A method for improving the efficiency of enzymatic preparation of L-rhamnose by regulating the substrate micellar state

Abstract

This invention discloses a method for improving the efficiency of enzymatic preparation of L-rhamnose by controlling the micellar state of the substrate. The method involves dissolving and diluting the rhamnolipid substrate using a pretreatment medium to dissociate the rhamnolipid micelle structure, obtaining a highly dispersed substrate pretreatment solution. This pretreatment solution is then introduced into an acidic or neutral reaction system containing α-L-rhamnosidase for enzymatic hydrolysis, releasing free L-rhamnose. This achieves a synergistic effect in promoting micellar dissociation and enzymatic hydrolysis efficiency. Under optimal conditions, the enzymatic hydrolysis efficiency of L-rhamnose increased from 10.65% in the pure water treatment control group to 30.25%, an increase of 1.84 times. Further optimization of the hydrolysis conditions resulted in a final conversion rate of 97.78% for the rhamnolipid substrate. This method effectively overcomes the problem of rhamnolipid micelles hindering enzymatic reactions, providing a theoretical basis and technical foundation for the green preparation of L-rhamnose.

Description

Technical Field

[0001] This invention belongs to the field of biomanufacturing and enzyme catalysis technology, specifically involving a pretreatment process that significantly improves the enzymatic hydrolysis conversion efficiency by regulating the micellar dissociation state of rhamnolipid substrates through physical-chemical means. Background Technology

[0002] L-Rhamnose, a rare natural deoxyhexose, is an indispensable raw material in food sweeteners, pharmaceutical synthesis intermediates (such as precursors for cardiovascular and antitumor drugs), and high-end daily chemical products. Currently, L-Rhamnose production mainly relies on strong acid and alkali hydrolysis extraction from natural plants (such as oak bark). This traditional chemical method suffers from environmental and economic problems, including crude processes, numerous side reactions, large amounts of acid and alkali waste, and high purification costs. Direct microbial fermentation for the synthesis of free rhamnose faces bottlenecks such as long metabolic pathways, severe feedback inhibition, and extremely low yields. In contrast, using rhamnolipids derived from biofermentation as precursors and employing specific α-L-rhamnosidase for targeted biocatalytic hydrolysis offers significant advantages, including mild reaction conditions, fewer byproducts, and environmental friendliness. However, a significant physical bottleneck hinders industrial application in actual enzymatic conversion processes: due to their amphiphilic structure, rhamnolipid molecules readily self-assemble into large and dense micelle structures in aqueous systems. This self-aggregation effect buries the glycosidic bonds within the macromolecule deep within the micelle core or tight interface, preventing exogenous α-L-rhamnosidases (such as GH106 family enzymes) from effectively contacting the substrate (i.e., extremely poor "substrate accessibility"). This results in a conversion rate typically below 11% in conventional aqueous systems. Therefore, designing a safe, efficient, and enzyme-indestructible substrate pretreatment process to completely dissociate rhamnolipid micelles and release reaction sites has become a key technological gap in overcoming the efficiency bottleneck of enzymatic L-rhamnose preparation. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing technologies where the self-aggregation of rhamnolipid substrates to form tight micelles hinders enzyme molecules from contacting substrate binding sites, resulting in low L-rhamnose hydrolysis conversion. This invention provides a highly efficient and green substrate micelle regulation pretreatment method that can dissociate substrate molecules from a large-size micelle state into highly dispersed monomers or small molecule aggregates, thereby increasing the hydrolysis efficiency by 1.07 to 1.84 times.

[0004] The technical solution adopted in this invention is as follows: A method for improving the efficiency of enzymatic preparation of L-rhamnose by controlling the micelle state of the substrate, the method comprising the following steps: (1): Substrate dissociation pretreatment: Rhamnollipid is dissolved in a pretreatment medium for predissociation to obtain a highly dispersed substrate pretreatment solution; the pretreatment medium is selected from either methanol aqueous solution or alkaline buffer solution; (2): Enzymatic hydrolysis reaction: The substrate pretreatment solution obtained in step (1) is introduced into the enzymatic hydrolysis reaction system, α-L-rhamnosidase is added, and the reaction is carried out by shaking to release free L-rhamnose.

