Mannase gene and application of mannase gene in production of mannose oligomer by converting citrus peel

Abstract

The invention relates to mannase, an amino acid sequence of a mutant of mannase, a coding gene and application, and belongs to the technical field of bioengineering. The invention provides mannase derived from tritypha globosa and a coding gene and an amino acid sequence of a mutant of the mannase. The mannase can be used for preparing mannose oligomer with the polymerization degree of 2-5 by converting mannan from citrus peel in a high-specificity manner. The sequence is obtained through whole genome sequencing of wild type microorganisms, the activity and stability of the enzyme are improved through a protein engineering design method, and practical application of the enzyme to conversion of waste biomass citrus peels into mannose oligomer is facilitated. According to the method, the waste biomass citrus peel can be converted into the mannan-oligosaccharide with a physiological function by using a small amount of enzyme through an enzyme hydrolysis method, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to the gene and amino acid sequence of a mannanase mutant derived from Trichosporonoides oedocephalis, and application of the enzyme in converting citrus peel mannan to produce oligomannosaccharides. Background Art

[0002] Citrus fruits are the most cultivated fruit category in the world and are grown in more than 140 countries and regions around the world. China is the main origin of citrus fruits in the world. According to reports, the total cultivated area of ​​citrus fruits in my country is about 2.5 million hectares, and the total output ranks first in the world (Journal of Huazhong Agricultural University (2021) 40(1): 58-69). Fresh fruit is the main consumption channel for citrus fruits (55%). In recent years, the proportion of processed products such as canned food and juice has also increased rapidly (35%-40%). A large amount of peel waste is generated in the production of fresh fruit and commercial canned food and citrus juice, which can easily cause environmental pollution such as soil and water. Under the guidance of the garbage classification and recycling policy, related research on the reuse of citrus peels has also attracted widespread attention. At present, the main methods for utilizing this type of waste are as a raw material for extracting pectin, as a raw material for animal feed, and as a raw material for extracting active substances such as essential oils, limonene, proteins and biological enzymes (Chinese patents CN105541687, CN111500371, etc.). Due to the complex composition of citrus peel, common utilization methods cannot make comprehensive use of citrus peel. Citrus peel plant fiber is rich in polysaccharides, including mannan, which will become an obstacle to its high-value utilization if it cannot be degraded. Studies have shown that mannan is a common anti-nutritional factor in animal feed. It cannot be hydrolyzed by the digestive enzymes secreted by the animal itself. It easily absorbs water and swells in the digestive tract to form a gel, increasing the viscosity of the chyme and affecting the digestive efficiency of the animal (China Animal Husbandry Journal (2014) 50(01): 54-58).

[0003] Mannan is a highly branched polysaccharide that is mainly found in the cell walls of higher plants. It is an important structural component of hemicellulose and one of the media for plant energy storage. There are a variety of mannanases in nature that can hydrolyze the mannan in citrus peel into oligomannos, which have important nutritional value (Journal of Applied and Environmental Biology (2021) 27(06): 1441-1447). Studies have shown that oligomannos can competitively bind to mannosyl lectins, hinder the binding of pathogenic bacteria to lectins, and maintain the balance of human intestinal flora. At present, there have been many reports on mannanases derived from microorganisms and their use in hydrolyzing mannans, but there are no reports on their hydrolysis of citrus peel mannans (Chinese patents CN116286751, CN114437938, etc.). The reason may be that the existing mannanases are difficult to form effective adsorption on the complex structure of citrus peel, and some mannanases are not stable enough in the reaction system, making it difficult to maintain high activity during long-term hydrolysis. The applicant discovered in the study that wild-type Trichoderma sphaerocephala can efficiently degrade citrus peel mannan to prepare oligomannan. Therefore, the applicant studied the mannanase in it through whole genome sequencing, cloning, heterologous expression and protein engineering, and it has good application prospects in the comprehensive utilization of citrus peel. Summary of the invention

[0005] The purpose of the present invention is to provide an amino acid and gene sequence of a mannanase derived from Trisporum sphaerocephalum, a related recombinant expression vector, a recombinant expression strain and its application in transforming citrus peel to prepare oligomannosaccharides with a degree of polymerization of 2-5.

[0006] Further, the mannanase gene is characterized by being one of the following sequences: (1) the amino acid sequence shown in SEQ ID No. 1 in the sequence listing; (2) replacing the tyrosine at position 672 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with alanine; (3) replacing the glycine at position 160 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with leucine, isoleucine, valine or alanine; (4) replacing the glutamic acid at position 379 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with glutamine; (5) replacing the tryptophan at position 322 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with phenylalanine; (6) A combination of the point mutations described in (2) to (5) above;

[0007] Furthermore, the nucleic acid encodes the mannanase according to claim 1 or 2.

