Bienzyme System Research Articles

Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis.

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.

Textile-Based Membraneless Microfluidic Double-Inlet Hybrid Microbial-Enzymatic Biofuel Cell.

This study reports the development of a textile-based colaminar flow hybrid microbial-enzymatic biofuel cell. Shewanella MR-1 was used as a biocatalyst on the anode, and bienzymatic system catalysts based on glucose oxidase and horseradish peroxidase were applied on an air-breathing cathode to address the overpotential loss in a body-friendly way. A single-layer Y-shaped channel configuration with a double-inlet was adopted. Microchannels of biofuel cells were patterned by silk screen printing with Ecoflex to maintain the flexibility of textile substrates without harm to the human body. The electrodes were fabricated with poly(3,4-ethylenedioxythiophene):polystyrene sulfonate and a mixture of multiwalled carbon nanotubes and single-walled carbon nanotubes by screen printing. The effects of electrode materials, catalyst type, catalyst concentration, and glucose concentration in the catholyte were investigated to optimize the fuel cell performance. The peak power density (44.9 μW cm-2) and maximum current density (388.9 μA cm-2) of the optimized hybrid biofuel cell were better than those of previously reported textile- or paper-substrate microscale single microbial fuel cells. The developed biofuel cell will be a useful platform as a microscale power source that is harmless to the environment and living organisms.

An efficient approach to produce flavonoid monophosphate by a coupled bienzymatic system

Flavonoids, with therapeutic potential, often encounter challenges due to poor aqueous solubility, which limits their oral bioavailability and efficacy. Phosphorylation presents a promising solution, yet current chemical methods require protective agents to mitigate side product formation. Here, we developed a coupled bienzymatic system using an ATP-dependent dikinase, flavonoid phosphate synthetase (BsFPS), for selective synthesis of flavonoid monophosphates. This system integrates a class III polyphosphate kinase 2 (PPK2-III) for ATP regeneration from AMP, thereby reducing costly ATP consumption and enhancing economic viability. Through PPK2-III screening, ErPPK was identified as a compatible candidate, facilitating a streamlined one-pot system. Structural analysis revealed that the residue at the equivalent site of Arg75ErPPK influences the binding pocket entry distance and flavonoid derivative inhibition propensity on PPK2-III, confirming the compatibility of ErPPK and providing structural insights for identifying PPK2-III with reduced inhibition likelihood. Further optimization strategies, including balancing Mg2+ and polyphosphate ratios, incorporating Tween surfactants for enhanced substrate solubilization without compromising enzyme stability, and increasing the rate-limiting enzyme amount, significantly elevated the conversion rate. Notably, pH control was crucial due to pH decline caused by BsFPS catalysis. pH-stat conversion achieved a 99.0 % conversion of luteolin, the flavonoid target, within 8 h, yielding up to 21.5 mM (7.9 g/L) luteolin monophosphates with up to 95 % reduction in ATP requirement. This enzymatic phosphorylation approach shows promise for advancing polyphenolic monophosphate bioproduction and transforming their functional applications.

The use of bioluminescent enzyme bioassay for the analysis of human saliva: Advantages and disadvantages.

The purpose of the work was to find optimal conditions for bioluminescent enzymatic analysis of saliva (based on the use of NADH:FMN oxidoreductase + luciferase) and then to determine the biological effect of using bioluminescence assay of saliva to study the physiological state of the body under normal and pathological conditions. The saliva of snowboarders and students were studied in the "rest-training" model. The saliva of patients diagnosed with acute pharyngitis was examined in the "sick-healthy" model. Bioluminescence assay was performed with a lyophilized and immobilized bi-enzyme system using cuvette, plate, and portable luminometers. The concentrations of secretory immunoglobulin A (sIgA) and cortisol were determined by enzyme immunoassay, and the total protein content was measured by spectrophotometric method. The activity of the bioluminescent system enzymes increased as the amount and volume of saliva in the sample was decreased. The cuvette and plate luminometers were sensitive to changes in the luminescence intensity in saliva assay. Luminescence intensity correlated with the concentrations of sIgA and cortisol. The integrated bioluminescent index for saliva was reduced in the "rest-training" model and increased in the "sick-healthy" model. Thus, the non-invasive bioluminescent saliva analysis may be a promising tool for assessing the health of the population.

