CN117568422A - Method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate - Google Patents

Method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate Download PDF

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CN117568422A
CN117568422A CN202311263887.9A CN202311263887A CN117568422A CN 117568422 A CN117568422 A CN 117568422A CN 202311263887 A CN202311263887 A CN 202311263887A CN 117568422 A CN117568422 A CN 117568422A
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ribose
allose
hydrolysate
ccdpease
accegi
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许敬亮
吕永坤
董寒玉
赵安琪
熊文龙
张申
阿拉牧
陈黎
崔凤霞
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Zhengzhou University
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Abstract

The invention provides a method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate. The corn stalk hydrolysate is prepared by low-alkali pretreatment and enzyme hydrolysis of corn stalk raw materials, and mainly comprises the following components: d-glucose, D-xylose and L-arabinose. The invention constructs engineering bacteria BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI carrying D-glucose isomerase (AcceGI) gene, D-psicose-3-epimerase (CcDPease) gene and L-rhamnose isomerase (Bs-L-RhI) gene. The bacterium contains an intact pathway for the conversion of D-xylose to D-ribose and D-glucose to D-allose. Experimental results show that the strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI can be used as a whole-cell catalyst to convert D-xylose in corn straw hydrolysate into D-ribose and D-glucose into D-allose. The research provides a new idea for comprehensive utilization of corn stalk hydrolyzed sugar, and further conversion process optimization is expected to greatly improve the comprehensive utilization rate of corn stalk hydrolyzed sugar.

Description

Method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate
Technical Field
The invention relates to the field of biochemical engineering, in particular to a method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate.
Background
Lignocellulose is the most abundant biomass resource on earth, considered as a renewable, environmentally friendly and sustainable carbon resource on earth, mainly referring to non-grain forestry and agricultural residues such as: corn stalks, wheat stalks, rice stalks, and the like. In composition, lignocellulose is mainly composed of three parts of cellulose (38% -50%), hemicellulose (23% -32%) and lignin (15% -30%), cellulose is composed of thousands of D-glucose, is combined by beta-1, 4 bonds, and a plurality of linear cellulose molecules are tightly combined through intermolecular hydrogen bonds and van der Waals bonds to form a highly crystalline superfine fiber structure. Hemicellulose is a heteropolymer composed of several different types of monosaccharides linked by (1, 4) -glycosidic bonds, such as xylose, arabinose, mannose and galactose, and the degradation of lignocellulose is generally carried out by cellulolytic enzymes. Corn stover, one of the lignocellulosic materials, can be hydrolyzed to produce glucose and xylose.
D-ribose is a simple monosaccharide or pentose containing 5 carbon atoms, is an important component substance of genetic material in organisms, namely ribonucleic acid (RNA), exists in all cells, is an important component of nucleotide, various coenzymes and genetic material nucleic acid, is also a structural component of energy substance Adenosine Triphosphate (ATP), and has important physiological functions and application prospects. D-ribose is widely applied to the fields of medicine, food and beverage, nutrition, health care, clinical nutrition and the like. In the field of medicine, D-ribose is an important intermediate and starting material of various kinds of medicines such as nucleosides, antitumor medicines and the like, and nearly 50% of antiviral medicines used clinically at present are nucleoside medicines such as capecitabine, ticagrelor and adefovir, and D-ribose is used as the intermediate and starting material.
The earliest methods for the synthesis of D-ribose involved enzymatic hydrolysis, or chemical synthesis from glucose, arabinose, gluconic acid and xylose, but these methods had the disadvantages of high cost, low conversion rate, complicated purification, easy environmental pollution, etc., and thus have not been commercially successful to a great extent. Biosynthesis of D-ribose occurs in the Pentose Phosphate Pathway (PPP). Deletion of the transketolase gene tkt results in accumulation of D-ribose in a variety of microorganisms including Saccharomyces cerevisiae, E.coli, B.pumilus and B.subtilis. The PPP approach has a plurality of fermentation byproducts, ATP is consumed for synthesizing the D-ribose, the product is difficult to separate, the industrial production is not facilitated, and only the transketolase defective bacillus strain can produce the D-ribose.
D-allose is an important D-type rare sugar with 80% sweetness compared with sucrose, but extremely low calorie, and is an ideal sucrose substitute, and the rare sugar has been demonstrated to exert a number of beneficial physiological functions including anti-tumor, anti-cancer, anti-inflammatory, anti-freeze, anti-osteoporosis, antihypertensive, neuroprotection, immunosuppressant, etc. In addition, it exhibits antioxidant properties by regulating the generation of Reactive Oxygen Species (ROS), and thus D-allose has great potential for clinical application as a pharmaceutical agent.