[0008] Furthermore, the recombinant expression vector comprises the nucleic acid as claimed in claim 3.

[0009] Furthermore, the recombinant expression transformant comprises the recombinant expression vector as claimed in claim 4.

[0010] Further, the Trisporus sphericalensis mannanase catalyst is any one of the following forms: (1) culturing the recombinant expression transformant according to claim 5, and isolating transformant cells containing the mannanase; (2) culturing the recombinant expression transformant as claimed in claim 5, isolating a crude enzyme liquid containing the mannanase, and drying the obtained crude enzyme powder; (3) culturing the recombinant expression transformant as claimed in claim 5, isolating a pure enzyme liquid containing the mannanase, and drying the obtained pure enzyme powder; (4) culturing the recombinant expression transformant according to claim 5, isolating the mannanase-containing transformant and immobilizing the obtained immobilized enzyme; (5) Cultivating the recombinant expression transformant according to claim 5, isolating and immobilizing transformant cells containing the mannanase.

[0011] Furthermore, the technical route of the conversion process is that the citrus peel is pretreated with steam distillation, and then added to a reaction solution containing Trisporus sphaerocephalus mannanase for reaction for 2-24 hours to obtain a reaction solution containing oligomannosaccharides.

[0012] Furthermore, the pretreatment process is a steam distillation pretreatment at a temperature of 100-110° C. for 0.5-2 hours, and after the treatment, the pretreatment is dried and ground to a particle size of 100-140 meshes.

[0013] Furthermore, the temperature of the conversion process is 40-60°C and the pH is 5.5-9.5.

[0014] Furthermore, in the conversion process, the amount of citrus peel added is 1-10% (mass ratio), and the amount of catalyst (protein) added is 1-10% (mass ratio) of the substrate mass. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the result of protein recombinant expression of mannanase in the present invention.

[0016] Figure 2 This is the optimum temperature measurement result of the mannanase in the present invention.

[0017] Figure 3 This is the optimum pH measurement result of the mannanase in the present invention. DETAILED DESCRIPTION

[0018] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0019]

[0043] "Embodiments" as referred to herein are intended to refer to a particular feature, structure, or characteristic that may be included in at least one implementation of the invention.

[0020] Example 1 Preparation of recombinant mannanase expression vector

[0021] According to the whole genome sequencing results of Trichoderma sphaerocephala ATCC 16958 (J Microbiol Biotechnol (2016) 26 (4): 700-709 and Food Sci Technol Res (2017) 23 (6): 793-800, etc.), the mannanase gene (as shown in Seq ID No. 2) and the expression vector pET-22b (+) were double-digested with restriction endonucleases EcoR I and Hind III, and recovered by agarose gel electrophoresis. The recovered target gene fragments were ligated with T4 DNA ligase, and the whole product (10 μL) was added to 100 μL Escherichia coli DH5α competent cells, and placed on ice for 20 minutes, in a 42°C water bath for 1.5 minutes, and placed on ice for 3 minutes. Add 400 μL LB medium and culture at 37°C shaking table for 60 minutes, centrifuge at 10000 rpm / min for 5 minutes, and spread the precipitate on LB medium containing ampicillin (Amp) resistance. After 16 hours of culture, pick the transformant for sequencing verification. The successfully transformed strain was inoculated in LB medium with Amp resistance, cultured overnight at 37°C shaking table, and the recombinant plasmid was extracted. The recombinant plasmid was added to the competent cells of Escherichia coli BL21 (DE3) for transformation, and the positive clones were screened on the plate to obtain the genetically engineered strain capable of expressing the mannanase gene.

[0022] Example 2 Cultivation of Escherichia coli containing the target gene and heterologous expression of recombinant mannanase

[0023] The recombinant BL21 (DE3) Escherichia coli was inoculated into a 5mL Amp-resistant LB shaking tube for overnight culture. 500μL was inoculated into LB medium with Amp resistance for expansion culture, and cultured in a 37℃ shaking incubator for 3h. When the OD600 value was greater than 0.6, 400μL IPTG (isopropylthiogalactoside, final concentration 0.8mmol / L) was added to the clean bench. Expression was induced in a 20℃ constant temperature shaking incubator for 20h. The bacterial liquid was collected in a 50mL centrifuge tube, centrifuged at 8000rpm / min in a 4℃ refrigerated centrifuge for 10min, the bacteria were collected, washed 3 times with phosphate buffer solution, 3mL of cell lysis solution was added to the bacteria, ultrasonically broken, and the supernatant was collected by centrifugation as a crude mannanase enzyme solution. Further purification was performed using nickel ion purification resin to obtain a pure mannanase enzyme solution.