Monolayer textile-based co-laminar flow biocompatible enzymatic biofuel cell

In this study, a body-friendly textile-based enzymatic glucose biofuel cell, which is applicable to wearable electronics, was developed. The microchannel of the biofuel cell was patterned by silk screen printing using Ecoflex. To develop this body-friendly wearable enzymatic fuel cell, a bi-enzyme system composed of glucose oxidase-catalase and glucose oxidase-horseradish peroxidase was applied to the anode and cathode, respectively. The catholyte mixed with glucose was saturated with O2 to enhance the fuel cell performance. The cell performance was affected by concentrations of enzyme biocatalyst and glucose. The best performance of the newly developed fuel cell was the peak power density of 43.6 ± 1.97 μW cm−2. The fuel cell was tested in an electrolyte physiological condition, and its power density was reduced to 25 %. Compared to the paper-based enzymatic fuel cell, the power density was approximately 11 % lower; however, the duration (11 h) was significantly longer. The performance of the proposed fuel cell was slightly lower than that of traditional enzymatic biofuel cells. Nevertheless, it is expected that the proposed body-friendly enzymatic biofuel cell has future promise as a power source for flexible bioelectronics.

Turning waste into treasure: Biosynthesis of value-added 2-O-α-glucosyl glycerol and d-allulose from waste cane molasses through an in vitro synthetic biology platform

The efficient and economical conversion of agricultural waste into glycosides and rare sugars is challenging. Herein, an in vitro synthetic bienzyme system consisting of sucrose phosphorylase and d-allulose 3-epimerase was constructed to produce 2-O-α-glucosyl glycerol and d-allulose from cane molasses. Lactic acid in the cane molasses significantly induced sucrose phosphorylase to hydrolyze sucrose instead of glycosylation. Notably, lactic acid significantly inhibited the catalytic performance of d-allulose 3-epimerase only in the presence of Na+ and K+, with an inhibition rate of 75%. After removing lactic acid and metal ions, 116 g/L 2-O-α-glucosyl glycerol and 51 g/L d-allulose were synthesized from 500 mM sucrose in the treated cane molasses with a sucrose consumption rate of 97%. Our findings offer an economically efficient and environmentally friendly pathway for the industrial production of glycosides and rare sugars from food industry waste.

Bi-enzymatic chemo-mechanical feedback loop for continuous self-sustained actuation of conducting polymers

Artificial actuators have been extensively studied due to their wide range of applications from soft robotics to biomedicine. Herein we introduce an autonomous bi-enzymatic system where reversible motion is triggered by the spontaneous oxidation and reduction of glucose and oxygen, respectively. This chemo-mechanical actuation is completely autonomous and does not require any external trigger to induce self-sustained motion. The device takes advantage of the asymmetric uptake and release of ions on the anisotropic surface of a conducting polymer strip, occurring during the operation of the enzymes glucose oxidase and bilirubin oxidase immobilized on its surface. Both enzymes are connected via a redox polymer at each extremity of the strip, but at the opposite faces of the polymer film. The time-asymmetric consumption of both fuels by the enzymatic reactions produces a double break of symmetry of the film, leading to autonomous actuation. An additional break of symmetry, introduced by the irreversible overoxidation of one extremity of the polymer film, leads to a crawling-type motion of the free-standing polymer film. These reactions occur in a virtually unlimited continuous loop, causing long-term autonomous actuation of the device.

Designed Synthesis of Compartmented Bienzyme Biocatalysts Based on Core–Shell Zeolitic Imidazole Framework Nanostructures (Small 7/2023)

Compartmented Bienzyme Biocatalysts Biomimetic mineralization and slow seed-induced nucleation strategy enables the synthesis of compartmented bienzyme system based on core-shell [email protected] in aqueous solution. Bienzyme can be accurately localized into the shell and core with uniform distribution. The optimization of pore structure and bienzyme positional assembly provides a guidance for preparing efficient multi-enzyme biocatalysts. More details can be found in article number 2206606 by Liang Qi, Hongsheng Liu, and co-workers.

