Method for preparing mannose from konjac flying powder

Abstract

The invention discloses a method for preparing mannose from konjak flying powder, which comprises the following steps: treating the konjak flying powder with amylase and glucoamylase to obtain glucose, treating the glucose with glucose isomerase to obtain fructose, and treating the fructose with mannose isomerase to obtain the mannose. The mannose isomerase is expressed by an optimized coding gene, and the sequence of the optimized coding gene is as shown in SEQ ID NO. 1.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing D-mannose by taking konjac fly flour as a raw material, so as to realize the high value of konjac waste fly flour. Background Art

[0002] The tubers of konjac mainly contain glucomannan, in addition to starch, alkaloids, cellulose and minerals. Konjac fly flour is the leftover material of konjac refined flour, the main processed product of konjac, accounting for 30% to 40% of the refined flour mass. The carbohydrate content in konjac fly flour is about 50%, including 22.81% starch, 8.95% water-soluble sugar, 2.46% water-soluble reducing sugar (water-soluble sugar refers to glucose and mannose), and 1.67% cellulose. Its starch content is comparable to that of potatoes (21.0%) and sweet potatoes (26.4%), and has a high utilization value. At present, the utilization of konjac is mainly in the form of refined flour. Since konjac fly flour contains compounds such as alkaloids and tannins, and contains astringent substances, it has poor palatability and has not been directly utilized by the food industry. A very small amount of fly flour is sold as low-priced feed or desiccant, and most of it is discarded. This discarded powdery substance also pollutes the environment. That is, the current utilization rate of konjac fly flour is extremely low. Therefore, it is of great practical significance to utilize my country's abundant konjac fly flour resources and turn waste into treasure.

[0003] Mannose is a natural hexose and an isomer of glucose. It often appears as a monomer of the polysaccharide mannan. Studies have confirmed that mannose has broad application prospects in the food, cosmetics, and pharmaceutical industries. Mannose can be used as a sweetener for food and is also an effective animal feed additive that can inhibit the growth of intestinal pathogens. In medicine, it has health care effects, such as prebiotic and anti-inflammatory effects, anti-diabetic function, prevention of urinary tract infections, and treatment of phosphomannose isomerase deficiency. In addition, it is also a key component or intermediate for the synthesis of anti-tumor drugs, immunostimulants, auxiliary moisturizers, mannitol, and vitamins.

[0004] At present, the production methods of D-mannose include natural extraction, chemical synthesis and biotransformation. However, due to the high crystallinity and polymerization in the cell walls of the extracted plants, the natural extraction method requires strict conditions such as high temperature to extract D-mannose from mannan-containing materials using acid hydrolysis and thermal hydrolysis, which greatly increases the extraction cost. The chemical synthesis of D-mannose involves complex reactions, which are usually carried out under high temperature and strong acidic environment, and require the participation of one or more catalysts, which increases the production of by-products and harmful pollutants during the reaction. The biotransformation method is increasingly receiving widespread attention due to its mild reaction conditions and fewer harmful by-products. At present, the isomerization reaction can produce higher-value sugars by converting cheaper monosaccharides, so it can be used as an alternative method for industrial production of important sugars.

[0005] In summary, using konjac fly starch, the byproduct of konjac processing, as raw material and converting it into high-value mannose through biological enzymatic hydrolysis is a reliable way to realize the resource utilization of konjac and promote the healthy development of the konjac industry. At present, there is no relevant research on the efficient conversion of konjac fly starch into high-value D-mannose. Summary of the invention

[0006] The present invention aims to provide a method for preparing mannose from konjac fly flour in view of the defects of effective development and utilization of components in konjac fly flour and the above-mentioned deficiencies in the methods for preparing mannose in the prior art.

[0007] In order to achieve the above object, the technical solution provided by the present invention is:

[0008] The method for preparing mannose from konjac fly flour is to treat the konjac fly flour with amylase and saccharifying enzyme to obtain glucose, then treat the glucose with glucose isomerase to obtain fructose, and then treat the fructose with mannose isomerase to obtain mannose; the mannose isomerase is expressed by an optimized encoding gene, and the optimized encoding gene sequence is shown in SEQ ID NO.1.

