Cloning and functional analysis of IPI gene from Fritillaria unibracteata Hsiao et K. C. Hsia / 药学学报

An open reading frame (ORF) of isopentenyl-diphosphate delta isomerase gene (FuIPI) was cloned from Fritillaria unibracteata Hsiao et K. C. Hsia. (F. unibracteata). Furthermore, the bioinformatics and functional analyses of FuIPI were performed in this study. The result showed that, the ORF of FuIPI gene was 825 bp, encoding a polypeptide of 274 amino acids in length, with a relative molecular mass of about 31 kD and a theoretical isoelectric point of 5.61. Sequence analysis showed that FuIPI contained conserved structural domains and key residues involved in the catalyzing process. The phylogenetic analysis exhibited that FuIPI was closely related to IPIs of Dendrobium officinale and Musa acuminate. Real-time PCR analysis showed that FuIPI was distributed in different tissues of F. unibracteata, but had the highest transcriptional level in leaves, followed by stems, bulbs, and flowers. Furthermore, the FuIPI protein was successfully expressed in Escherichia coli BL21(DE3). The purified FuIPI protein successfully catalyzed the conversion from isopentenyl diphosphate (IPP) to dimethylallyl pyrophosphate (DMAPP). The above results provided a theoretical basis for further investigation of the molecular role of FuIPI in the biosynthesis of alkaloids.

Advances in research methods for biosynthetic pathway analysis of active ingredients in traditional Chinese medicine / 中国中药杂志

The active ingredients in traditional Chinese medicine(TCM)are the foundation for the efficiency of TCM and the key to the formation of Dao-di herbs. It is of great significance to study the biosynthesis and regulation mechanisms of these active ingredients for analyzing the formation mechanism of Daodi herbs and providing components for the production of active ingredients in TCM by synthetic biology. With the advancements in omics technology, molecular biology, synthetic biology, artificial intelligence, etc., the analysis of biosynthetic pathways for active ingredients in TCM is rapidly progressing. New methods and technologies have promoted the analysis of the synthetic pathways of active ingredients in TCM and have also made this area a hot topic in molecular pharmacognosy. Many researchers have made significant progress in analyzing the biosynthetic pathways of active ingredients in TCM such as Panax ginseng, Salvia miltiorrhiza, Glycyrrhiza uralensis, and Tripterygium wilfordii. This paper systematically reviewed current research me-thods for analyzing the biosynthetic functional genes of active ingredients in TCM, elaborated the mining of gene elements based on multiomics technology and the verification of gene functions in plants in vitro and in vivo with candidate genes as objects. Additionally, the paper summarized new technologies and methods that have emerged in recent years, such as high-throughput screening, molecular probes, genome-wide association studies, cell-free systems, and computer simulation screening to provide a comprehensive reference for the analysis of the biosynthetic pathways of active ingredients in TCM.

Lysohexaenetides A and B, linear lipopeptides from Lysobacter sp. DSM 3655 identified by heterologous expression in Streptomyces / 中国天然药物

Lysobacter harbors a plethora of cryptic biosynthetic gene clusters (BGCs), albeit only a limited number have been analyzed to date. In this study, we described the activation of a cryptic polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) gene cluster (lsh) in Lysobacter sp. DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp. S001. As a result of this methodology, we were able to isolate two novel linear lipopeptides, lysohexaenetides A (1) and B (2), from the recombinant strain S001-lsh. Furthermore, we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs. This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes, particularly in the absence of genetic manipulation tools.

Heterologous production of bioactive xenoacremone analogs in Aspergillus nidulans / 中国天然药物

Tyrosine-decahydrofluorene derivatives are a class of hybrid compounds that integrate the properties of polyketides and nonribosomal peptides. These compounds feature a [6.5.6] tricarbocyclic core and a para-cyclophane ether moiety in their structures and exhibit anti-tumor and anti-microbial activities. In this study, we constructed the biosynthetic pathway of xenoacremones from Xenoacremonium sinensis ML-31 in the Aspergillus nidulans host, resulting in the identification of four novel tyrosine-decahydrofluorene analogs, xenoacremones I-L (1-4), along with two known analogs, xenoacremones A and B. Remarkably, compounds 3 and 4 contained a 12-membered para-cyclophane ring system, which is unprecedented among tyrosine-decahydrofluorene analogs in X. sinensis. The successful reconstruction of the biosynthetic pathway and the discovery of novel analogs demonstrate the utility of heterologous expression strategy for the generation of structurally diverse natural products with potential biological activities.