Heterogeneous biocatalytic reduction of 5-(hydroxy)methyl furfural using two co-immobilised alcohol dehydrogenases

Cofactor self-sustained reduction of HMF to BHMF catalyzed by a co-immobilized bi-enzymatic system, capable of in situ recycling NADH along the process.

Dual-target affinity analysis and separation of α-amylase and α-glucosidase inhibitor from Morus alba leaves by magnetic bifunctional immobilized enzyme system.

Morus alba leaves is a natural product with great anti-diabetic potential. However, the therapeutic efficacy of natural product is usually achieved through the interaction of active compounds with specific targets. Among them, active compounds with multi-target therapeutic functions are more effective than single target enzyme. In this study, a bienzyme system was constructed by co-immobilizing α-amylase and α-glucosidase onto Fe3 O4 for affinity screening of dual-target active component in the complex extract from M. alba leaves. As a result, a potential active compound was selectively screened by ligand fishing, separated by HSCCC using a solvent system of ethyl acetate-n-butanol-water (3:2:5, v/v), and identified as rutin. In addition, the result of molecular docking showed that rutin could interact with the active center of α-amylase and α-glucosidase through multiple hydrogen bonds, van der Waals forces, etc. to play an inhibitory role. These results demonstrated the effectiveness of polydopamine magnetically immobilized bienzyme system for dual-target affinity screening of active substance. This study not only revealed the chemical basis of the antidiabetic activity of M. alba leaves from a dual-target perspective, but also promoted the progress of multi-target affinity screening.

Designed Synthesis of Compartmented Bienzyme Biocatalysts Based on Core-Shell Zeolitic Imidazole Framework Nanostructures.

For complex cascade biocatalysis, multienzyme compartmentalization helps to optimize substrate transport channels and promote the orderly and tunable progress of step reactions. Herein, a simple and general synthesis strategy is proposed for the construction of a multienzyme biocatalyst by compartmentalizing glucose oxidase and horseradish peroxidase (GOx and HRP) within core-shell zeolite imidazole frameworks (ZIF)-8@ZIF-8 nanostructures. Owing to the combined effects of biomimetic mineralization and the fine regulation of the ZIF-8 growth process, the uniform shell encloses the seed (core) surface by epitaxial growth, and the bienzyme system is accurately localized in a controlled manner. The versatility of this strategy is also reflected in ZIF-67. Meanwhile, with the ability to covalently bind divalent metal ions, lithocholic acid (LCA) is used as a competitive ligand to improve the pore structure of the ZIF from a single micropore to a hierarchical micro/mesopore network, which greatly increases mass transfer efficiency. Furthermore, the multienzyme cascade reaction is exemplified by the oxidation of o-phenylenediamine (OPD). The findings show that the bienzyme assembly strategy significantly affects the biocatalytic efficiency mainly by influencing the utilization efficiency of the intermediate (Hydrogen peroxide, H2 O2 ) between the step reactions. This study sheds new light on facile synthetic routes to constructing in vitro multienzyme biocatalysts.

Preparation of magnetic microcapsules of α-amylase and α-glucosidase for dual-target affinity screening of active components from Toona sinensis

Natural products are an important source of drug development and have good prospects in the treatment of many diseases. However, the efficacy of many natural drugs is achieved through multi-targets, which poses a huge obstacle to their development. In this study, a bienzyme microcapsule system based on α-amylase and α-glucosidase was constructed for the ligand fishing of natural compounds. The microcapsules were formed on the surface of calcium carbonate particles by self-polymerization of dopamine, which physically encapsulated iron oxide and α-glucosidase. Then, α-amylase was immobilized on the surface of magnetic material with glutaraldehyde as crosslinking agent, and calcium carbonate template was removed by EDTA to form a bienzyme system for affinity screening of active components from Toona sinensis. The results showed that two compounds, 1,2,3,4,6-penta-O-galloyl-β-D-glucose and quercitrin were affinity screened from the extracts of Toona sinensis. In vitro activity experiment proved that they were α-amylase and α-glucosidase inhibitors, respectively. Moreover, both of them were mixed type inhibitors that can interact with active centers of corresponding enzymes through multiple hydrogen bonds, van der Waals forces, etc. This study not only proved the effectiveness of the bienzyme system, but also provided a successful example for multi-target screening of natural products.

