for the high-value utilization of waste shrimp shell resources.

[0006] Another purpose of the present application is to provide the application of the chitinase CtChi70 produced by the engineered strain in the conversion of chitin from shrimp shells into chitobiose.

[0007] The object of the present application is achieved by the following technical solutions. The present application discloses a Pichia pastoris engineering bacterium for efficiently expressing chitinase CtChi70, wherein the Pichia pastoris engineering bacterium is a host bacterium of Pichia pastoris, and at least one copy of CtChi70 gene including a secretion signal peptide is expressed in the host bacterium.

[0008] Further, the secretion signal peptide is alpha-factor, alphaMF1, Msb2, pre-Ost1-pro-alphaf(I) or alpha-factor (V50A), etc.

[0009] Specifically, the nucleotide sequence of the CtChi70 gene is shown in SEQ ID NO: 2, and the amino acid sequence encoded by the nucleotide sequence is shown in WP_027093193.1 39-691aa or SEQ ID NO: 1.

[0010] Specifically, the amino acid sequence of alpha-factor is shown in QIM58710.1 1-89aa, and the nucleotide sequence is shown in MN267804.1 1-267bp; the amino acid sequence of alphaMF1 is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4; the amino acid sequence of Msb2 is shown in XP_002489622.1 1-31aa, and the nucleotide sequence is shown in XM_002489577.1 1-93bp; the amino acid sequence of pre-Ost1-pro-alphaf(I) is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6; the amino acid sequence of alpha-factor (V50A) is obtained by mutating V50A in QIM58710.1 1-89aa, and the nucleotide sequence is obtained by mutating V50A: codon from GTT to GCT in MN267804.1 1-267bp.

[0011] Further, in the heterologous expression, the above-mentioned genes are constructed into a gene expression cassette by using an inducible promoter AOX1.

[0012] Further, the Pichia pastoris engineering bacterium further over-expresses any one or more of the following genes: Hac1 gene, KEX2 gene, PDI gene, ERO1 gene, BiP gene.

[0013] Further, in the overexpression, the above-mentioned genes are constructed into gene expression cassettes by using PGK1 promoter or inducible promoter AOX1; the PGK1 promoter is a PGK1 promoter derived from Pichia pastoris.

[0014] The Hac1 gene, the KEX2 gene, the PDI gene, the ERO1 gene, and the BiP gene are all endogenous genes of Pichia pastoris.

[0015] Specifically, the amino acid sequence of the Hac1 gene is shown in WJE88854.1, and the nucleotide sequence is shown in OQ326497.1; the amino acid sequence of the KEX2 gene is shown in XP_002491199.1, and the nucleotide sequence is shown in XM_002491154.1; the amino acid sequence of the PDI gene is shown in ACF17572.1, and the nucleotide sequence is shown in EU805807.1; the amino acid sequence of the ERO1 gene is shown in XP_002489645.1, and the nucleotide sequence is shown in XM_002489600.1; and the amino acid sequence of the BiP gene is shown in XP_002491027.1, and the nucleotide sequence is shown in XM_002490982.1.

[0016] Further, the Pichia pastoris includes but is not limited to Pichia pastoris GS115 and the like.

[0017] Specifically, the Pichia pastoris engineering strain for efficiently expressing chitinase CtChi70 is obtained by performing the following operations on Pichia pastoris: (a) heterologous expression of the CtChi70 gene including a secretion signal peptide; or (b) heterologous expression of 2 copies of the CtChi70 gene including a secretion signal peptide; or (c) heterologous expression of the CtChi70 gene including a secretion signal peptide, and overexpression of the Hac1 gene, the KEX2 gene, the PDI gene, the ERO1 gene, or the BiP gene; (d) heterologous expression of 2 copies of the CtChi70 gene including a secretion signal peptide, and overexpression of the Hac1 gene, the KEX2 gene, the PDI gene, the ERO1 gene, or the BiP gene.