Advances in heterologous expression, structural elucidation and molecular modification of pullulanase / 生物工程学报

Starch is composed of glucose units linked by α-1, 4-glucoside bond and α-1, 6-glucoside bond. It is the main component of foods and the primary raw material for starch processing industry. Pullulanase can effectively hydrolyze the α-1, 6-glucoside bond in starch molecules. Combined with other starch processing enzymes, it can effectively improve the starch utilization rate. Therefore, it has been widely used in the starch processing industry. This paper summarized the screening of pullulanase-producing strain and its encoding genes. In addition, the effects of expression elements and fermentation conditions on the production of pullulanase were summarized. Moreover, the progress in crystal structure elucidation and molecular modification of pullulanase was discussed. Lastly, future perspectives on pullulanase research were proposed.

Expression, purification, and functional identification of immunoglobulin degrading enzyme IdeS in Escherichia coli / 药学学报

In the process of evolution, pathogenic Streptococcus pyogenes secretes an immunoglobulin G-degrading enzyme IdeS which can specifically cleave the hinge region of immunoglobulin G in order to escape the immune response against the host. On the one hand, IdeS can be used for IgG fingerprinting as a tool enzyme combined with mass spectrometry technology. On the other hand, IdeS can be used to treat the antibody-responsive diseases produced by autoimmunity as a therapeutic protein. In this study, the backbone of plasmid pCold was used to construct two expression vectors of recombinant protein IdeS, which were heterologously expressed in Escherichia coli Shuffle T7. After purification by affinity chromatography, the recombinant IdeS activity was detected and their activity differences between the two were compared. Among them, the yield of the recombinant IdeS containing the His6-tag at the N-terminus was 4 mg·L-1, and the cleavage reaction with antibody IgG1 at 1∶200 (m/m) at 37 ℃ for 30 min could complete. However, the yield of the recombinant IdeS containing both the N-terminal His6 tag and the C-terminal silica affinity tag (silica bing peptide, SiBP) is 1.5 mg·L-1, and the degradation reaction with antibody IgG1 at 1∶20 (m/m) at 37 ℃ for 30 min could reach the end. The C-terminal fusion peptide has a great influence on the yield and activity of IdeS, which is not conducive to subsequent application in drug development. Above all, the recombinant IdeS containing the His6-tag at the N-terminus expressed by this system has high activity and can fully meet the needs of antibody drug development and mapping analysis of IgG.

Enhanced heterologous expression of the cytochrome c from uncultured anaerobic methanotrophic archaea / 生物工程学报

Cytochrome c is a type of heme proteins that are widely distributed in living organisms. It consists of heme and apocytochrome c, and has potential applications in bioelectronics, biomedicine and pollutant degradation. However, heterologous overexpression of cytochrome c is still challenging. To date, expression of the cytochrome c from uncultured anaerobic methanotrophic archaea has not been reported, and nothing is known about the function of this cytochrome c. A his tagged cytochrome c was successfully expressed in E. coli by introducing a thrombin at the N-terminus of CytC4 and co-expressing CcmABCDEFGH, which is responsible for the maturation of cytochrome c. Shewanella oneidensis, which naturally has enzymes for cytochrome c maturation, was then used as a host to further increase the expression of CytC4. Indeed, a significantly higher expression of CytC4 was achieved in S. oneidensis when compared with in E. coli. The successful heterologous overexpression of CytC4 will facilitate the exploitation of its physiological functions and biotechnological applications.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)

Reconstitution of biosynthetic pathway for mushroom-derived cyathane diterpenes in yeast and generation of new "non-natural" analogues

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities. To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity,

Expression and characterization of β-N-acetylglucosaminidases from Bacillus coagulans DSM1 for N-acetyl-β-D glucosamine production / 生物工程学报

β-N-acetylglucosaminidases (NAGases) can convert natural substrates such as chitin or chitosan to N-acetyl-β-D glucosamine (GlcNAc) monomer that is wildly used in medicine and agriculture. In this study, the BcNagZ gene from Bacillus coagulans DMS1 was cloned and expressed in Escherichia coli. The recombinant protein was secreted into the fermentation supernatant and the expression amount reached 0.76 mg/mL. The molecular mass of purified enzyme was 61.3 kDa, and the specific activity was 5.918 U/mg. The optimal temperature and pH of the BcNagZ were 75 °C and 5.5, respectively, and remained more than 85% residual activity after 30 min at 65 °C. The Mie constant Km was 0.23 mmol/L and the Vmax was 0.043 1 mmol/(L·min). The recombinant BcNagZ could hydrolyze colloidal chitin to obtain trace amounts of GlcNAc, and hydrolyze disaccharides to monosaccharide. Combining with the reported exochitinase AMcase, BcNagZ could produce GlcNAc from hydrolysis of colloidal chitin with a yield over 86.93%.