The synthetic route of D-allose has a chemical method and a biological method, wherein the chemical method usually generates various byproducts, and the harmless treatment of the generated waste is a challenge. Currently, most of the research on D-allose bio-production is based on the izumarig enzymatic cascade as a strategy for the synthesis of rare sugars by enzyme-catalyzed epimerization between monosaccharides. Although reversible epimerization leads to low substrate conversion, this method is still the best choice for preparing rare sugars and has been successfully applied in the industrial production of D-psicose from D-fructose.
Disclosure of Invention
Accordingly, the invention aims to provide a method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate, wherein a co-expression plasmid is constructed by using D-glucose isomerase (AcceGI, SEQ ID NO: 1), D-allose-3-epimerase (CcDPease, SEQ ID NO: 2) and L-rhamnose isomerase (Bs-L-RhI, SEQ ID NO: 3), and engineering bacteria for producing D-ribose are constructed, so that the direct synthesis of D-ribose and D-allose by using low-cost substrate corn straw hydrolysate is realized.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate, wherein the method for synthesizing the D-ribose and the D-allose uses escherichia coli as chassis cells, and a whole cell catalyst is constructed to convert the corn straw hydrolysate into the D-ribose and the D-allose.
Optionally, the preparation method of the corn stalk hydrolysate comprises the following steps: airing corn straw to make the moisture less than or equal to 15%, then crushing the corn straw by a crusher and screening the corn straw by a 60-mesh sieve plate. Straw was mixed with 2% (w/v) NaOH in a 1:20 (w/w) system and autoclaved at 80℃for 2h under normal pressure. Washing the straw residue subjected to alkali steaming with 20% (v/v) acetic acid to neutrality, and drying in an oven at normal pressure and 105 ℃ for later use. 2g of dried straw residues are weighed and placed in a 100mL blue cap bottle, 20mL of acetic acid-sodium acetate buffer solution with the pH value of 4.8 and 10FPU/g of cellulase as a substrate are added, and the blue cap bottle is placed in a constant temperature shaking table with the temperature of 50 ℃ and the speed of 200rpm for enzymolysis for 3 days. And centrifuging the product after enzymolysis, and collecting supernatant to obtain the corn stalk hydrolysate.
Alternatively, the escherichia coli is escherichia coli BL21 (DE 3).
Optionally, the whole cell catalyst construction method comprises the following steps: the D-glucose isomerase-encoding gene (AcceGI, SEQ ID NO: 4) from Acidothermus cellulolyticus B and the D-psicose-3-epimerase-encoding gene (CcDPease, SEQ ID NO: 5) from Clostridium cellulolyticum H B were cloned between BamHI and HindIII sites of vector pET28a (PB) using a one-step cloning kit (Nanjinovirginia Biotech Co., ltd.) respectively to obtain recombinant plasmid pET28a (PB) N-CcDPease-AcceGI; PCR amplification of isomerase-encoding genes: (Bs-L-RhI, SEQ ID NO: 6), pET28a (PB) -CcDPease-AcceGI was digested with NheI, dpnI was digested (Bs-L-RhI, SEQ ID NO: 6), and the digested (Bs-L-RhI, SEQ ID NO: 6) was cloned into the NheI site of vector pET28a (PB) -CcDPease-AcceGI (see Table 1 for primers used) using a one-step cloning kit (Nannonfirazan Biotech Co., ltd.) to obtain recombinant plasmid pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI. And (3) transforming the recombinant plasmid into escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI. The recombinant strain can be used for preparing a whole-cell catalyst, synthesizing D-xylose into D-ribose, and synthesizing D-glucose into D-allose.
Optionally, the conditions for converting the corn stalk hydrolysate into the D-ribose and the D-allose by the recombinant strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI are as follows: the recombinant strain was cultured overnight in LB medium at 37℃and 200rpm, transferred to a 250mL Erlenmeyer flask containing 25mL of fresh LB medium at 2% (v/v) and cultured to logarithmic phase at 37℃and 200 rpm. IPTG was added at a final concentration of 500. Mu.M, and the culture was continued at 25℃and 200rpm for 12 hours to express the gene of the D-ribose synthesis pathway from D-xylose. Centrifuging the induced system at 4deg.C and 4000rpm for 5min, collecting thallus, washing thallus with sterilized ultrapure water to remove culture medium, and re-suspending thallus with 20mM Phosphate Buffer (PBS) with pH of 7.5 to obtain resting cell which can be used as whole cell catalyst for subsequent reaction.