[0005] After the reaction was completed, the content of L-rhamnose released in the reaction solution was quantitatively determined by 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization high performance liquid chromatography (HPLC), and the conversion rate was calculated.

[0006] Rhamnol was diluted to a concentration of 5-15 g / L and then pre-dissolved. When the pretreatment medium is a methanol-water solution, its volume fraction is 10%-70%; preferably 10%-40%; and most preferably 40%. In this case, the enzymatic hydrolysis system in step 2) is at pH 4.5-5.5 and temperature 35-45℃, and the enzymatic hydrolysis reaction is carried out by shaking.

[0007] When the pretreatment medium is an acid-base buffer solution, the acid-base buffer solution is a citrate-phosphate buffer solution with a pH of 3-8, or a Tris-HCl buffer solution with a pH of 8-9, or a glycine-NaOH buffer solution with a pH of 9-12.

[0008] Preferably, a glycine-NaOH buffer solution with a pH of 9-12 is used; More preferably, it is a glycine-NaOH buffer solution with pH 11.

[0009] Using a pH 11.0 buffer solution to dissolve rhamnolipids and introducing the alkaline-soluble substrate into the enzymatic hydrolysis system will inevitably cause a change in the pH of the system, which will cause α-L-rhamnosidase (RHA-Bra) to deviate from its optimal catalytic conditions. In this case, the reaction system is preferably a citrate-phosphate buffer system with a pH of 3 to 7; more preferably, a citrate-phosphate buffer system with a pH of 4 is selected.

[0010] Rhamnolipin was dissolved in citrate-phosphate buffer (pH 3-8), Tris-HCl buffer (pH 8-9), and glycine-NaOH buffer (pH 9-12) at different pH values. Under higher pH conditions, the hydrophilic terminal carboxyl group (-COOH) of the rhamnolipin molecule completely dissociated into a carboxylic acid anion (-COO). - The molecules generally carry a negative charge on their surface. Due to the electrostatic repulsion between like charges, the micelle structure of rhamnolipids is effectively disrupted, making the monomers easier for enzymes to recognize and act on, thereby greatly improving the hydrolysis efficiency.

[0011] The final concentration of the substrate rhamnolipid in the reaction system was controlled to be 0.2 g / L to 0.5 g / L.

[0012] The α-L-rhamnosidase is an enzyme capable of specifically hydrolyzing the α-L-rhamnosidic bond at the end of natural glycosides, catalyzing the shedding of bound rhamnosine to generate free rhamnosine; preferably, it is RHA-Bra from the GH106 family; RHA-Bra from the GH106 family; enzyme dosage is 1000 U / mL to 25000 U / mL.

[0013] The preferred reaction temperature for step 2) is 35°C, and the enzyme dosage is 25000 U / mL.

[0014] Beneficial effects:

[0015] This invention breaks down the inherent hydrophobic interactions and steric hindrance between rhamnolipid molecules through targeted pretreatment of the substrate. Under optimal conditions of methanol pretreatment (40%) and alkaline pretreatment (pH 11), the enzymatic hydrolysis efficiency of L-rhamnose jumped from 10.65% in the pure water control group to 22.53% (an increase of 1.12 times) and 30.25% (an increase of 1.84 times), respectively.

[0016] This invention employs a dilution strategy that combines high-concentration pre-dissolution with low-concentration reaction. The medium-to-high concentration methanol (e.g., 40%) or strong base (pH 11) used in the pretreatment is greatly diluted and neutralized after entering the weakly acidic main reaction system, thereby protecting the protein spatial conformation of α-L-rhamnosidase and achieving a perfect balance between disrupting substrate micelles and maintaining enzyme activity.

[0017] Green, economical, and easy to scale up industrially: The methanol and alkaline buffer solutions used are inexpensive and can be easily recovered through simple distillation. No complex toxic additives are introduced, which is in line with the industrial principles of green manufacturing and high-value utilization. Attached Figure Description

[0018] Figure 1: Schematic diagram of the formation of rhamnolipin micelles and their dissociation by methanol and pH (a. molecular structure; b. self-assembled micelles; c. dissociation by methanol; d. dissociation by pH).