[0009] Preferably, the method specifically comprises the following steps:

[0010] (1) dissolving konjac powder in water, and placing in a boiling water bath and heating for 25 min until gelatinization, cooling to 60° C., adjusting the pH value to 6, then adding a calcium chloride solution having a calcium chloride concentration of 0.01 mol / L (the presence of calcium ions can keep the α-amylase active and stable during the hydrolysis process), and then adding 2000 U / g of amylase to make the enzyme concentration in the reaction system reach 10 U / mL, stirring and mixing, and then placing in a 90° C. constant temperature water bath for 1 min to obtain a reaction solution A; the mass percentage of the konjac powder in water is 30%, the volume percentage of the calcium chloride solution and water is 0.2%, and the mass percentage of the amylase and water is 0.5%;

[0011] (2) adjusting the pH value of the reaction solution A to 4.2-4.5 with hydrochloric acid, and rapidly cooling the reaction solution A to 60° C., then adding 100,000 U / mL of saccharifying enzyme, stirring and mixing evenly, and keeping the mixture at 60° C. for 1 hour to obtain a glucose solution; the volume percentage of the saccharifying enzyme to the reaction solution A is 0.5%;

[0012] (3) adjusting the pH value of the glucose solution to 8-9 with a dilute alkaline solution, standing for 0.5-1h, and obtaining a mixture of glucose and mannose; adding a mixture of glucose isomerase and mannose isomerase at a weight of 0.05% of the mixed solution, adjusting the temperature to 25°C and the pH value to 8, and reacting for 0.5-1h to obtain a reaction solution B, wherein the mass ratio of the glucose isomerase to the mannose isomerase is 1:3 to 1:7; performing quantitative and qualitative analysis of the mannose in the reaction solution B by high performance liquid chromatography, separating and purifying the reaction solution B through a six-column sequential simulated moving bed filled with a calcium type cationic ion resin as a separation material, and obtaining a mannose-rich component A and other components B; and subjecting component A to centrifugal spray drying to obtain a mannose powder, wherein the centrifugal spray drying parameters are: an inlet air temperature of 100-140°C, an outlet air temperature of 30-60°C, and a feed mass percentage concentration of 60%.

[0013] Preferably, the dilute alkaline solution in step (3) is a 0.05 M glycine-sodium hydroxide buffer.

[0014] More preferably, in step (3), the pH value of the glucose solution is adjusted to 8.5 using 0.05 M glycine-sodium hydroxide buffer.

[0015] Preferably, the mannose isomerase in step (3) is prepared based on the amino acid sequence of the D-MIase gene of Bifidobacterium (NCBI accession number: WP_010080894.1), and then the coding gene is optimized by codons and artificially synthesized. The synthesized gene is inserted into the pCold-TF expression vector by homologous recombination using ECoR I restriction endonuclease to construct the recombinant plasmid pColdTF-BifiMIaseA, and the mannose isomerase expressed in Escherichia coli BL21 is introduced; the optimized coding gene sequence is shown in SEQ ID NO.1.

[0016] Preferably, the mass ratio of glucose isomerase to mannose isomerase in step (3) is 1:5.

[0017] Preferably, the other component B obtained in step (3) is concentrated and crystallized to separate glucose and fructose.

[0018] Preferably, the mannose is D-mannose.

[0019] The present invention will be further described below:

[0020] In a dilute alkaline solution, glucose can be converted through an enol intermediate to obtain a mixture containing glucose, mannose and fructose, and this conversion is reversible between the two, and finally reaches a dynamic equilibrium. It is conventionally known to those skilled in the art that the conversion of glucose to fructose and mannose is more obvious when sodium hydroxide is used as a pH regulator and the pH value is greater than 10. However, the present invention creatively uses the treated fructose and glucose mixture, and selects a specific pH regulator (0.05M glycine-sodium hydroxide buffer). It is found that in this case, it can make good use of the reversible conversion between the three to obtain a higher content of mannose, and cooperate with subsequent enzymatic hydrolysis treatment to convert fructose and glucose into mannose to the greatest extent.

[0021] The purity of the mannose product obtained by the method of the invention reaches more than 99%, realizing the high-value utilization of konjac waste fly powder, and has the advantages of high comprehensive utilization rate and reduced pollution. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, but not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are conventional biochemical reagents and can be obtained from commercial sources unless otherwise specified.