Tricarbocyclic core formation of tyrosine-decahydrofluorenes implies a three-enzyme cascade with XenF-mediated sigmatropic rearrangement as a prerequisite

Tyrosine-decahydrofluorene derivatives feature a fused [6.5.6] tricarbocyclic core and a 13-membered

Construction and application of recombinant human UDP-glucuronosyltransferases expression systems / 药学学报

In the research and development of new drugs, it is very important to investigate the in vitro metabolism of candidate drugs. Traditional models such as liver microsomes have many limitations, while the in vitro model of recombinant human drug metabolizing enzymes is considered as an important and useful approach because of its convenient access, stable activity and low cost. In this study, six major human UDP-glucuronosyltransferases (UGTs) genes (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) were cloned from human liver cDNA and heterologously expressed in Saccharomyces cerevisiae and baculovirus-infected insect cell. UGT1A1, 1A3, 1A6 and 1A9 were successfully expressed in yeast and showed glucuronidation activity against a variety of different structural types of substrates, but their activities were low. All six UGTs were successfully expressed and exhibited significantly improved glucuronidation activity when Trichopolusia ni cells BTI-TN5B1-4 (High Five) were used as the host. The recombinant human UGTs expressed in insect cells can catalyze the glucuronidation of their specific substrates, and the glucuronidation products were synthesized at milligram-scale with yields of 13%-66% for the first time, of which the structures were identified via MS, 1H NMR, and 13C NMR spectroscopic analysis. Above all, the recombinant human UGTs yeast and insect cell expression systems constructed in this study can be used for in vitro metabolism evaluation in the early stage of new drugs research and development, and also provide a new tool for the synthesis of glucuronide metabolites.

Pathway design and key enzyme analysis of diosgenin biosynthesis / 生物工程学报

As a naturally occurring steroid sapogenin, diosgenin acts as the precursor of hundreds of steroid medicines, and thereby has important medicinal value. Currently, industrial production of diosgenin relies primarily on chemical extraction from plant materials. Clearly, this strategy shows drawbacks of excessive reliance on plant materials and farmland as well as environment pollution. Due to development of metabolic engineering and synthetic biology, bio-production of diosgenin has garnered plenty of attention. Although the biosynthetic pathways of diosgenin have not been completely identified, in this review, we outline the identified biosynthetic pathways and key enzymes. In particular, we suggest heterologous biosynthesis of diosgenin in Saccharomyces cerevisiae. Overall, this review aims to provide valuable insights for future complete biosynthesis of diosgenin.

Modification and Expression of Beta-1,4-Endoglucanase encoding sequences of fungal origin in Escherichia coli BL21 / Modificación y expresión de secuencias codificantes de Beta-1,4-Endoglucanasa de origen fúngico en Escherichia coli BL21

ABSTRACT Lignocellulose is the main and most abundant component of biomass. Annually, 200 million tons are generated in the world. Colombia has a high production of lignocellulosic residues that can be used in many industrial processes such as bioethanol produc-tion, promoting the bioeconomy. The objective of the present work was to express lignocellulolytic enzymes of eukaryotic origin in Escherichia coli BL21 (DE3). Initially, endoglucanase eukaryotic genes were selected and modified using bioinformatics methods for their production in E. coli BL21 (DE3) and saccharification of pure cellulose substrates. The gene selected for its modification and expression was egl B from the fungus Aspergillus nidulans. Subsequently the enzyme integrity was tested by 3D modeling and molecular docking, as well as the conformation of its active site and its affinity for substrates of interest. Finally, cloning of the modified gene in plasmid pET151 TOPO was made and transformed in the strain E. coli BL21 (DE3) where several lignocellulose degradation tests were carried out using semiquantitative methods for the enzyme activity in carboxymethylcellulose. The presence of the three genes of interest within the plasmid pET151 TOPO and within the transformed cells of E. coli TOP10 and E. coli BL21 (DE3) was verified by colony PCRs performed. The presence of this gen was corroborated by sequencing. Expression of the modified endoglucanase enzyme was achieved in E. coli BL21 (DE3) expression cells, in soluble and functional form, demonstrated by the hydrolysis of the CMC substrate.