The role of lignin in the conversion of wheat straw to cellobionic acid by Neurospora crassa HL10

An engineered Neurospora crassa strain (HL10) naturally produces cellulases, cellobiose dehydrogenase (CDH), and heterologously expresses laccase. It can convert Avicel (cellulose) to cellobionic acid (cellobionate) without any enzyme addition at high yields. However, the addition of a catalytic amount of artificial redox mediators was required to achieve a high yield conversion. When a lignocellulosic substrate like wheat straw was used as the carbon source, it was found that adding exogenous artificial redox mediators did not improve cellobionate yield. We hypothesized that lignin and lignin degradation products were able to serve as redox mediators for the CDH-laccase conversion system. Experiments were designed to prove the hypothesis. When N. crassa HL10 was grown on Avicel, the addition of enzyme hydrolyzed lignin (EHL) can achieve a similar level of cellobionate production as with ABTS addition. The addition of EHL can increase the cellobionate production rate in the CDH-laccase bi-enzyme system using cellobiose as the substrate. The formation of lignin radicals, and the quenching of lignin radicals by reduced CDH, were verified by an electron paramagnetic resonance (EPR) experiment, providing further evidence that lignin radicals can serve as the electron acceptor of reduced CDH. Hence, lignocellulosic biomass is a self-sufficient substrate for the production of cellobionate. The conversion of the lignocellulosic substrate to cellobionate using N. crassa HL10 can be achieved without any exogenous enzyme addition or redox mediator addition.

Surfactant-assisted synthesis of colloidosomes for positional assembly of a bienzyme system

In this study, using sorbitan monooleate (Span-80), we developed a new strategy to achieve the positional assembly of a horse radish peroxidase/glucose oxidase bienzyme system within the zeolitic imidazolate framework-8 (ZIF-8)-based colloidosomes. Span-80 with self-adsorption property, was used as an auxiliary stabilizer to endow the enzyme-loaded ZIF-8 (enzyme@ZIF-8) with proper wettability in the formation of water–in–oil Pickering emulsion templates. Benefited from this, simultaneous immobilization of the bienzymes in the lumen and shell of the colloidosomes could be achieved. Meanwhile, Span-80 was found to act as a mediator to induce and control the overgrowth of the ZIF-8 crystallites on the enzyme@ZIF-8 layers, by which a “bricklaying” solidification protocol was established for the shell preparation. The resulting colloidosomes enabled the flexible positional change of the bienzymes, its shell exhibited the monolayer structure with increased compactness and homogenized micropores. Furthermore, the multi-enzyme cascade reaction was exemplified with the oxidization of o-phenylenediamine. An in-depth investigation showed that the assembly strategies of the bienzymes significantly affected their biocatalytic efficiency by influencing the utilization efficiency of the H2O2 intermediate between the step reactions. This study provides an insight towards a facile synthetic route for constructing multi-enzyme biocatalyst.

Bilirubin oxidase as a single enzymatic oxygen scavenger for the development of reductase-based biosensors in the open air and its application on a nitrite biosensor