Optionally, the corn stalk hydrolysate has the components of (D-glucose 49.81g/L and D-xylose 15.27 g/L).
Alternatively, the conversion conditions are 65 ℃ for 8-48h.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention uses the cheap corn stalk hydrolysate as a substrate, greatly reduces the production cost of D-ribose and D-allose, and realizes the high-value utilization of lignocellulose raw materials.
(2) Compared with the pentose phosphate way for producing the D-ribose, the invention has single reaction process, no energy consumption and less reaction byproducts, and is convenient for subsequent separation and purification.
(3) Compared with the enzymatic method, the whole cell transformation avoids a series of problems of complicated enzyme purification steps, cofactors and the like, and the reaction condition is mild, so that the method is more suitable for industrial production.
Drawings
FIG. 1 is a schematic diagram of the synthetic pathway of whole cell conversion of corn stalk hydrolysate to D-ribose and D-allose. The pathway consists of D-glucose isomerase, D-psicose-3-epimerase and L-rhamnose isomerase.
FIG. 2 (A) plasmid map of recombinant plasmid pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI. The recombinant plasmid contains a D-glucose isomerase coding gene (AcceGI, SEQ ID NO: 4), a D-psicose-3-epimerase coding gene (CcDPease, SEQ ID NO: 5) and a D-ribose-5-phosphatase isomerase coding gene (Bs-L-RhI, SEQ ID NO: 6), the expression of the genes is controlled by a T7 promoter and a T7 terminator, and the genes are connected in series in a monocistronic mode to form a conversion path from D-xylose to D-ribose and D-glucose to D-psicose. (B) Recombinant strain protein expression profiling
FIG. 3 is a component analysis of the corn stalk hydrolysate. The corn stalk hydrolysate mainly comprises D-glucose, D-xylose and L-arabinose
FIG. 4 ion chromatography analysis of whole cell transformed products. The corn stalk hydrolysate is used as a substrate, and D-ribose and D-allose are synthesized after the whole cell transformation of the recombinant strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI.
FIG. 5 shows the yield map of the transformed D-ribose and D-allose from the whole cell of strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI. The reaction conditions are as follows: the concentration of inducer IPTG is 0.05mM, the induction temperature is 25 ℃, the induction time is 12h, the reaction temperature is 65 ℃, the reaction pH is 7.5, the corn stalk hydrolysate (D-glucose 49.81g/L, D-xylose 15.27 g/L.)
Detailed description of the preferred embodiments
The invention provides a method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate, and the synthesis method can directly convert the corn straw hydrolysate into the D-ribose and the D-allose by constructing recombinant escherichia coli engineering strains.
The method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn stalk hydrolysate and the application thereof provided by the invention are described in detail below with reference to examples, but are not to be construed as limiting the scope of the invention.
Detailed Description
Example 1: preparation of whole cell catalyst
The D-glucose isomerase-encoding gene (AcceGI, SEQ ID NO: 4) from Acidothermus cellulolyticus B and the D-psicose-3-epimerase-encoding gene (CcDPease, SEQ ID NO: 5) from Clostridium cellulolyticum H B were cloned between BamHI and HindIII sites of vector pET28a (PB) using a one-step cloning kit (Nanjinovirginia Biotech Co., ltd.) respectively to obtain recombinant plasmid pET28a (PB) N-CcDPease-AcceGI; PCR amplification of isomerase-encoding genes: (Bs-L-RhI, SEQ ID NO: 6), pET28a (PB) -CcDPease-AcceGI was digested with NheI, dpnI was digested (Bs-L-RhI, SEQ ID NO: 6), and the digested (Bs-L-RhI, SEQ ID NO: 6) was cloned into the NheI site of vector pET28a (PB) -CcDPease-AcceGI (see Table 1 for primers used) using a one-step cloning kit (Nannonfirazan Biotech Co., ltd.) to obtain recombinant plasmid pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI (FIG. 2A). And (3) transforming the recombinant plasmid into escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI. The recombinant strain can be used for preparing a whole-cell catalyst, and the corn stalk hydrolysate is converted into D-ribose and D-allose.
TABLE 1 primers used in the present invention
Recombinant E.coli BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI was cultured overnight in LB medium at 37℃and 200rpm, transferred to a 250mL Erlenmeyer flask containing 25mL fresh LB medium at 2% (v/v), and cultured to logarithmic phase at 37℃and 200 rpm. IPTG with a final concentration of 500. Mu.M was added, and the culture was continued at 25℃and 200rpm for 12 hours to express the genes of the pathway for synthesizing D-ribose from D-xylose and D-allose from D-glucose.