[0018] The construction method of the Pichia pastoris engineering strain for efficiently expressing chitinase CtChi70 includes the following steps: (1) heterologous expression of the CtChi70 gene including a secretion signal peptide on the basis of Pichia pastoris to construct a recombinant strain; (2) heterologous expression of the CtChi70 gene including a secretion signal peptide on the basis of Pichia pastoris, and then obtaining a high-copy recombinant strain by a high-copy strain screening method; (3) Based on the recombinant strain in step (1) or (2), overexpress the Hac1 gene, KEX2 gene, PDI gene, ERO1 gene or BiP gene to construct a recombinant strain.

[0019] Furthermore, the preferred method for screening high-copy strains is the plate gradient antibiotic concentration PTVA method.

[0020] The application of the above-mentioned engineered Pichia pastoris strain is one of the following applications: (I) Application in the efficient expression of chitinase CtChi70; (II) Application in the conversion of chitin to chitobiose; (III) Application in the high-value utilization of shrimp shells.

[0021] The application (I) involves inoculating engineered Pichia pastoris bacteria into a fermentation medium for fermentation culture and inducing it with methanol to achieve efficient expression of chitinase.

[0022] Furthermore, the fermentation conditions are 30±1℃, 250±10 rpm for 96-120h; and the amount of methanol used is 0.5-1.5% (preferably 1%).

[0023] Furthermore, the fermentation medium is BMMY medium.

[0024] Furthermore, in application (II), the chitin includes at least one of shrimp shell-derived chitin and colloidal chitin.

[0025] Application (II) involves producing chitinase by high-density fermentation of Pichia pastoris engineered strains in a fermenter, concentrating and freeze-drying the chitinase preparation; then mixing the chitinase preparation with chitin and performing enzymatic hydrolysis; after enzymatic hydrolysis, inactivating the enzyme, separating the solid and liquid, collecting the supernatant, and obtaining chitobiose.

[0026] Furthermore, the fermentation tank is preferably a 7L glass fermentation tank.

[0027] Furthermore, the concentration is preferably carried out using hollow fiber column ultrafiltration.

[0028] Furthermore, the freeze-drying is achieved by adding a preservative to the concentrate and then freeze-drying it. Preferably, the preservative is glycine, and the dosage is 1.5%. The freeze-drying time is 24 ± 4 h.

[0029] Furthermore, the CtChi70 protein in the chitinase preparation accounts for ≥90% of the total protein content.

[0030] Furthermore, the chitin derived from the shrimp shell is prepared using a mild extraction method, specifically by demineralizing with citric acid and deproteinizing with acidic protease.

[0031] Preferably, the demineralization condition is 50-60℃ for 4-5h.

[0032] Preferably, the deproteinization condition is 45-50℃ water bath stirring for 4-5h; further 50℃ water bath stirring for 4h.

[0033] Further, the enzymolysis system is as follows: chitinase enzyme preparation 500-1500 U / L, chitin 8-12 g / L, solvent is 0.1M, pH 4.5-5.5 sodium citrate buffer; Further preferably, the enzymolysis system is as follows: chitinase enzyme preparation 1000 U / L, chitin 10 g / L, solvent is 0.1M, pH 5.0 sodium citrate buffer; Further, the enzymolysis condition is as follows: 45±2℃ for 24h.

[0034] Further, the solid-liquid separation mode is centrifugation.

[0035] The centrifugation condition is 8000-12000g centrifugation for 5-15min; more preferably 10000g centrifugation for 10min.

[0036] The supernatant is selectively subjected to the following steps: concentration, drying, to obtain chitobiose solid.

[0037] The present application has the following advantages and effects relative to the prior art: (1) The present application uses a synergistic optimization method to significantly improve the expression amount of chitinase CtChi70. Through signal peptide screening, high-copy recombinant strain screening, Hac1 gene overexpression and high-density fermentation in a fermenter (expression amount of 5.72g / L), a foundation is laid for large-scale industrial application.

[0038] (2) Simplify the enzyme preparation process and reduce production costs: CtChi70 accounts for more than 90% of the total protein in the fermentation broth obtained by high-density fermentation, so high-purity chitinase can be obtained without complex purification steps. By concentrating the fermentation broth and freeze-drying, high-purity enzyme preparation can be obtained, greatly saving the labor, time and material costs required in the traditional purification process.