[0019] Figure 2 α-L-rhamnosidase activity in methanol and ethanol.

[0020] Figure 3: Effect of adding different concentrations of methanol to pretreat the substrate on the hydrolysis efficiency of α-L-rhamnosidase (RHA-Bra).

[0021] Figure 4The effect of pretreatment of substrates with buffer solutions of different pH values ​​on the hydrolysis efficiency of α-L-rhamnosidase (RHA-Bra) is shown in the figure.

[0022] Figure 5 The effect of different pH values ​​on the catalytic efficiency of RHA-Bra.

[0023] Figure 6 The effect of reaction temperature on the catalytic efficiency of RHA-Bra.

[0024] Figure 7 Effects of different enzyme dosages on the catalytic efficiency of RHA-Bra. Detailed Implementation

[0025] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0026] The recombinant Pichia pastoris engineered strain and its expressed α-L-rhamnosidase, disclosed in the Chinese patent (CN119464254A) authorized by our laboratory, with its amino acid sequence as shown in SEQ ID NO.1 and the nucleotide sequence encoding the α-L-rhamnosidase as shown in SEQ ID NO.2, are used as the preferred source of the enzyme preparation of the present invention. The preparation method is described in the aforementioned patent.

[0027] Unless otherwise specified, all experimental materials used in the embodiments of this invention can be obtained from commercially available sources.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Rhamnose glycolipids (≥95%) were obtained from Xi'an Ruijie Biotechnology Co., Ltd.

[0031] Rhamnolipid concentration was determined using the anthrone-sulfuric acid method, and the sulfuric acid-anthrone colorimetric method was used to quantitatively determine the rhamnolipids and their theoretical maximum rhamnose yield in the samples. The specific procedures are as follows: (1) Sample extraction and redissolution: The pH of the sample to be tested was adjusted to 2.0 with HCl, and then extracted three times with an equal volume of ethyl acetate. The collected extract was dried in an oven to remove the solvent, and then 1 mL of deionized water was accurately added to fully redissolve the sample.

[0032] (2) Colorimetric reaction and determination: Take 500 μL of the reconstituted sample, add 2 mL of sulfuric acid-anthrone reagent, and shake thoroughly to mix. Place the mixture in a boiling water bath for 5 min, and after cooling, use a spectrophotometer to measure the absorbance of the reaction solution at 620 nm. Finally, calculate the actual concentration of rhamnose in the sample based on the pre-plotted rhamnose standard curve. C Rha Based on the initial substrate amount, the theoretical concentration of rhamnose that can be released when rhamnolipids are completely degraded was calculated. C theo The rhamnolipin calculation is performed by substituting the following formula.

[0033] The rhamnose conversion rate (Y) is calculated as follows:

[0034] C RL Rhamnose lipid concentration; C Rha The actual concentration of rhamnose in the enzymatic hydrolysis solution was determined by HPLC. C theo The theoretical concentration of rhamnose that can be released when the initial rhamnose lipolipide substrate is completely hydrolyzed.

[0035] The standard curve obtained using the anthrone-sulfuric acid method is y = 0.002379x + 0.105532 (R²). 2 =0.999424), calculated using the conversion factor 3 for this rhamnolipid, the rhamnosine content in the sample is 103.70 g / L, which is converted to a rhamnolipid content of 311.10 g / L. The rhamnosine conversion rate in subsequent enzymatic hydrolysis condition optimization experiments was calculated using this measured data.

[0036] Rhamnolipin is viscous. The mother liquor of rhamnolipin was diluted 50-fold directly. Specifically, 1 mL of the mother liquor was precisely pipetted into a 50 mL centrifuge tube using a pipette tip with a scissor tip. 49 mL of methanol-water solution or alkaline buffer was added, and the pipette tip was repeatedly rinsed with the solvent to wash away any residual mother liquor. The sample was then vortexed for 1–2 min and sonicated at room temperature for 5–10 min until the solution was clear and homogeneous. Relevant information was then labeled and the solution was ready for use. At this point, the concentration of rhamnolipin was 6.22 g / L.

