INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
·
2022
·
IF 5.911
Wenguang Fan,Baoyu Li,Nana Du,Tian Hui,Yingying Cao,Xin Li,Haiwei Ren
3-phenyllactic acid (PLA) has broad anti-fungal activity, however, target sites of PLA on fungal cells and its anti-fungal mechanism of action have been poorly studied. In this study, we explored the inhibition mechanism of Rhizopus oryzae ( R. oryzae ) on rotten lily bulbs by PLA. The minimum inhibitory concentration value of PLA against R. oryzae was 8 mg/mL. We observed the ultrastructure of R. oryzae by scanning electron microscopy and transmission electron microscopy which indicated that PLA did not damage the cell membrane, but destroyed the mitochondria and other organelles. Tandem mass tag proteomes showed that PLA significantly down-regulated ( P < 0.05) the expression of hexokinase (HK), phosphofructokinase (PFK), a-ketoglutarate dehydrogenase (a-KGDH), adenylate kinase (ADK1), Cytochrome C oxidase and NADH dehydrogenase, and up-regulated ( P < 0.05) the expression of mitochondrial ADP/ATP carrier proteins (AAC) and subunit IV (CCIO IV) in glycolysis, tricarboxylic acid cycle or oxidative phosphorylation metabolism. Following these findings, down-regulated HK and a-KGDH activity of aforementioned pathways was shown by enzyme activity assay, and regulated gene expression of ADK1, AAC, CCIO IV and NADH dehydrogenase was further confirmed by real-time quantitative PCR. Central carbon metabolomics showed that citric acid, cis-Aconitic acid, isocitric acid, alpha-Ketoglutaric acid, succinate, fumarate and malic acid of the tricarboxylic acid cycle metabolites were significantly down-regulated ( P < 0.05), and ATP production by oxidative phosphorylation was also significantly reduced ( P = 0.02), resulting in insufficient energy production. Thus, ROS levels increased by 141% of the control values and cytochrome C was released, resulting in gradual cell apoptosis. All data indicated that energy metabolism was the target of PLA against R. oryzae . This was the first study to show that energy metabolism could be the target of PLA against R. oryzae , which could provide a theoretical basis to study the mechanism of fungal inhibition.
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Microbial Cell Factories
·
2023
·Vol. 22
·期 1
·1-14
·
IF 6.4
Xu Lili,Qin Yimin,Song Yufeng,Tang Aixing,Liu Youyan
Background Existing methods for alkene epoxidation are based on lipase-catalysed perhydrolysis. However, the inactivation of the expensive lipase enzyme is problematic for enzymatic epoxidation at large scales due to the use of hydrogen peroxide and peracids at high concentrations in the reaction. The immobilisation of whole cells appears to be a promising approach to alleviate this problem.Results A green oxidation system containing hydrogen peroxide, Na3C6H5O7, an acyl donor, and glutaraldehyde (GA)-crosslinked cells of Rhizopus oryzae was developed for the epoxidation of alkenes. GA-crosslinked cells of Rhizopus oryzae were adopted as a biocatalyst into the epoxidation system. A variety of alkenes were oxidised with this system, with a 56–95% analytical yield of the corresponding epoxides. The catalytic performance of the crosslinked treated cells was substantially improved compared to that of the untreated cells and the initial reaction rate increased from 126.71 to 234.72 mmol/L/h, retaining 83% yields even after four batches of reactions. The addition of 3.5 mmol Na3C6H5O7 not only acts as an acid-trapping reagent to eliminate the negative effect of the carboxylic acid on the alkene oxide but also forms a saturated salt solution with the aqueous phase, affecting the concentration of H2O2 in the three phases and thus the epoxidation reaction. Organic solvents with a logP value > 0.68 were good at producing hydroxy peracids; however, this method is only suitable for oxidation in a two-liquid phase.Conclusion sCompared with other lipase biocatalysts, the GA-crosslinked whole-cell biocatalyst is inexpensive, readily available, and highly stable. Therefore, it can be considered promising for industrial applications.