[0039] (3) The present application establishes a combined system from shrimp shell chitin extraction to chitobiose production, relying on mild extraction and enzyme-catalyzed conversion, to convert low-cost, abundant and usually discarded shrimp shell waste into high-value, potentially functional chitobiose products. This not only improves resource utilization, but also has good environmental friendliness and economic sustainability. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 Figure 4 is a CtChi70 production graph of chitinase expression strains with optimized signal peptides in shake flask culture; wherein, Ost1 refers to pre-Ost1-pro-alf (I) signal peptide, V50A refers to alpha-factor (V50A) signal peptide; alpha-factor represents strain M01, alphaMF1 represents strain M02, Msb2 represents strain M03, Dan4 represents strain M04, Ost1 represents strain M05, V50A represents strain M06. P <0.001.

[0041] Figure 2 Figure 5 is a CtChi70 enzyme activity graph of chitinase expression strains with optimized signal peptides in shake flask culture; wherein, Ost1 refers to pre-Ost1-pro-alf (I) signal peptide, V50A refers to alpha-factor (V50A) signal peptide; alpha-factor represents strain M01, alphaMF1 represents strain M02, Msb2 represents strain M03, Dan4 represents strain M04, Ost1 represents strain M05, V50A represents strain M06.

[0042] Figure 3 Figure 6 is a CtChi70 production graph of further constructed high-copy chitinase expression strains in shake flask culture; wherein, M02 refers to CtChi70 one-copy strain, M07 refers to CtChi70 two-copy strain. P <0.001.

[0043] Figure 4 Figure 7 is a CtChi70 enzyme activity graph of further constructed high-copy chitinase expression strains in shake flask culture; wherein, M02 refers to CtChi70 one-copy strain, M07 refers to CtChi70 two-copy strain. P <0.01.

[0044] Figure 5 Figure 8 is a CtChi70 production graph of chitinase expression strains after further overexpression of auxiliary factors in shake flask culture; wherein, ERO1 represents strain M08, KEX2 represents strain M09, BiP represents strain M10, CPR5 represents strain M11, Hac1 represents strain M12, PDI represents strain M13. P <0.01.

[0045] Figure 6Figure 6 is a CtChi70 enzyme activity chart of the chitinase expression strain further overexpressing the auxiliary factor in the shake flask culture; wherein ERO1 represents the strain M08, KEX2 represents the strain M09, BiP represents the strain M10, CPR5 represents the strain M11, Hac1 represents the strain M12, and PDI represents the strain M13. P <0.05.

[0046] Figure 7 Figure 7 is the yield and enzyme activity of CtChi70 under the fermentation amplification conditions in the fermenter; wherein Methanol refers to the consumption of methanol in the fermentation process.

[0047] Figure 8 Figure 8 is the effect of different treatment temperatures on the demineralization effect of shrimp shells.

[0048] Figure 9 Figure 9 is a TLC chromatogram analysis of chitobiose produced by enzymatic hydrolysis of shrimp shell chitin by CtChi70; wherein L1 is a standard containing GlcNAc, L2 is a standard containing (GlcNAc)2, L3 is the supernatant of shrimp shells enzymatically hydrolyzed by CtChi70, and L4 is the supernatant of shrimp shells without enzyme treatment. DETAILED DESCRIPTION

[0049] The present application will be further described in conjunction with the following examples and drawings, but the embodiments of the present application are not limited thereto. In the following examples, the test methods are generally carried out according to the conventional experimental conditions or according to the experimental conditions suggested by the manufacturer, unless otherwise specified. The materials, reagents, etc. used are commercially available reagents and materials, unless otherwise specified.

[0050] The promoters and gene information involved in the examples are as follows: Table 1 Promoter and gene information

[0051] Example 1 Construction of recombinant strain for efficient expression of chitinase CtChi70 (1) Construction of CtChi70 recombinant expression vector The CtChi70 gene was synthesized by Suzhou Jinyuzhi Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO: 2. Subsequently, the CtChi70 gene was inserted into the pPIC9K vector by the Gibson Assembly method, thereby constructing the recombinant plasmid pPIC9K-CtChi70 (named A01) as the basic expression vector for expressing CtChi70 in Pichia pastoris.