RESUMEN La lignocelulosa es el componente principal y más abundante de la biomasa. Anualmente se generan 200 millones de toneladas en el mundo. Colombia tiene una alta producción de residuos lignocelulósicos que pueden ser utilizados en muchos procesos industriales como la producción de bioetanol, promoviendo la bioeconomía. El objetivo del presente trabajo fue expresar enzimas lignocelulolíticas de origen eucariota en Escherichia coli BL21 (DE3). Inicialmente, los genes eucariotas de endoglucanasa se seleccionaron y modificaron mediante métodos bioinformáticos para su producción en E. coli BL21 (DE3) y la sacarificación de sustratos de celulosa. El gen seleccionado para su modificación y expresión fue eglB del hongo Aspergillus nidulans. Posteriormente se evaluó la integridad de la enzima mediante modelado 3D y acoplamiento molecular, así como la conformación de su sitio activo y su afinidad por sustratos de interés. Finalmente, se realizó la clonación del gen modificado en el plásmido pET151 TOPO y se transformó en la cepa E. coli BL21 (DE3) donde se realizaron varios ensayos de degradación de lignocelulosa utilizando métodos semicuantita-tivos para la actividad enzimática en carboximetilcelulosa. La presencia del gen de interés dentro del plásmido pET151 TOPO y dentro de las células transformadas de E. coli TOP10 y E. coli BL21 (DE3) se verificó mediante PCR de colonia. La presencia de este gene se corroboró por secuenciación eglB. La expresión de la enzima endoglucanasa modificada se logró en células de E. coli BL21 (DE3), en forma soluble y funcional, demostrada por la hidrólisis del sustrato de CMC.

Overexpression of Chitinase A Gene from Serratia marcescens in Bacillus subtilis and Characterization of Enhanced Chitinolytic Activity

Abstract Chitinase enzymes possess various usages in agriculture, biotechnology and medicine due to their chitin degrading property. Thus, efficient production of chitinase enzymes with desired properties has importance for its use. In this study, chitinase A (chiA) gene from Serratia marcescens Bn10 was cloned and heterologously overexpressed using pHT43 vector in Bacillus subtilis 168. The recombinant chitinase was characterized in terms of temperature, pH, and various effectors. The extracellular chitinase activity in recombinant B. subtilis was found 2.15-fold higher than the parental strain after 2 h of IPTG induction. Optimum temperature and pH for the extracellular chitinase activity in the recombinant B. subtilis were determined as 60 oC and pH 9.0, respectively. NaCl, Ca2+, Mn2+, Cu2+, Zn2+, sodium dodecyl sulfate (SDS), Tween-20, and ethanol increased the chitinase activity whereas Mg2+ caused an inhibition. The most notable increment on the chitinase activity was provided by Zn2+ (3.2 folds) and then by SDS (2.9 folds). The chitinase, overproduced by the recombinant B. subtilis 168 heterologously expressing chiA, was determined to have optimum activity at high temperature and alkaline conditions as well as various effectors increase its activity. The extracellular chitinase of recombinant B. subtilis might be a promising source for agricultural, biotechnological and medical applications.

Expression optimization and molecular modification of heparin C5 epimerase / 生物工程学报

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.

Characterization of a sucrose phosphorylase from Leuconostoc mesenterides for the synthesis of α-arbutin / 生物工程学报

Sucrose phosphorylase (SPase) gene from Leuconostoc mesenteroides ATCC 12291 was synthesised after codon optimization, and inserted into pET-28a plasmid to generate pET-28a-spase. The recombinant strain Escherichia coli BL21 (DE3)/pET-28a-spase was induced for Spase expression. The recombinant protein Spase was purified and characterized. The specific enzyme activity of SPase was 213.98 U/mg, the purification ratio was 1.47-fold, and the enzyme activity recovery rate was 87.80%. The optimal temperature and the optimal pH of the SPase were identified to be 45 °C and 6.5 respectively, and Km, Vmax and kcat of the SPase for sucrose was 128.8 mmol/L, 2.167 μmol/(mL·min), and 39 237.86 min-1. The recombinant SPase was used for α-arbutin production from hydroquinone and the reaction process was evaluated. The optimal conditions for synthesis of α-arbutin by SPase were 40 g/L hydroquinone, 5:1 molar ratio of sucrose and hydroquinone, and 250 U/mL recombinant SPase at pH 7.0 and 30 °C for 24 h in the dark, and then 500 U/mL glucoamylase was added at 40°C for 2.5 h. Under the optimized process, the yield of α-arbutin reached 98 g/L, and the hydroquinone conversion rate was close to 99%. In summary, the recombinant SPase was cloned and characterized, and its application for α-arbutin production was feasible.