[0037] Enzyme activity was measured using pNPR as a substrate, and quantification was achieved by detecting the change in absorbance of the product p-nitrophenol at 405 nm. The standard reaction system (100 μL) contained: 75 μL acetate-sodium acetate buffer, 20 μL 5 mM pNPR solution, and 5 μL appropriately diluted enzyme solution. The system was incubated in a 40 °C water bath for 20 min, followed by quenching the reaction with 100 μL 2 M sodium carbonate solution for 5 min. The absorbance at 405 nm was read using a microplate reader.

[0038] like Figure 1 As shown, substrate micelle modulation pretreatment can dissociate substrate molecules from a large-size micelle state into highly dispersed monomers or small molecule aggregates.

[0039] Example 1: Methanol pretreatment to dissociate substrate micelles 10 μL, 20 μL, 30 μL, and 40 μL of methanol or ethanol were added to the reaction system, respectively, to achieve a final alcohol concentration (volume fraction) of 10%–40%, followed by enzyme activity detection. Results are as follows: Figure 2 As shown, α-L-rhamnosidase has a relatively high enzyme activity in methanol.

[0040] Rhamnollipids were dissolved in 0% (pure water control), 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80% methanol aqueous solutions, respectively.

[0041] Catalytic reaction: Accurately transfer 71.40 μL of the pretreated rhamnolipin solution to a reaction system containing pH 5.0 citrate-phosphate buffer, bringing the total reaction volume to 1 mL; the final concentration of rhamnolipin in the system is 0.44 g / L. Add 1000 U / mL of purified α-L-rhamnosidase (RHA-Bra). Incubate at 40 °C with shaking for 3 h.

[0042] Sample detection (PMP pre-column derivatization): Take 200 μL of the enzymatic hydrolysis product, add 200 μL of 0.30 mol / L NaOH and 200 μL of 0.50 mol / L PMP methanol solution, and react in a water bath at 70℃ for 30 min. After cooling to room temperature, neutralize with 200 μL of 0.30 mol / L HCl, and extract three times with 800 μL of chloroform to remove excess PMP. Take the upper aqueous phase, filter it through a 0.22 μm filter membrane, and perform HPLC analysis. The results are as follows. Figure 3 As shown.

[0043] This study used L-rhamnose conversion rate as the core indicator for evaluating enzymatic hydrolysis efficiency. The enzymatic hydrolysis efficiency of the pure water control group (0% methanol) was only 10.65%. The enzymatic hydrolysis efficiency of the 10% methanol pretreatment group reached 22.09%; the 40% methanol pretreatment group reached a peak of 22.53%. In the 80% methanol group, due to the excessive organic solvent introduced into the reaction system, enzyme activity was slightly inhibited, and the efficiency decreased to 19.04%, but it was still significantly higher than that of the control group.

[0044] Example 2: Pretreatment of substrate micelles with different pH buffer solutions Rhamnollipids were dissolved in pure water, citrate-phosphate buffer (pH 3-8), Tris-HCl buffer (pH 8-9), and glycine-NaOH buffer (pH 9-12), respectively.

[0045] Catalytic reaction: Accurately transfer 71.40 μL of the pretreated rhamnolipin solution to a reaction system containing pH 5.0 citrate-phosphate buffer, bringing the total reaction volume to 1 mL; the final concentration of rhamnolipin in the system is 0.44 g / L. Add 1000 U / mL of purified α-L-rhamnosidase (RHA-Bra). Incubate at 40 °C with shaking for 3 h.

[0046] Sample detection (PMP pre-column derivatization): Take 200 μL of the enzymatic hydrolysis product, add 200 μL of 0.30 mol / L NaOH and 200 μL of 0.50 mol / L PMP methanol solution, and react in a water bath at 70℃ for 30 min. After cooling to room temperature, neutralize with 200 μL of 0.30 mol / L HCl, and extract three times with 800 μL of chloroform to remove excess PMP. Take the upper aqueous phase, filter it through a 0.22 μm filter membrane, and perform HPLC analysis. The results are as follows. Figure 4 As shown, pretreatment with a glycine-NaOH buffer solution at pH 11 resulted in better enzymatic hydrolysis of rhamnolipids.