Protein gel electrophoresis (SDS-PAGE) was used to examine the protein expression. The protein gel electrophoresis test method for protein expression comprises the following steps: the protein gel electrophoresis model is a Siemens flight polyacrylamide protein gel electrophoresis apparatus, and a voltage of 150V is used for 30min. The protein gel electrophoresis analysis result shows that the D-xylose isomerase protein: acceGI, D-psicose-3-epimerase protein: ccDPEase, D-ribose-5-phosphate isomerase: the corresponding molecular weights of Bs-L-RhI are 53.0kDa, 36.3kDa, 41.5kDa, respectively.
The blank (E.coli BL21/pET28a (PB) N) had no obvious protein band; the experimental group had a distinct protein band (fig. 2B). Indicating that the three genes are expressed.
Centrifuging the induced system at 4deg.C and 4000rpm for 5min, collecting thallus, washing thallus with sterilized ultrapure water to remove culture medium, and re-suspending thallus with 20mM Phosphate Buffer (PBS) with pH of 7.5 to obtain resting cell.
Example 2: preparation of straw hydrolysate
Airing corn straw to make the moisture less than or equal to 15%, then crushing the corn straw by a crusher and screening the corn straw by a 60-mesh sieve plate. Straw was mixed with 2% (w/v) NaOH in a 1:20 (w/w) system and autoclaved at 80℃for 2h under normal pressure. Washing the straw residue subjected to alkali steaming with 20% (v/v) acetic acid to neutrality, and drying in an oven at normal pressure and 105 ℃ for later use. 2g of dried straw residues are weighed and placed in a 100mL blue cap bottle, 20mL of acetic acid-sodium acetate buffer solution with the pH value of 4.8 and 10FPU/g of cellulase as a substrate are added, and the blue cap bottle is placed in a constant temperature shaking table with the temperature of 50 ℃ and the speed of 200rpm for enzymolysis for 3 days. And centrifuging the product after enzymolysis, and collecting supernatant, namely corn stalk hydrolysate, which can be used as a substrate for producing D-ribose and D-allose.
The corn stalk hydrolysate is analyzed, and the detection result is shown in figure 3 by using ion chromatography, wherein the corn stalk hydrolysate contains 51.15g/L of D-glucose, 15.683g/L of D-xylose and 2.305g/L of L-arabinose.
Example 3: preparation of D-ribose and D-allose from corn stalk hydrolysate
On the basis of example 2, recombinant E.coli BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI was cultured overnight in LB medium at 37℃and 200rpm, transferred to a 250mL Erlenmeyer flask containing 25mL of fresh LB medium at 2% (v/v), and cultured to logarithmic phase at 37℃and 200 rpm. IPTG with a final concentration of 500. Mu.M was added, and the culture was continued at 25℃and 200rpm for 12 hours to express the genes of the pathway for synthesizing D-ribose from D-xylose and D-allose from D-glucose. Centrifuging the induced system at 4deg.C and 4000rpm for 5min, collecting thallus, washing thallus with sterilized ultrapure water to remove culture medium, and re-suspending thallus with 20mM Phosphate Buffer (PBS) with pH of 7.5 to obtain resting cell.
Adding corn stalk hydrolysate with final concentration (D-glucose 49.81g/L, D-xylose 15.27 g/L) and 1mM CoCl into the reaction system 2 20mM MgCl 2 The reaction was carried out at 65 ℃. The reaction supernatant was taken and detected by ion chromatography. Coli BL21/pET28a (PB) N with empty plasmid pET28a (PB) N was used as a blank, otherwise the same procedure was followed.
The method for analyzing the D-ribose by ion chromatography is as follows: ion chromatograph model is the Siemens flight ICS-6000 high pressure ion chromatograph, the detector used is electrochemical detector, chromatographic column model is PA-1, mobile phase A is ultrapure water, mobile phase B is 200mM NaOH aqueous solution, gradient elution is (B%): 0min40%,15min40%,15.1min 100%,25min100%,25.1min 40%,35min40%, constant flow rate 0.8mL/min, sample injection amount 25 μl, column temperature box and detector temperature of 30deg.C. Ion chromatography analysis results (shown in FIG. 4).
The results indicate that when reacting to 48h (as shown in FIG. 5). The bacterial strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI takes corn straw hydrolysate as a substrate (D-glucose 49.81g/L, D-xylose 15.27 g/L) to catalyze and synthesize 2.66 g/LD-allose, the conversion rate is 5.34%, the catalytic synthesis rate is 2.23 g/LD-ribose, and the conversion rate is 14.6%.
The method for synthesizing the D-ribose and the D-allose by using the whole cells of the corn straw hydrolysate, which is constructed by the invention, has great application potential.