Expression, purification and characterization of catalase from Corynebacterium glutamicum / 生物工程学报

Catalase catalyzes the decomposition of H₂O₂ to H₂O and O₂, and has a wide range of industrial applications. However, most catalases used in the textile and paper industries are often subjected to high-alkaline challenges which makes it necessary to develop alkaline catalase. In this study, a catalase from Corynebacterium glutamicum was expressed in Escherichia coli, and the expression conditions were optimized. The recombinant catalase was purified by Ni-chelating affinity chromatography, and the recombinant enzyme was characterized. The optimal conditions of producing the recombinant catalase were: an IPTG concentration of 0.2 mmol/L, a culturing temperature of 25 °C and a culturing time of 11 h. The purified catalase had a specific activity of 55 266 U/mg, and it had a high activity in the pH range of 4.0 to11.5, with the highest activity at pH 11.0. When treated in pH 11.0 for 3 h, the enzyme retained 93% of its activity, indicating that the enzyme was qualified with a favorable stability under high-alkaline condition. The recombinant catalase had maximal activity at 30 °C, and showed a satisfactory thermal stability at a range of 25 °C to 50 °C. The apparent Km and Vmax values of purified catalase were 25.89 mmol/L and 185.18 mmol/(minmg), respectively. Besides, different inhibitors, such as sodium dodecyl sulfate (SDS), urea, NaN₂, β-mercaptoethanol, and EDTA had different degrees of inhibition on enzyme activity. The catalase from C. glutamicum shows high catalytic efficiency and high alkaline stability, suggesting its potential utilization in industrial production.

Expression and purification of the transcription factor StMsn2 from Setosphaeria turcica in Escherichia coli

Background: In Saccharomyces cerevisiae, Msn2, which acts as a key transcription factor downstream the MAPKHOG cascade pathway, also regulates the expression of genes related to stress responses. However, little is known about the regulation mechanisms of the transcription factor in Setosphaeria turcica. Results: In this study, a zinc finger DNA-binding protein, designated as StMSN2, was cloned from S. turcica. Sequencing results showed that StMSN2 had a 1752 bp open reading frame (ORF), which was interrupted by an intron (135 bp) and encoded a putative 538-amino acid protein. Phylogenetic analysis further revealed that StMsn2 was more closely related to Msn2 of Aspergillus parasiticus. StMSN2 was cloned into the pET-28a vector with His (Histidine) tags and induced with 1 mM IPTG (isopropyl-ß-D-thiogalactoside) at 37°C. The recombinant His-tagged StMsn2 was purified, and a band of size approximately 58.8 kDa was obtained. The high specificity of the polyclonal antibody Msn2-2 was detected with the StMsn2 protein from S. turcica and prokaryotic expression system, respectively. Conclusions: A new gene, named StMSN2, with 1617 bp ORF was cloned from S. turcica and characterized using bioinformatics methods. StMsn2 was expressed and purified in a prokaryotic system. A polyclonal antibody, named Msn2-2, against StMsn2 with high specificity was identified.

Heterologous expression and characterization of Aspergillus oryzae acidic protease in Pichia pastoris / 生物工程学报

Acid protease, an important aspartic protease, has been widely used in food, pharmaceutical and tanning industries. To promote the research and application of acid protease, an acid protease gene (pepA) from Aspergillus oryzae was obtained from fermented soy based on metagenome sequencing, and then cloned and transformed into Pichia pastoris GS115 for heterologous expression. The characteristic of recombinant PepA was also investigated. The activity of acid protease in the culture supernatant of P. pastoris was 50.62 U/mL. The molecular mass of PepA was about 50 kDa, and almost no other proteins in the supernatant were observed, as shown by SDS-PAGE. The optimum pH and temperature of PepA were determined as pH 4.5 and 50 ℃. Mn²⁺ and Cu²⁺ enhanced the activity of PepA, whereas Fe³⁺, Fe²⁺ and Ca² had inhibitory effects on its activity. The above findings can provide guidance for heterologous expression and industrial application of acid protease from Aspergillus oryzae.