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2024
·
IF 5.7
Yong Wang,Ziyuan Wang,Huifen Yu,Haidong Teng,Jianping Wu,Jiaqi Xu,Lirong Yang
Lipase from Rhizopus oryzae (ROL) exhibits remarkable sn-1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and N-glycosylation mutants are then recombinantly expressed and screened in Pichia pastoris, the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.
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LWT-FOOD SCIENCE AND TECHNOLOGY
·
2024
·
IF 6
Zihan Li,Wei Su,Yingchun Mu,Qi Qi,Li Jiang
Protein degradation occurs during the fermenting of traditional mutton sausages, which influences on the flavor and quality of products. In this study, HS-SPME-GC-MS and Sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to study the degradation kinetics of myofibrillar protein (MP) and sarcoplasmic protein (SP) during fermentation and ripening of fermented sausages by single and mixed starters ( Pediococcus acidilactici CICC 10344 and Rhizopus Oryzae ATCC 96382). It was shown that flavor formation and protein degradation are related. Adding mixed starters effectively reduced the loss of sulfhydryl content compared to single starter. Moreover, cathepsin B and cathepsin L activities were continuously enhanced during sausage fermentation. Analysis of SDS-PAGE showed processing affected protein degradation, and the degree of degradation was increased with increasing fermentation time. Meanwhile, the inoculation of mature sausage samples with mixed starters accelerated the production of water-soluble flavor compounds (e.g., free amino acids and TCA-soluble peptides). The mixed starters group had more amino acids presenting the umami-free amino acids. Based on the ROAV values for odor thresholds, we identified 12 key volatile flavor compounds. The correlation heat map showed higher concentrations of key volatile flavor compounds in the mixed starters. Overall, Pediococcus acidilactici and Rhizopus Oryzae addition may effectively improve the flavor quality of fermented sausage products during fermentation.
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BIORESOURCE TECHNOLOGY
·
2024
·
IF 9.7
Lili Xu,Qingqing Zhao,Haibo Liu,Dankui Liao,Aixing Tang,Youyan Liu
Limonene dioxide (LDO) is essential for manufacturing bio-based polycarbonate and non-isocyanate polyurethanes. Herein, we report a strategy for the chemoenzymatic epoxidation of (R)-(+)-limonene to LDO with high selectivity using Rhizopus oryzae whole cells. The presence of sufficient excess acid in the system is essential, in addition to overcoming the hydrolysis of the intermediate product, 1,2-limonene oxide, to accomplish the double epoxidation of limonene. When using a high concentration of H 2 O 2 and trisodium citrate, the conditions were effectively established, even when Novozym 435 was substituted, which had previously been reported to be ineffective in achieving high-yield double epoxidation of limonene. The transfer of H 2 O 2 and peracids between the cell and solvent slowed production. The total amount of peracids generated remained constant, but their concentrations remained moderate, favoring the double epoxidation of limonene. An LDO yield of 93 % and no intermediate product, limonene oxide, were obtained under optimized conditions within 20 h.
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RSC Advances
·
2018
·Vol. 8
·期 72
·41247-41254
·
IF 2.936
Jiong-feng Zhao,Zhe Wang,Fei-long Gao,Jian-ping Lin,Li-rong Yang,Mian-bin Wu
Rhizopus oryzae lipase (ROL) is important because of its extreme sn-1,3-regioselectivity, but it shows poor thermostability, which severely restricts its application. In this work, the thermostability of ROL was greatly enhanced by rational design. First, several sites that may affect the thermostability of ROL were identified by multiple-sequence alignment. The half-lives of mutants V209L and D262G at 55 °C were about 4.38- and 4.2-times those of the wild-type, respectively. Then, a disulfide bond was introduced between positions 190 and 238 based on the prediction of Disulfide by Design 2, which greatly improved the thermostability of the protein. The activity of variant E190C/E238C retained about 58.2% after incubation at 55 °C for 720 min, whereas the half-life of wild type ROL was only about 11.7 min. On the basis of the results obtained by the two methods, we carried out a combined mutation. Quadruple mutant V209L/D262G/E190C/E238C was constructed and the thermostability was improved even further. The half-lives at 55 °C and 65 °C were 102.5- and 20-times those of the wild-type ROL. This improvement in thermostability will give ROL wider industrial applicability, especially in the preparation of structured lipids.