The commercialization of amperometric or voltammetric biosensors that operate at potentials lower than −0.2 V vs SHE has been hindered by the need for anoxic working conditions due to the interference of molecular oxygen, whose electrochemical reduction can potentially mask other redox processes and generate reactive oxygen species (ROS). A deoxygenation step must be thus integrated into the analytical process. To this end, several (bio)chemical oxygen scavenging systems have been proposed, such as the bi-enzyme system, glucose oxidase/catalase. Still, a few issues persist owing to enzyme impurities and the formation of oxygen reactive species.Here in, we propose a new mono-enzymatic oxygen scavenging system composed of a multicopper oxidase as a single biocatalytic oxygen reducer. As a model, we used bilirubin oxidase (BOD), which catalyzes the direct reduction of oxygen to water in the presence of an electron donor substrate, without releasing hydrogen peroxide. Both the direct electron transfer and mediated electrochemical approach using different co-substrates were screened for the ability to promote the enzymatic reduction of oxygen. An optimal combination of BOD with sodium ascorbate proved to be quick (5 min) and effective. It was subsequently employed, as a proof-of-concept, in a voltammetric biosensor based on a multiheme cytochrome c nitrite reductase, which performs the reduction of nitrite to ammonia at potentials below −0.3 V vs SHE. The nitrite biosensor performed well under ambient air, with no need for a second enzyme to account for the build-up of oxygen reactive intermediaries.

Immobilization of a Bienzymatic System via Crosslinking to a Metal-Organic Framework

A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic linkers and inorganic nodes, MOFs feature empty void space with large surface areas and have the ability to be modified post-synthesis. Our target enzyme system for immobilization is glucose oxidase (GOx) and chloroperoxidase (CPO). Glucose oxidase catalyzes the oxidation of glucose and is used for many applications in biosensing, biofuel cells, and food production. Chloroperoxidase is a fungal heme enzyme that catalyzes peroxide-dependent halogenation, oxidation, and hydroxylation. These two enzymes work sequentially in this enzyme system by GOx producing peroxide, which activates CPO that reacts with a suitable substrate. This study focuses on using a zirconium-based MOF, UiO-66-NH2, to immobilize the enzyme system via crosslinking with the MOF’s amine group on the surface of the MOF. This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS), providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond CPO and GOx onto UiO-66-NH2, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx is discussed through assays and characterization analyses. The DSG-crosslinked composites displayed enhanced activity relative to the free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability, with at least 30% of the activity being retained after four catalytic cycles. The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability.

Sequentially co-immobilized PET and MHET hydrolases via Spy chemistry in calcium phosphate nanocrystals present high-performance PET degradation

Accumulation of polyethylene terephthalate (PET) has brought an enormous threat to the ecosystem. The recently reported PET hydrolase (DuraPETase) and MHET hydrolase (MHETase) can synergistically catalyze the complete PET degradation. Hence, this work was designed to develop a bienzymatic cascade catalysis by co-immobilizing the two enzymes for PET biodegradation. DuraPETase and MHETase were sequentially co-immobilized in calcium phosphate nanocrystals (CaP) through SpyTag/SpyCatcher system. MHETase-SpyCatcher was first embedded inside the nanocrystals via biomimetic mineralization, and DuraPETase-SpyTag was then conjugated on the outlayer (~1.5 µm). The bienzyme compartmentalization facilitated DuraPETase interaction with the solid substrate, and the layered structures of the nanocrystals protected the enzymes, thus enhancing their stability. The high specific surface area of the nanocrystals and the proximity effects from the bienzymatic cascade were beneficial to the improved enzyme activity. Experimental data and molecular dynamics simulations revealed the activation effect of Ca2+ on DuraPETase. Taken together, the final results indicate that the PET degradation efficiency of DuraPETase-MHETase@CaP increased by 6.1 and 1.5 times over the free bienzyme system within 10 d at 40 °C and 50 °C, with weight losses at 32.2% and 50.3%, respectively. The bienzymatic cascade with DuraPETase-MHETase@CaP can completely degrade PET, contributing to the recycling of PET.

