Microbial Cell Factories
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2023
·Vol. 22
·期 1
·1-14
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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.
研究领域:
生物化学
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酶工程
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DOI
PubMed
高影响因子
RSC Advances
·
2018
·Vol. 8
·期 72
·41247-41254
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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.
研究领域:
生物化学
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酶工程
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PubMed
高影响因子
BIORESOURCE TECHNOLOGY
·
2015
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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.
研究领域:
生物工程
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代谢生物学
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DOI
PubMed
高影响因子
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.
研究领域:
生物化学
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有机合成
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酶工程
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DOI
PubMed
高影响因子
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.
研究领域:
生物工程
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微生物学
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2019
·Vol. 188
·期 3
·677-689
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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.
研究领域:
生物化学
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酶工程
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食品科学
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DOI
PubMed
高影响因子
JOURNAL OF FOOD SCIENCE AND TECHNOLOGY-MYSORE
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2021
·Vol. 59
·期 6
·2339-2350
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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.
研究领域:
食品科学
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微生物学
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分析化学
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PubMed
高影响因子
Molecular Catalysis
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2024
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IF 3.9
Xiangsheng Zheng,Xiaohong Hao,Yan Wang,Siyu Gao,Dantong Wen,Jinchuan Wang
The development of novel biocatalysts is essential to promote the commercialization of biodiesel production by transesterification reaction. In this paper, Rhizopus oryzae lipase (ROL) was immobilized on an amino-functionalized zirconium-based metal organoskeleton by interfacial adsorption. The immobilization conditions were optimized and the enzymatic properties were tested, and the resulting novel biocatalysts exhibited higher stability and heat resistance. SEM, XRD and BET analyses were used to characterize the biocatalysts and carrier materials. The catalytic performance of ROL@UiO-66-NH 2 in the production of biodiesel by transesterification reaction was explored, and the production process was optimized by response surface method. The results showed that the conversion rate of FAEE reached 82.05% at molar ratio of ethanol/oil of 15.43:1, reaction temperature of 50.28°C, reaction time of 120.9 min, DES addition of 48.08 wt%, biocatalyst addition of 3 wt%, and ultrasonic power of 90 W. In addition, ROL@UiO-66-NH 2 demonstrated good recyclability, with the catalytic efficiency remaining at 71.87% after five cycles.
研究领域:
生物化学
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催化材料
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可再生能源
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DOI
高影响因子
INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY
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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.
研究领域:
微生物学
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真菌学
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生物化学
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分子生物学
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DOI
PubMed
高影响因子
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.
研究领域:
生物技术
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酶学
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蛋白质工程
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生物化学
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DOI
PubMed
高影响因子
LWT-FOOD SCIENCE AND TECHNOLOGY
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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.
研究领域:
食品科学
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微生物学
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蛋白质化学
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风味化学
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产品关联
DOI
高影响因子
LANGMUIR
·
2021
·
IF 3.882
Yixian Luo,Dou Jin,Wenjin He,Jianzhong Huang,Aicheng Chen,Feng Qi
The opal-like SiO2 microcarriers with different pore diameters named opal-SiO2I and opal-SiO2II were synthesized and utilized as microcarriers to immobilize Rhizopus oryzae lipase (ROL) and Aspergillus oryzae α-amylases (AOA). ROL and AOA can be more stably immobilized on the cross-linked SiO2 opals by neopentyl glycol diglycidyl ether (NGDE), which is the first attempt to use it as a cross-linking agent compared with glutaraldehyde. According to the morphology analysis, multiple layers of SiO2 monodisperse microspheres were regularly packed and formed an opal-like structure, and enzymes were well scattered and immobilized throughout the SiO2 opals. The results showed that the performance of enzymes immobilized on opal-SiO2II with a larger specific surface area was much better than that of opal-SiO2I. The enzyme activity of ROL@opal-SiO2II and AOA@opal-SiO2II cross-linked with 1% NGDE increased 5.32 and 9.32 times compared with their free counterpart, respectively. Furthermore, pH and thermal stability and reusability of ROL/AOA@opal-SiO2II were significantly improved and higher than those of ROL/AOA@opal-SiO2I and free enzymes. This study provides an easily obtained microcarrier opal-SiO2II, which shows potential for efficient different enzyme immobilizations and further industrial applications.
研究领域:
生物化学
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材料科学
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酶工程
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DOI
PubMed
高影响因子
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