[0052] (2) Construction and expression of the initial expression strain M01 The A01 plasmid was linearized by Xba I enzyme digestion, and the digestion product was recovered. Then, it was electroporated into the host strain Pichia pastoris GS115 competent cells at a condition of 0.75 kV. After transformation, the cells were plated on histidine-deficient MD plates and cultured at 30°C for 2 days. Several single colonies were picked and lysed using 0.1% NaOH. The lysate was used as a template for yeast colony PCR identification. The positive recombinant strain was named M01 and used for subsequent shake flask induction expression.

[0053] The specific expression process was as follows: a single colony was inoculated in 3 mL of YPD medium and cultured at 30°C and 250 rpm for 24 h. Then, it was transferred into 10 mL of BMGY medium at an inoculation amount of 1% (v / v) and cultured for about 11 h to make the OD 600 =2-6. Then, the culture was centrifuged at low temperature, and 10 mL of BMMY medium was added. Methanol was added at an amount of 1% (v / v) every 24 h for induction expression for 96 h. During the induction period, samples were taken every 24 h to determine the protein concentration and chitinase activity in the supernatant.

[0054] Chitinase activity determination: The chitinase enzyme activity was determined by the method of measuring the reducing sugar in the reaction product using DNS (3,5-dinitrosalicylic acid). A 2 wt% colloidal chitin solution was used as a substrate to prepare a 50 µL reaction system (reaction product: DNS reagent = 1:2, v / v). The system was boiled in a water bath for 5 min, cooled to room temperature, and then water was added to make the total volume 200 µL. After centrifugation, 100 µL of supernatant was taken and placed in a 96-well plate. The absorbance (OD 540 ) was measured at 540 nm. A standard curve was constructed using N-acetylglucosamine, and the enzyme activity unit was defined as 1 U, which was the production of 1 µM reducing sugar per minute. The protein concentration was determined by SDS-PAGE electrophoresis combined with a BSA standard curve.

[0055] After preliminary expression test, the expression amount of CtChi70 secreted by the strain M01 was about 32 mg / L, and the enzyme activity was about 41 U / L.

[0056] (3) Screening and optimization of secretory signal peptide To break through the limitation of the classical signal peptide on the secretion efficiency of the exogenous enzyme, we screened a signal peptide combination covering multiple optimization mechanisms. The combination not only contains the classical optimization variant α-factor (V50A), but also systematically incorporates the derived signal peptide αMF1 with innovative pro-peptide design, the natural signal peptides Msb2 and Dan4 derived from the highly efficient secretion proteins of Pichia pastoris, and the composite signal peptide pre-Ost1-pro-αf(I) composed of different functional domains. The α-factor (V50A) introduces a V50A mutation in the hydrophobic core region of the α-factor signal peptide, which may enhance its interaction efficiency with the Sec61 translocator and other secretion elements, and is a known relatively effective mutant; the core of αMF1 is a truncated and modified "pro-peptide" retained after the signal peptide (leader peptide), which can act as a chaperone to assist the target protein in correct folding after translation, delay premature aggregation, and possibly more efficiently guide the protein into the secretion pathway, thereby significantly improving the secretion yield; Msb2 and Dan4 are derived from the highly efficient secretion proteins of Pichia pastoris, and their advantage lies in co-evolution with the host's signal recognition particle, transport channel and processing protease system, with natural high compatibility; pre-Ost1-pro-αf(I) is a rationally designed composite structure, containing an enhanced leader sequence from Ost1, fused with a modified α-factor pro-peptide, which aims to combine the advantages of different functional units, such as stronger transport traction and better folding assistance.

[0057] Based on the construction of A01 plasmid, the original signal peptide α-factor was replaced with different candidate signal peptides, including αMF1, Msb2, Dan4, pre-Ost1-pro-αf(I), and α-factor (V50A), using Gibson Assembly method to construct recombinant plasmids A02, A03, A04, A05, and A06, respectively. The recombinant strains M02, M03, M04, M05, and M06 were obtained by colony PCR verification after electroporation of GS115 with the above plasmids. The above plasmids were linearized and electroporated into GS115, and the recombinant strains M02-M06 were obtained by colony PCR identification. Then, the same shake flask induction conditions as M01 were used for cultivation, and the concentration and enzyme activity of the supernatant were determined.