[0047] Based on the determined optimal substrate depolymerization pretreatment conditions (using glycine-NaOH buffer at pH 11), the effect of pH on rhamnose conversion was further investigated.

[0048] To eliminate the interference of highly alkaline substrate solutions on the pH of the reaction microenvironment and achieve strict control of conditions, 71.40 μL of rhamnolipin solution pretreated with glycine-NaOH buffer at pH 11 was accurately transferred and added to standardized enzymatic reaction systems containing citrate-phosphate buffers (pH) at different pH values. In a total reaction system of 1 mL, the final concentration of rhamnolipin was 0.44 g / L, and the enzyme addition for purifying RHA-Bra was 1000 U / mL. After thorough mixing, the reaction was incubated at 40 ℃ with shaking for 3 h, with other operating conditions identical to those described above. Results are as follows: Figure 5 As shown.

[0049] Since rhamnolipids require an alkaline environment (pH 11.0) to dissolve, introducing the alkaline-soluble substrate into the enzymatic hydrolysis system inevitably alters the system's pH, causing α-L-rhamnosidase (RHA-Bra) to deviate from its optimal catalytic conditions. Therefore, this study systematically investigated the effect of system pH on rhamnosine conversion using alkaline-soluble rhamnolipids as the substrate. The results are as follows: Figure 5 As shown, the effect of pH on rhamnose conversion was investigated in a citrate-phosphate buffer system (pH 3-7). The results showed that the rhamnose conversion rate first increased and then decreased: it reached its peak at pH 4 (32.05%), maintained a high level (>30%) in the slightly acidic pH range of 3-5, and then sharply decreased to 8.94% under neutral conditions (pH 7). This significant discrepancy between the optimal catalytic pH (4) and the optimal substrate solubility pH (11) stems from the difference between the enzyme's biochemical properties and the substrate's physical behavior: RHA-Bra, as a typical acidic glycosidase, has the most stable active site conformation and highest catalytic efficiency at pH 4; while rhamnose lipids require strongly alkaline conditions to effectively decompose micelles to improve substrate accessibility. To achieve efficient enzymatic hydrolysis, the pH dependence of substrate dispersion and catalytic reaction must be resolved. Therefore, to overcome this contradiction, this study proposes a strategy that separates physical dispersion from enzymatic catalysis: first, a highly dispersed rhamnolipin solution is prepared using an alkaline solution, and then a trace amount is added to an acidic buffer system for the reaction. This design ensures both the effective disassembly of rhamnolipin micelles and avoids damage to enzyme activity caused by drastic changes in the overall environment, achieving dual optimization of substrate dispersion and enzymatic catalysis.

[0050] As the pretreatment pH increased, the enzymatic conversion rate fluctuated: it was 27.05% at pH 5; it remained between 18.60% and 22.27% in the pH range of 6-10; when the pretreatment pH reached 11, the enzymatic conversion rate increased sharply to 30.25%, which was 1.84 times higher than that of the pure water group (10.65%); when the pH was 12, the enzymatic efficiency dropped sharply to 10.18% due to the hydrolysis and inactivation of rhamnolipid ester bonds caused by the extreme strong alkali.

[0051] Example 3 1. Effect of reaction system temperature on rhamnose conversion rate Based on the established optimal reaction pH of 4, the effect of reaction temperature on rhamnose conversion was further investigated. In a 1 mL total reaction system, citrate-phosphate buffer at pH 4 was used, with a final concentration of rhamnose lipids of 0.44 g / L and a final enzyme addition of 1000 U / mL for purified RHA-Bra. After thorough mixing, the reaction system was incubated at 25, 30, 35, 40, 45, 50, and 60 °C for 3 h with constant temperature and shaking. Other operating conditions were the same as described above.