An ultrasensitive and long-lasting chemiluminescence immunoassay for IP-10 detection based on a 4-bromophenol-reinforced bienzymatic system

The 10-kDa chemokine interferon-gamma-inducible protein 10 (IP-10) is considered one of the most promising biomarkers for diagnosing both tuberculosis and COVID-19 infections. The blood samples of patients at different disease states contain different levels of IP-10, which need to be detected in a rapid, specific and ultrasensitive manner. Here, we report a bienzymatic chemiluminescence sandwich immunoassay (BCSI) assay for the ultrasensitive and stable detection of IP-10. In this assay, IP-10 is first efficiently captured using a double-antibody sandwich strategy. The detection antibody is linked to catalase (CAT) via a streptavidin–biotin signal amplification system to achieve highly efficient conversion of hydrogen peroxide (H2O2) to oxygen and water. In the chemiluminescence (CL) reaction, horseradish peroxidase (HRP) acts as an efficient catalyst, and 4-bromophenol acts as an enhancer for the cyclic transition of HRP, which results in a strong and durable CL signal. The bienzymatic catalysis with CAT and HRP and the potentiation of 4-bromophenol enables the assay to be ultrasensitive and stable. The CL intensity was found to be well correlated with the detection of IP-10 at levels in the range of 0.71 to 125,000 pg/mL, which covers more than 6 orders of magnitude, with a detection limit of 0.63 pg/mL. The coefficient of variation was 1.49%, and the recovery range of IP-10 in serum was 86.21%-104.57%. This assay provides a wide linear range and high sensitivity and may be a promising method for the high-throughput detection of IP-10 in the diagnosis of tuberculosis and COVID-19.

α-Amylase Changed the Catalytic Behaviors of Amyloglucosidase Regarding Starch Digestion Both in the Absence and Presence of Tannic Acid

The courses of starch digestion with individual α-amylase (AA), amyloglucosidase (AMG), and AA/AMG bi-enzyme system were performed and analyzed by first-order-reaction equations in the absence and presence of tannic acid (TA). An antagonistic effect between AA and AMG occurred at the digestion phase of readily-digestible starch due to the higher catalytic efficiency of AMG for starchy-substrates with more complex structures. This effect caused a faster rate of glucose production with AMG than with AA/AMG bi-enzyme system at this phase both in the absence and presence of TA. TA had a higher binding affinity to AA than to AMG as accessed by several methods, such as inhibition kinetics, fluorescence quenching, isothermal titration calorimetry (ITC), and molecular docking. Besides, differential scanning calorimetry (DSC) indicated that the change in the thermal and structural stabilities of enzymes in the presence of TA was related to the enzyme residues involved in binding with TA, rather than the inhibitory effects of TA. The binding characters of TA to both enzymes resulted in more “free” AMG without TA binding in AA/AMG bi-enzyme system than that in individual AMG. This binding property caused more and faster rate of glucose production at the digestion phase of slowly digestible starch (SDS) in the bi-enzyme system.

Cytochrome P450 Monooxygenase/Cytochrome P450 Reductase Bi-Enzymatic System Isolated From Ilex asprella for Regio-Specific Oxidation of Pentacyclic Triterpenoids.

Ilex asprella is a plant from Aquifoliaceae. Its root is commonly used as folk medicinal materials in southern China. The chemical compositions of I. asprella are rich in pentacyclic triterpenoids, which show various biological activities and demonstrate a good prospect for drug development. The elucidation of biosynthesis mechanism of triterpenoids in I. asprella could lay important foundations for the production of these precious plant secondary metabolites by metabolic engineering. Our previous studies have revealed IaAO1 (a CYP716A210 homolog) responsible for the C-28 oxidation of α- and β-amyrin. Herein, we reported the identification of three more cytochrome P450 monooxygenase genes IaAO2 (a CYP716A212 homolog), IaAO4 (CYP714E88), IaAO5 (CYP93A220), and a cytochrome P450 reductase gene IaCPR by using Saccharomyces cerevisiae eukaryotic expression system and gas chromatography-mass spectrometry. Among them, the protein encoded by IaAO2 can catalyze the C-28 oxidation of α-amyrin and β-amyrin, IaAO4 can catalyze the C-23 oxidation of ursolic acid and oleanolic acid, while IaAO5 is responsible for the C-24 oxidation of β-amyrin. By introducing three genes IaAO1, IaAO4 and IaCPR into S. cerevisiae. We constructed an engineered yeast strain that can produce C-23 hydroxyl ursane-type triterpenoid derivatives. This study contributes to a thorough understanding of triterpenoid biosynthesis of medicinal plants and provides important tools for further metabolic engineering.