The foregoing is merely an alternative embodiment of the invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the principles of the invention, and such modifications and variations should also be considered as being within the scope of the invention.

Claims (7)

1. A method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn stalk hydrolysate. The method is characterized in that corn straw hydrolysate is used as a substrate, escherichia coli is used as chassis cells, and D-glucose isomerase (AcceGI, SEQ ID NO: 1), D-psicose-3-epimerase (CcDPease, SEQ ID NO: 2) and L-rhamnose isomerase (Bs-L-RhI, SEQ ID NO: 3) are utilized to directly convert the corn straw hydrolysate into D-ribose and D-psicose (the reaction process is shown in figure 1).
2. The transformation pathway of claim 1, wherein (1) the gene encoding D-glucose isomerase derived from Acidothermus cellulolyticus B (AcceGI, SEQ ID NO: 4); (2) The D-psicose-3-epimerase encoding gene (CcDPease, SEQ ID NO: 5) derived from Clostridium cellulolyticum H10; (3) The gene encoding L-rhamnose isomerase derived from Bacillus subtilis 168 (Bs-L-RhI, SEQ ID NO: 6).
3. The method for synthesizing D-ribose according to claim 1, wherein the recombinant plasmid pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI containing all the genes is constructed by using plasmid pET28a (PB) N (with the sequence shown as SEQ ID NO: 7) as the expression vector of the transformation path.
4. According to claim 1 and claim 3, the recombinant plasmid is transformed into the escherichia coli BL21 (DE 3) by taking the escherichia coli BL21 (DE 3) as an expression host, so as to obtain the engineering strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI containing all the path genes.
5. The corn stalk hydrolysate according to claim 1, wherein corn stalk is dried to a moisture content of 15% or less, and then crushed by a crusher and screened by a 60-mesh screen. Straw was mixed with 2% (w/v) NaOH in a 1:20 (w/w) system and autoclaved at 80℃for 2h under normal pressure. Washing the straw residue subjected to alkali steaming with 20% (v/v) acetic acid to neutrality, and drying in an oven at normal pressure and 105 ℃ for later use. 2g of dried straw residues are weighed and placed in a 100mL blue cap bottle, 20mL of acetic acid-sodium acetate buffer solution with the pH value of 4.8 and 10FPU/g of cellulase as a substrate are added, and the blue cap bottle is placed in a constant temperature shaking table with the temperature of 50 ℃ and the speed of 200rpm for enzymolysis for 3 days. Centrifuging the product after enzymolysis, collecting supernatant, namely corn straw hydrolysate, and analyzing the components of the corn straw hydrolysate by using ion chromatography, wherein the results show that the components of the corn straw hydrolysate mainly comprise: the D-glucose content is 51.15g/L, the D-xylose content is 15.683g/L, and the L-arabinose content is 2.305g/L. D-glucose and D-xylose in the corn stalk hydrolysate can be used as substrates for producing D-ribose and D-allose respectively.
6. The method for whole cell synthesis of D-ribose and D-allose according to claim 1 and the recombinant engineering strain according to claim 4, characterized in that the transformation conditions used are as follows: (1) Culturing recombinant engineering bacteria of escherichia coli in an LB culture medium until OD600 = 0.6-0.8, adding isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.5mM, and inducing gene expression for 12h under the conditions of 25 ℃ and 200 rpm; (2) Centrifuging at 4deg.C and 4000rpm to collect bacterial cells, and sterilizing with ultrapure waterWashing the thalli to remove the culture medium, and then using a Phosphate Buffer Solution (PBS) with the pH of 7.5 and 20mM to resuspend the thalli to obtain resting cells; (3) Corn stalk hydrolysate is used as substrate, and CoCl with the final concentration of 1mM is added 2 And 20mM MgCl 2 As catalytic ion, reacting at 65 ℃ for 8-48h; (4) The yields of D-ribose and D-allose were analyzed separately using ion chromatography.
7. According to claim 1 and claim 6, when reacting for 48 hours, the engineering strain BL21/pET28a (PB) N-CcDPease-AcceGI-Bs-L-RhI takes corn stalk hydrolysate as a substrate to catalyze and synthesize 2.66g/L D-allose with a conversion rate of 5.34 percent and 2.23g/L D-ribose with a conversion rate of 14.6 percent (D-glucose 49.81g/L and D-xylose 15.27 g/L).
CN202311263887.9A 2023-09-27 2023-09-27 Method for synthesizing D-ribose and D-allose by comprehensively utilizing whole cells of corn straw hydrolysate Pending CN117568422A (en)

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