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APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2019
·Vol. 188
·期 3
·677-689
·
IF 2.14
Zhao Jiong-feng,Lin Jian-ping,Yang Li-rong,Wu Mian-bin
Nano-sized Fe 3 O 4 was synthesized by chemical co-precipitation and subsequently modified with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde to introduce aldehyde group on its surface. With the help of “interface activation” by adding sucrose esters-11 as surfactant, lipase from Rhizopus oryzae was successfully immobilized onto the carrier with great enhancement of activity. The hydrolysis activity of immobilized enzyme were 9.16 times and 31.6 times of free enzyme when p-nitrophenol butyrate and p-nitrophenol palmitate were used as substrates. The thermo-stability of immobilized enzyme was also enhanced compared to free enzyme. The immobilized enzyme was successfully applied in synthesis of 1,3-diacyglycerols (1,3-DAG). The specific esterification activity of immobilized enzyme was about 1.5 times of the free enzyme. The immobilized enzyme showed good region-selectivity towards 1,3-diacyglycerols and retained nearly 80% of its activity after reused for 60 times, revealing a good industrial application prospect.
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BIOCHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 3.6
Peiye Yu,Lili Xu,Xin Zhang,Qingqing Zhao,Youyan Liu
Freeze-dried Rhizopus oryzae mycelium efficiently catalyzed chemoenzymatic epoxidation of methyl oleate • Solvent-free system improved conversion and epoxide yield. • Epoxy methyl oleate showed excellent plasticizing performance. Unsaturated fatty acid epoxidation reactants are widely regarded as a new generation of bio-based plasticizers with the potential to replace traditional plasticizers. Herein, a chemoenzymatic epoxidation process using methyl oleate (MO) as a model substrate was established based on the perhydrolytic activity of freeze-dried Rhizopus oryzae CGMCC 3.5040 mycelium. Moreover, better epoxy conversion and yield were achieved by adopting a solvent-free system compared to the solvent system. It was found that high concentrations of hydrogen peroxide favored the perhydrolysis reaction over hydrolysis, thereby increasing the purity of the product. After 16 hours of reaction, a conversion of methyl oleate of 94.37% was achieved, the total epoxide yield reached 88.93%, and epoxy methyl oleate (EME) was quantified at 63.54%. The reaction process was described more accurately using the Langmuir–Hinshelwood–Hougen–Watson (LHHW) kinetic model, which suggested that a peroxycarboxylic acid was generated at the lipase on the mycelia surface and then transferred to the oil phase to catalyze the epoxidation reaction. When the purified methyl oleate epoxide was used as a plasticizer in polyvinyl chloride (PVC), the elongation at break ( ε b ) was significantly increased from 189.88% to 406.23%, demonstrating excellent plasticizing properties. This study provides a feasible and green catalytic process for the epoxidation of unsaturated fatty acids. Download: Download high-res image (205KB) Download: Download full-size image
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BIORESOURCE TECHNOLOGY
·
2015
·
IF 4.494
Guanyi Wang,Di Huang,Yong Li,Jianping Wen,Xiaoqiang Jia
In this work, wheat bran (WB) was utilized as feedstock to synthesize fumaric acid by Rhizopus oryzae . Firstly, the pretreatment process of WB by dilute sulfuric acid hydrolysis undertaken at 100 °C for 30 min offered the best performance for fumaric acid production. Subsequently, through optimizing the seed culture medium, a suitable morphology (0.55 mm pellets diameter) of R. oryzae was obtained. Furthermore, a metabolic-based approach was developed to profile the differences of intracellular metabolites concentration of R. oryzae between xylose (the abundant sugar in wheat bran hydrolysate (WBH)) and glucose metabolism. The xylitol, sedoheptulose 7-phosphate, ribulose 5-phosphate, glucose 6-phosphate, proline and serine were responsible for fumaric acid biosynthesis limitation in xylose fermentation. Consequently, regulation strategies were proposed, leading to a 149% increase in titer (up to 15.4 g/L). Finally, by combinatorial regulation strategies the highest production was 20.2 g/L from WBH, 477% higher than that of initial medium.