[0023] Embodiment 1 Konjac fly powder is degraded into glucose

[0024] 1. Konjac fly powder is diluted with water, 30g konjac fly powder is weighed and dissolved in 100mL water, placed in a boiling water bath and heated for 25min, gelatinized, cooled to 60°C, adjusted pH to 6 (pH meter), added 0.01mol / L calcium chloride 0.2mL, added 0.5g amylase (2000U / g amylase), the enzyme concentration of the reaction solution reached 10U / mL, stirred and mixed, maintained 1min in 90°C constant temperature water bath, and obtained reaction solution A;

[0025] 2. The pH of the reaction solution A obtained in step 1 is adjusted to 4.2-4.5 with hydrochloric acid, and the temperature is rapidly lowered to 60° C.; then saccharifying enzyme (enzyme solution with an enzyme activity of 100,000 / mL, 0.5% of the volume of the reaction solution A) is added, stirred and mixed evenly, and kept at 60° C. for 1 hour to obtain a glucose solution;

[0026] Example 2 Transformation and efficient expression of mannose isomerase gene

[0027] 1. Obtaining the mannose isomerase gene and constructing the vector

[0028] The putative amino acid sequence of D-MIase from Bifidobacterium (NCBI accession number: WP_010080894.1) was retrieved from the GeneBank database. The coding gene was then obtained by codon optimization, and the gene sequence had Escherichia coli preferred codons and was artificially synthesized. The sequence of the mannose isomerase gene is shown in SEQ ID NO.1. Subsequently, the synthesized gene was inserted into the pCold-TF plasmid vector using ECoR I restriction endonuclease by homologous recombination to construct the recombinant plasmid pColdTF-BifiMIaseA.

[0029] 2. Transformation of mannose isomerase gene

[0030] After ice bathing the competent EP tube containing E coli BL21 (DE3) for 10 minutes, add 1 μL of mannose isomerase recombinant plasmid pColdTF-BifiMIaseA, ice bath for 15-30 minutes, heat shock at 42℃ for 90 seconds, ice bath for 2 minutes, add 500 μL LB liquid medium, culture at 37℃ for 40-50 minutes, centrifuge, discard the supernatant, keep 50 μL of bacterial solution, mix well and spread on LB plate containing 20 μg / mL kanamycin resistance. Pick a single white colony in 500 μL LB liquid medium with corresponding resistance, shake at 200rpm, 37℃ for 12 hours, identify positive clones by bacterial solution PCR, and detect by electrophoresis. The obtained correct recombinant E. coli was named BL21-BifiMIaseA.

[0031] 3. Expression and purification of mannose isomerase gene in Escherichia coli

[0032] The activated strain BL21-BifiMIaseA carrying the recombinant plasmid was inoculated into a new 50 mL LB liquid medium, 50 μL of the corresponding antibiotics were added, and after culturing to the logarithmic phase, it was placed on a shaker at 15°C for cold shock for 30 min. IPTG was then added and induced at 15°C for 24 h. The bacteria were collected by centrifugation, 5 mL of HEPES buffer was added, and the cells were broken by an ultrasonic disruptor. The supernatant and precipitate were collected by centrifugation, and the supernatant was the crude enzyme solution. The expression of the target gene was analyzed by SDS-PAGE.

[0033] Purification: The supernatant was heat treated in a 70°C water bath for 20 minutes, centrifuged at 4°C and 20,000 r / min for 45 minutes, and the supernatant was obtained to obtain mannose isomerase. After purification, 12% protein gel was prepared and SDS-PAGE electrophoresis was performed.

[0034] Example 3 Conversion of glucose into mannose

[0035] Take the glucose solution in Example 1, adjust the pH to 8.5 (pH meter) with glycine-sodium hydroxide buffer (0.05M), and stand for 0.5-1 hour to obtain a glucose-mannose mixed solution; add a mixture of glucose isomerase and the purified mannose isomerase successfully expressed in Example 2 (mass ratio 1:5) according to 0.05% of the weight of the mixed solution, and adjust the temperature to 25°C and pH8 accordingly, and react for 0.5-1 hour; quantitative and qualitative analysis of mannose is performed by high performance liquid chromatography, and the enzymatic hydrolyzate of the reaction is separated and purified by a six-column sequential simulated moving bed filled with a calcium-type cationic ion resin as a separation material to obtain a mannose-rich component A and other components B; component A is subjected to freeze centrifugal drying to obtain mannose powder, and the centrifugal spray drying parameters are: inlet air temperature 100-140°C, outlet air temperature 30-60, and feed mass percentage concentration of 60%. The purity of mannose powder and the total yield of the product were measured, and the purity of the obtained D-mannose finished product reached more than 99%, and the total yield of the product reached 42%.