[0058] The results showed that when using the αMF1 signal peptide (strain M02), the expression amount of CtChi70 was significantly improved, with an expression amount of about 156.4 mg / L ( Figure 1 ), and an enzyme activity of about 124.7 U / L ( Figure 2 ). This result is significantly better than the initial strain M01, indicating that selecting an appropriate secretion signal peptide has a significant impact on the secretion expression of CtChi70.

[0059] (4) Construction of high-copy recombinant strains To further enhance the expression level of CtChi70, high-copy recombinant strains were screened using the PTVA method. Specifically, the transformed strain M02 was inoculated onto plates containing the antibiotic G418 (the antibiotic concentration was gradually increased from 0.5 mg / mL to 2 mg / mL, and then to 4 mg / mL). Recombinant strains that could survive and grow at high G418 concentrations were screened, and the resulting recombinant strain was named M07.

[0060] The copy number of M07 was determined by qPCR, and the housekeeping gene GAPDH was selected as the internal reference gene. 2 -ΔΔCt The method determined that M07 was a strain with 2.37 copies of the chitinase gene, which was rounded to two copies.

[0061] M07 was subjected to the same shake-flask induction expression process as described above, and its expression level and enzyme activity were measured.

[0062] The results showed that the CtChi70 expression level of strain M07 reached approximately 432 mg / L ( Figure 3 The enzyme activity is approximately 155 U / L. Figure 4 The expression level of CtChi70 was significantly higher than that of strain M02, indicating that increasing the gene copy number can effectively enhance the secretory expression level of CtChi70.

[0063] (5) Overexpression of cofactors To significantly enhance the secretory expression level of the target enzyme in Pichia pastoris, this invention employs a multi-gene synergistic host cell engineering strategy. This strategy involves overexpressing six endogenous genes (ERO1, KEX2, BiP, CPR5, Hac1, and PDI) located at three key nodes in the secretory pathway.

[0064] To address the endoplasmic reticulum's (ER) folding capacity and redox balance, the molecular chaperone BiP was overexpressed to enhance the stability and transport of nascent peptide chains. Overexpression of the disulfide isomerase PDI or the ER oxidoreductase ERO1 was used to strengthen the efficient and correct formation of disulfide bonds, ensuring that newly generated proteins fold correctly and avoiding cell damage caused by misfolding. For specific biochemical steps in folding and processing, peptidyl prolyl cis-trans isomerase CPR5 was overexpressed to accelerate the rate-limiting folding step involving proline isomerization; and Golgi protease KEX2 was overexpressed to ensure the precise processing and maturation of protein precursors at the secretory terminal. To address the overall adaptability and expansion of the ER, the core transcription factor Hac1 of the unfolded protein response was overexpressed, aiming to globally upregulate the expression of ER-related genes, systematically expand the cell's folding and secretory capacity, and provide synergistic support for the function of the aforementioned specific factors.

[0065] These genes were inserted into pPGK1ZA vector by Gibson Assembly, respectively, to construct overexpression plasmids pPGK1ZA-ERO1, pPGK1ZA-KEX2, pPGK1ZA-BiP, pPGK1ZA-CPR5, pPGK1ZA-Hac1, pPGK1ZA-PDI (named B01-B06), respectively. After linearization of the plasmids by Sac I digestion, the linearized plasmids were transformed into the competent cells of the above-mentioned recombinant strain M07 by electroporation, and then the transformed cells were spread on YPD plates containing bleomycin and cultured at 30°C for 2 days. Single colonies were picked and lysed by 0.1% NaOH, and then positive colonies were identified by colony PCR, and were named M08-M13, respectively. The recombinant strains were subjected to shake flask methanol induction expression, and the expression amount of CtChi70 and enzyme activity were determined.

[0066] In the above-mentioned pPGK1ZA vector, the promoter AOX1 in the pPICZA vector was replaced by the promoter PGK1.