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JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY
·
2023
·
IF 2
Miao Qiao,Xin Hua,Yongjun Yuan,Xi Cao
This work attempts to achieve the enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in acylglycerols as well as biodiesel production by investigating the performance of various lipases in ethanolysis. The ethanolysis activities of five immobilized lipases in decreasing order were as follows: Lipozyme TL IM, Novozym 40086/Lipozyme RM IM, Novozym 435 and Rhizopus oryzae lipase immobilized on polypropylene (RO PP). EPA could be enriched to a certain content by Lipozyme TL IM, Novozym 40086, Lipozyme RM IM, and RO PP. The enrichment of DHA was achieved well by those four sn -1,3 regioselective lipases, and DHA contents in acylglycerols were the highest when Lipozyme TL IM and Novozym 40086 were used, reaching 2.87-fold and 2.81-fold of the initial content. However, Novozym 435 could not enrich EPA and DHA effectively. Fatty acid ethyl esters produced in this work contained abundant palmitic acid and palmitoleic acid, which could be ideal biodiesel considering their chain length. The competitive factors of five lipases towards DHA were all significantly higher than those towards EPA. When Lipozyme TL IM and Novozym 40086 achieved the maximum DHA contents, their DHA recoveries in acylglycerols reached 91.87% and 89.00%, respectively. The DHA recoveries were all significantly higher than the corresponding EPA recoveries in the reactions catalyzed by five lipases. Moreover, Lipozyme TL IM could be a potential lipase for the separation of EPA and DHA. This study may promote the full use of fish oil and broaden the application of lipases.
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BIORESOURCE TECHNOLOGY
·
2011
·
IF 4.365
Yuefang Deng,Shuang Li,Qing Xu,Min Gao,He Huang
A mutant strain with high glucoamylase activity and insensitive to catabolite repression was developed to produce fumaric acid by simultaneous saccharification and fermentation (SSF) of starch without additional commercial glucoamylase supplementation. A series of mutant strains resistant to the non-metabolizable and toxic glucose analog 2-deoxyglucose (2-DG) were obtained by implanting nitrogen ion (N + ) into Rhizopus oryzae ME-F12. Among them, the best mutant strain DG-3 produced 39.80 g/L fumaric acid, which is 1.28-fold of that produced by ME-F12, and exhibited higher glucoamylase activity during SSF. Higher fumaric acid production (44.10 g/L) was achieved when the initial total sugar concentration of cornstarch was 100 g/L. During SSF of cheap, raw bioresource-degermed corn powder (100 g/L total sugar) by DG-3, the maximum fumaric acid concentration and productivity were 32.18 g/L and 0.44 g/(L h), respectively.
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JOURNAL OF FOOD SCIENCE AND TECHNOLOGY-MYSORE
·
2021
·Vol. 59
·期 6
·2339-2350
·
IF 2.701
Yang Yurong,Zhong Haiyan,Yang Ning,Xu Shuaizhe,Yang Tao
The pure culture fermentation has led to less flavorful rice wine and relatively lower bioactive substance level compared to traditional mixed culture fermentation; however, a pure strain is easily controlled by industrialized producers. The purpose of the present research was to screen a species of Rhizopus for improving the flavor deficiency and antioxidant function of sweet rice wine. Seven rice wine samples fermented with isolated strains were analyzed for their total phenolics, total flavonoids, peptide and antioxidant activity using spectrophotometry, as well as ethanol, ethyl acetate, β-phenethyl alcohol, and volatile alcohol contents measured by headspace gas chromatography (HS-GC), the further principal component analysis determined Rhizopus delemar rice wine better on aroma and antioxidant capacity. A comparison of phenolics profile between R. delemar and R. oryzae rice wines was made based on the measurement data of ultrahigh-performance liquid chromatography coupled with Q-exactive orbitrap mass spectrometry (UHPLC-QE-MS) data. Thirty-two phenolics were identified in sweet rice wine. Overall, the results presented in this study showed that a strain of R. delemar is available for the improvement of flavor and antioxidant activity in sweet rice wine, which has the great potential to be applied to industrialized products.
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