[0024] Example 3 Determination of oligomannosaccharide content

[0025] Accurately weigh 0.5g of oligomannosaccharide, dissolve it in 100mL acetic acid-sodium acetate buffer (pH 5.0) to prepare oligomannosaccharide stock solution. Pipette 0.00, 2.00, 4.00, 6.00, 8.00, 10.00mL of oligomannosaccharide stock solution in a 50mL volumetric flask, and dilute to the mark with water. Pipette 1.5mL of the diluted oligomannosaccharide stock solution, add 1.5mL of acetic acid-sodium acetate buffer and 2mL of DNS reagent, heat in a boiling water bath for 5min, rinse with tap water to room temperature immediately, and measure the absorbance value at 570nm using an enzyme marker. According to the above values, draw a standard curve. After the actual sample is processed according to the above steps, the content of oligomannosaccharide is calculated according to the standard curve.

[0026] Example 4: Preparation of oligomannosaccharides from citrus peel using recombinant mannanase

[0027] The target gene-containing Escherichia coli was inoculated into a seed culture medium composed of: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, K 2 HPO 4 0.5g / L, MgSO 4 7H 2 O 0.5g / L, FeSO 4 7H 2 O 0.1g / L, 30℃ shaker 200rpm culture for 2 days. The logarithmic phase seed liquid was inoculated into the fermentation medium at a 10% inoculum, which consisted of: citrus peel treated material 100g / L, yeast powder 20g / L, K 2 HPO 4 1g / L, NaCl 1g / L, MgSO 4 7H 2O 0.5g / L, FeSO 4 7H 2 O 0.1g / L, cultured at 30℃ with a shaker at 200rpm for 24 hours, samples were taken every hour to measure the oligomannan content, and the oligomannan production reached 14.2g / L after 16 hours.

[0028]

Claims

1. A mannanase derived from Trichosporonoides oedocephalis, characterized in that It can convert mannan in citrus peel into oligomannosaccharides with a degree of polymerization of 2-5.

2. The Trisporus sphericalensis mannanase according to claim 1, characterized in that is one of the following sequences: (1) the amino acid sequence shown in SEQ ID No. 1 in the sequence listing; (2) replacing the tyrosine at position 672 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with alanine; (3) replacing the glycine at position 160 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with leucine, isoleucine, valine or alanine; (4) replacing the glutamic acid at position 379 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with glutamine; (5) replacing the tryptophan at position 322 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing with phenylalanine; (6) A combination of the point mutations described in (2) to (5) above.

3. An isolated nucleic acid, characterized in that The nucleic acid encodes the mannanase according to claim 1 or 2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 3.

5. A recombinant expression transformant, characterized in that: The recombinant expression transformant comprises the recombinant expression vector according to claim 4.

6. A biocatalyst, characterized in that The Trisporus sphericalensis mannanase catalyst is any one of the following forms: (1) culturing the recombinant expression transformant according to claim 5, and isolating transformant cells containing the mannanase; (2) culturing the recombinant expression transformant as claimed in claim 5, isolating a crude enzyme liquid containing the mannanase, and drying the obtained crude enzyme powder; (3) culturing the recombinant expression transformant as claimed in claim 5, isolating a pure enzyme liquid containing the mannanase, and drying the obtained pure enzyme powder; (4) culturing the recombinant expression transformant according to claim 5, isolating the mannanase-containing transformant and immobilizing the obtained immobilized enzyme; (5) Cultivating the recombinant expression transformant according to claim 5, isolating and immobilizing transformant cells containing the mannanase.

7. A method for preparing oligomannosaccharides using the mannanase of Trichoderma sphaerocephalum as claimed in claim 8, characterized in that: After being pre-treated by steam distillation, the citrus peel is added into a reaction solution containing Trisporus sphaerocephalus mannanase and reacted for 2-24 hours to obtain a reaction solution containing oligomannosaccharides.

8. The method according to claim 7, characterized in that The temperature of the steam distillation pretreatment is 100-110°C, the time is 0.5-2 hours, and after the treatment, it is dried and ground to a particle size of 100-140 meshes.

9. The method according to claim 7, characterized in that The temperature of the conversion process is 40-60°C and the pH is 5.5-9.

5.

10. The method according to claim 7, characterized in that The amount of citrus peel added is 1-10% (mass ratio), and the amount of catalyst (protein) added is 1-10% (mass ratio) of the substrate mass.

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