[0067] Among these co-factors, the overexpression of Hac1 (strain M12) had the best effect. The expression amount of secreted CtChi70 of strain M12 was about 561 mg / L ( Figure 5 ), and the enzyme activity was about 181 U / L ( Figure 6 ), which was significantly better than that of strain M07 without overexpression of co-factors.

[0068] To test whether the use of a stronger promoter for overexpression of the Hac1 gene can further improve the effect, the PGK1 promoter in plasmid B05 was replaced by the AOX1 promoter to obtain B07 vector, which was transformed into strain M07 to obtain strain M14. The same shake flask induction expression test was performed on strain M14, and the expression amount of CtChi70 (514 mg / L) and enzyme activity (161.2 U / L) of strain M14 were slightly lower than those of strain M12. This indicates that, in this case, the PGK1 promoter for Hac1 co-factor is sufficient, and excessive enhancement of expression intensity will not bring greater secretory expression advantage.

[0069] In summary, through signal peptide screening, high-copy strain construction and overexpression of co-factors (Hac1), the recombinant Pichia pastoris strain M12 with high secretory CtChi70 was successfully obtained, which laid a foundation for subsequent industrial scale expression.

[0070] Example 2 Fermentation tank high-density fermentation for producing chitinase CtChi70 and preparing enzyme preparation (1) High-density fermentation Basic salt medium (1 L) contains: 26.7 mL phosphoric acid (85%), 0.93 g calcium sulfate, 18.2 g potassium sulfate, 14.9 g magnesium sulfate heptahydrate, 4.13 g potassium hydroxide, 40 g glycerol, add water to 1 L.

[0071] PTM1 (1 L) contains: 6.0 g copper sulfate pentahydrate, 0.08 g sodium iodide, 3.0 g manganese sulfate monohydrate, 0.2 g sodium molybdate dihydrate, 0.02 g boric acid, 0.5 g cobalt chloride, 20.0 g zinc chloride, 65.0 g ferrous sulfate heptahydrate, 0.2 g biotin, 5 ml sulfuric acid, add water to 1 L.

[0072] Take a single colony of recombinant yeast strain M12 and inoculate it into 10 mL of YPD medium. Incubate at 30°C and 250 rpm for 24 h. Then inoculate 1% into 200 mL of BMGY medium and incubate at 30°C and 250 rpm until the OD 600 = 2-6 is reached. Obtain the seed liquid.

[0073] Inoculate the seed liquid into sterilized 3 L of basic salt medium (containing PTM1) and perform fermentation in a 7 L glass fermenter.

[0074] The fermentation tank culture process is divided into three stages: The first stage is the glycerol batch stage. The temperature is controlled at 30°C, the pH is maintained at 5.0, the aeration rate is 2vvm, and the stirring speed is 500 rpm. Incubate for 18-24 h until the glycerol is consumed, at which time the dissolved oxygen suddenly rises, indicating that the glycerol in the fermentation tank has been consumed. Take a sample and prepare for the next stage.

[0075] The second stage is the glycerol feeding stage. Start feeding 50% glycerol (containing PTM1) at a speed linked to the rotation speed, maintaining the dissolved oxygen between 20%-40%. When the OD 600 of the cells reaches 250, stop feeding glycerol, record the glycerol consumption, and observe that the dissolved oxygen suddenly rises. After about 30 min of starvation, take a sample and prepare for the next stage.

[0076] The third stage is the methanol feeding stage. Adjust the pH to 6.0 with ammonia water and slowly feed methanol (containing PTM1) at a speed linked to the rotation speed, controlling the dissolved oxygen between 20%-40%. Take a sample every 12 h to measure the OD 600 , CtChi70 expression level, and enzyme activity, and record the methanol consumption. When the fermentation reaches 108 h, the expression level and enzyme activity begin to decrease, and the fermentation is stopped at this time. The maximum OD6 00The maximum methanol consumption was 2500 mL, reaching 474. The maximum CtChi70 expression level was 5.72 g / L at 96 h of the entire fermentation cycle, and the maximum CtChi70 enzyme activity was 2151 U / L at 96 h. Figure 7 ).