[0052] 2. Effect of enzyme dosage on rhamnose conversion rate Based on the determination of optimal substrate pretreatment conditions, optimal reaction pH (4), and optimal temperature (35 ℃), the effect of RHA-Bra enzyme addition on rhamnose conversion was further investigated. In a 1 mL total reaction system, citrate-phosphate buffer at pH 4 was used, and the final mass concentration of rhamnose lipids was kept constant at 0.44 g / L. Purified RHA-Bra was added to the system to achieve final enzyme additions of 0, 1000, 2000, 4000, 5000, 6000, 8000, 12000, 15000, 20000, 25000, and 30000 U / mL, respectively. After thorough mixing, the reaction system was incubated at 35 ℃ with shaking for 3 h. Other operating conditions were the same as described above.

[0053] like Figure 6The effect of reaction temperature on the catalytic efficiency of RHA-Bra exhibits a typical bell-shaped curve. Within the range of 25–60 °C, after 3 h of reaction, the rhamnose conversion rate of the enzyme initially increased and then decreased with increasing temperature, reaching a peak of 34.77% at 35 °C, followed by a significant decrease in the high-temperature region, dropping to 12.31% at 60 °C. This variation is essentially the result of the mutual constraint between the thermodynamics of the enzyme-catalyzed reaction and the conformational stability of the protein: at low temperatures (<35 °C), the thermal motion of the system molecules is slow and the enzyme activity is not fully activated, limiting the effective collision frequency between the enzyme and the substrate; when the temperature exceeds the optimal equilibrium point of 35 °C, the excessive heat energy destroys the non-covalent bonds that maintain the higher-order structure of the enzyme protein, inducing irreversible thermal denaturation and inactivation of the catalytic active center.

[0054] like Figure 7 As shown, within an enzyme dosage gradient of 0–30,000 U / mL, the rhamnolipid conversion exhibited typical saturation kinetics. With increasing enzyme concentration, the rhamnolipid conversion rate continuously increased, reaching a peak value (97.78%) at 25,000 U / mL. Subsequently, further increasing the enzyme activity to 30,000 U / mL resulted in a stable conversion rate (97.69%), with no further significant increase. This "increase-then-stable" trend reveals the dynamic switching mechanism of the rate-limiting factor in the reaction system: in the low and medium enzyme activity stages, the number of catalytically active sites of the enzyme is the bottleneck of the reaction, and increasing the enzyme concentration can greatly promote substrate conversion; however, when the enzyme concentration reaches 25,000 U / mL, the free rhamnolipids in the system are fully bound and almost completely hydrolyzed.

Claims

1. A method for improving the efficiency of enzymatic preparation of L-rhamnose by regulating the micelle state of the substrate, characterized in that, Includes the following steps: 1) The rhamnolipin substrate was dissolved and diluted using a pretreatment medium to dissociate the rhamnolipin micelle structure and obtain a highly dispersed substrate pretreatment solution; 2) The substrate pretreatment solution is introduced into an acidic or neutral reaction system containing α-L-rhamnosidase to carry out enzymatic hydrolysis and release free L-rhamnose.

2. The method according to claim 1, characterized in that: In step 1), the pretreatment medium is a methanol aqueous solution, and the volume fraction of methanol in the methanol aqueous solution is 10%~70%.

3. The method according to claim 1, characterized in that: In step 1), the pretreatment medium is an alkaline buffer solution with a pH value of 11.

0.

4. The method according to claim 3, characterized in that: The alkaline buffer solution is a glycine-NaOH buffer solution.

5. The method according to claim 1, characterized in that: In step 2), the final concentration of the substrate rhamnolipid in the enzymatic hydrolysis reaction system is 0.2~0.5 g / L.

6. The method according to claim 1, characterized in that: In step 2), the amount of α-L-rhamnosidase added to the reaction system is 1000~30000U / mL.

7. The method according to claim 1, characterized in that: In step 2), the reaction temperature of the enzymatic hydrolysis reaction system is 35~45℃, and the reaction pH is 3~7.

8. The method according to claim 1, characterized in that: In step 2), the reaction time for the enzymatic hydrolysis is 3 hours.

9. The method according to claim 3, characterized in that: In step 1), the alkaline buffer is a glycine-NaOH buffer with pH=11, and in step 2), the enzymatic hydrolysis reaction system is a citrate-phosphate buffer with pH=4.

10. The method according to claim 2, characterized in that: The volume fraction of methanol in the methanol-water solution is 10% to 40%.

Images