Claims

1. A method for preparing mannose from konjac fly starch, characterized in that: The method comprises the following steps: treating konjac fly flour with amylase and saccharifying enzyme to obtain glucose, then treating glucose with glucose isomerase to obtain fructose, and then treating fructose with mannose isomerase to obtain mannose; the mannose isomerase is expressed by an optimized coding gene, and the sequence of the optimized coding gene is shown in SEQ ID NO.

1.

2. The method for preparing mannose from konjac fly starch as claimed in claim 1, characterized in that: The method specifically comprises the following steps: (1) Dissolve konjac powder in water and heat in a boiling water bath for 25 minutes until gelatinization, cool to 60°C, adjust the pH value to 6, then add a calcium chloride solution with a calcium chloride concentration of 0.01 mol / L, and then add 2000 U / g of amylase to make the enzyme concentration in the reaction system reach 10 U / mL, stir and mix evenly, and place in a constant temperature water bath at 90°C for 1 minute to obtain a reaction solution A; the mass percentage of the konjac powder in water is 30%, the volume percentage of the calcium chloride solution and water is 0.2%, and the mass percentage of the amylase and water is 0.5%; (2) adjusting the pH value of the reaction solution A to 4.2-4.5 with hydrochloric acid, and rapidly cooling the reaction solution A to 60° C., then adding 100,000 U / mL of saccharifying enzyme, stirring and mixing evenly, and keeping the mixture at 60° C. for 1 hour to obtain a glucose solution; the volume percentage of the saccharifying enzyme to the reaction solution A is 0.5%; (3) adjusting the pH value of the glucose solution to 8-9 with a dilute alkaline solution, and standing for 0.5-1h to obtain a mixed solution of glucose and mannose; adding a mixture of glucose isomerase and mannose isomerase to the mixed solution at a weight of 0.05% of the mixed solution, adjusting the temperature to 25°C and the pH value to 8, and reacting for 0.5-1h to obtain a reaction solution B, wherein the mass ratio of the glucose isomerase to the mannose isomerase is 1:3 to 1:7; performing quantitative and qualitative analysis of the mannose in the reaction solution B by high performance liquid chromatography, separating and purifying the reaction solution B by passing through a six-column sequential simulated moving bed filled with a calcium type cationic ion resin as a separation material to obtain a mannose-rich component A and other components B; and subjecting component A to centrifugal spray drying to obtain a mannose powder, wherein the centrifugal spray drying parameters are: an inlet air temperature of 100-140°C, an outlet air temperature of 30-60°C, and a feed mass percentage concentration of 60%.

3. The method for preparing mannose from konjac fly flour as claimed in claim 2, characterized in that, The dilute alkaline solution in step (3) is a 0.05 M glycine-sodium hydroxide buffer.

4. The method for preparing mannose from konjac fly flour as claimed in claim 3, characterized in that: In step (3), the pH value of the glucose solution is adjusted to 8.5 using 0.05 M glycine-sodium hydroxide buffer.

5. The method for preparing mannose from konjac fly starch as claimed in claim 2, characterized in that, The mannose isomerase in step (3) is prepared based on the inferred amino acid sequence of the D-MIase gene of Bifidobacterium, and then the coding gene is optimized by codons and artificially synthesized. The synthesized gene is inserted into the pCold-TF expression vector by homologous recombination using ECoR I restriction endonuclease to construct a recombinant plasmid pColdTF-BifiMIaseA, and the mannose isomerase expressed in Escherichia coli BL21 is introduced; the optimized coding gene sequence is shown in SEQ ID NO.

1.

6. The method for preparing mannose from konjac fly starch as claimed in claim 5, characterized in that: The mass ratio of glucose isomerase to mannose isomerase in step (3) is 1:

5.

7. The method for preparing mannose from konjac fly starch as claimed in claim 2, characterized in that: The other component B obtained in step (3) is concentrated and crystallized to separate glucose and fructose.

8. The method for preparing mannose from konjac fly starch according to any one of claims 1 to 6, characterized in that: The mannose is D-mannose.