[0077] (2) Enzyme preparation The fermentation broth obtained from the fermenter was centrifuged to remove the cell precipitate, and the supernatant was collected. The supernatant was first clarified by passing it through a 750 kDa hollow fiber column, and then concentrated by ultrafiltration through a 7 kDa hollow fiber column, followed by liquid replacement, to obtain a concentrated solution. SDS-PAGE analysis of the concentrated solution showed that CtChi70 protein accounted for more than 90% of the total protein, indicating that no further purification (such as column chromatography) was required to obtain a high-purity enzyme preparation.

[0078] The concentrate was treated with 1.5% glycine as a preservative, pre-frozen at -80°C, and then freeze-dried for 24 hours to obtain high-purity CtChi70 freeze-dried powder. This freeze-dried powder is stable, easy to store and transport, and suitable for subsequent industrial applications.

[0079] Example 3: Mild extraction of chitin from shrimp shells and its specific conversion of chitobiose under the action of CtChi70 (1) Mild extraction of chitin from shrimp shells The waste shrimp shells are washed and dried to remove moisture, and then ground using a cryogenic grinder to obtain shrimp shell powder that can pass through a 100-mesh sieve.

[0080] Demineralization of shrimp shell powder: 0.1 g of shrimp shell powder was added to 2 mL of 10% (w / v) citric acid solution and allowed to stand for 4 h at different temperatures (20℃, 30℃, 40℃, 50℃, 60℃). After treatment, the solid was recovered by vacuum filtration, dried, and then ashed in a muffle furnace at 600℃ for 3 h to determine the ash content and calculate the demineralization rate. The demineralization rate reached 92.8% after treatment at 60℃ for 4 h. Figure 8 ).

[0081] To further demineralize, a treatment at 60℃ for 4 hours under stirring was tested, and the demineralization rate reached 97.1%.

[0082] To further remove protein, 100,000 U of acidic protease was dissolved in 200 mL of 0.1% citric acid solution. 0.22 g of shrimp shell powder, demineralized using the above method, was added to this solution, and the mixture was stirred in a 50°C water bath for 4 hours for protease treatment. After treatment, the liquid was removed by filtration again, and the resulting solid was washed with water and dried to obtain chitin powder extracted from shrimp shells, i.e., shrimp shell chitin.

[0083] (2) CtChi70 catalyzes the conversion of chitin in shrimp shells into chitobiose. The extracted shrimp shell chitin was dispersed in a 0.1 M sodium citrate buffer solution at pH 5.0, and the substrate concentration was adjusted to 10 g / L. An appropriate amount of CtChi70 enzyme preparation (1000 U / L) was added, and the mixture was reacted at 45 °C for 24 h. After the reaction was completed, the mixture was heated at 98 °C for 5 min to inactivate CtChi70, and then centrifuged (10000 g, 10 min), and the supernatant was collected as the reaction solution.

[0084] Thin-layer chromatography (TLC) analysis was performed on the supernatant: 2 µL of the reaction solution was spotted onto a silica gel plate. A developing solvent of n-butanol:glacial acetic acid:water = 8:3:2 (v / v / v) was used. After development, the plate was dried and developed using aniline-diphenylamine colorimetric solution (0.4 g diphenylamine, 0.4 mL aniline, 20 mL acetone, 3.0 mL 85% phosphoric acid). The silica gel plate was heated for 1–3 min after color development.

[0085] Results of TLC analysis ( Figure 9 The results showed that during the 24-hour enzymatic hydrolysis reaction, after CtChi70 hydrolysis, the chitin from the shrimp shell was mainly hydrolyzed to produce products mainly composed of chitobiose (GlcNAc)2. This indicates that the CtChi70 produced by this invention has a high specific hydrolysis ability for chitin from shrimp shells, and successfully realizes the conversion of waste shrimp shell resources into high-value-added chitobiose.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A Pichia pastoris engineered strain that efficiently expresses chitinase CtChi70, characterized in that: The Pichia pastoris engineered strain uses Pichia pastoris as the host strain and heterologously expresses at least one copy of the CtChi70 gene, which includes a secretion signal peptide, in the host strain. The amino acid sequence encoded by the CtChi70 gene is shown in SEQ ID NO: 1; The secretion signal peptides α-factor, αMF1, Msb2, pre-Ost1-pro-αf(I), or α-factor (V50A); The amino acid sequence of α-factor is shown as 1-89aa in QIM58710.1; the amino acid sequence of αMF1 is shown as SEQ ID NO: 3; the amino acid sequence of Msb2 is shown as 1-31aa in XP_002489622.1; the amino acid sequence of pre-Ost1-pro-αf(I) is shown as SEQ ID NO: 5; the amino acid sequence of α-factor (V50A) is obtained by mutating 1-89aa in QIM58710.1 to V50A.

2. The Pichia pastoris engineered strain that efficiently expresses chitinase CtChi70 according to claim 1, characterized in that: The Pichia pastoris engineered strain also overexpresses any one or more of the following genes: Hac1 gene, KEX2 gene, PDI gene, ERO1 gene, and BiP gene; The Hac1 gene, KEX2 gene, PDI gene, ERO1 gene, and BiP gene are all endogenous genes of Pichia pastoris.

3. The Pichia pastoris engineered strain that efficiently expresses chitinase CtChi70 according to claim 2, characterized in that: The nucleotide sequence of the CtChi70 gene is shown in SEQ ID NO: 2; The Pichia pastoris includes, but is not limited to, Pichia pastoris GS115.

4. The Pichia pastoris engineered strain that efficiently expresses chitinase CtChi70 according to claim 2, characterized in that: The CtChi70 gene expression cassette was constructed using the inducible promoter AOX1. The Hac1, KEX2, PDI, ERO1, and BiP genes all utilize the PGK1 promoter or the inducible promoter AOX1 to construct gene expression cassettes. The PGK1 promoter or the inducible promoter AOX1 are both derived from Pichia pastoris.

5. The method for constructing the Pichia pastoris engineered strain that efficiently expresses chitinase CtChi70 according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Based on Pichia pastoris, the CtChi70 gene, which includes a secretion signal peptide, was heterologously expressed to construct a recombinant strain; (2) Based on Pichia pastoris, the CtChi70 gene, which includes secretion signal peptide, was heterologously expressed, and then high-copy recombinant strains were obtained by screening high-copy strains. (3) Based on the recombinant strain in step (1) or (2), overexpress the Hac1 gene, KEX2 gene, PDI gene, ERO1 gene or BiP gene to construct a recombinant strain.

6. The application of the Pichia pastoris engineered strain that efficiently expresses chitinase CtChi70 according to any one of claims 1 to 4, characterized in that: For one of the following applications: (I) Application in the efficient expression of chitinase CtChi70; (II) Application in the conversion of chitin to chitobiose; (III) Application in the high-value utilization of shrimp shells.

7. The application according to claim 6, characterized in that: The application (I) involves inoculating engineered Pichia pastoris bacteria into a fermentation medium for fermentation culture and inducing it with methanol to achieve efficient expression of chitinase.

8. The application according to claim 6, characterized in that: In application (II), the chitin includes at least one of shrimp shell-derived chitin and colloidal chitin.

9. The application according to claim 8, characterized in that: Application (II) involves producing chitinase by high-density fermentation of Pichia pastoris engineered strains in a fermenter, concentrating and freeze-drying the chitinase preparation; then mixing the chitinase preparation with chitin and performing enzymatic hydrolysis; after enzymatic hydrolysis, inactivating the enzyme, separating the solid and liquid, collecting the supernatant, and obtaining chitobiose.

10. The application according to claim 9, characterized in that: The concentration is achieved using hollow fiber column ultrafiltration. The freeze-drying process involves adding a preservative to a concentrated liquid and then freeze-drying it. The chitin derived from shrimp shells is prepared using a mild extraction method, specifically by demineralizing with citric acid and deproteinizing with acidic protease. The enzymatic hydrolysis system is as follows: 500-1500 U / L of chitinase preparation, 8-12 g / L of chitin, and 0.1M sodium citrate buffer solution with pH 4.5-5.5 in the solvent. The enzymatic hydrolysis conditions are as follows: reaction at 45±2℃ for 24 h.

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