APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2024
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IF 3.1
Zhang Yueting,Nie Hongmei,Zhang Fei,Jin Mengmeng,Wang Zhao,Zheng Jianyong
Aspergillus oryzae is an ideal cell factory for protein expression with powerful protein processing and secretion capabilities. The current study aimed to explore the homologous expression of A. oryzae lipase AOL (GenBank: KP975533) by constructing an auxotrophic A. oryzae △ pyrG △ nptB and subsequently characterizing the immobilization and catalytic properties of recombinant lipase. Initially, the pyrG gene knocked out in wild-type A. oryzae by homologous recombination, followed by the creation of a uridine/uracil auxotroph transformation. Through this system, the protease gene nptB was precisely knocked out, leading to a substantial decrease in extracellular (39.04%) and intracellular (90.07%) protease activity. The A. oryzae △ nptB △ pyrG strain was used as host for homologous expression of lipase AOL. After transformation of linearized lipase-expression cassette, the engineered A. oryzae AOL-8 was screened out with the lipase gene copy number of 14, exhibiting extracellular and intracellular lipase activities of 1.75 U/mL and 46.4 U/g, respectively. Subsequently, the production and immobilization of the recombinant lipase, via physical adsorption on macroporous resin XRZ04B, were achieved through submerged fermentation of the AOL-8 strain. The results of esterification catalytic properties of immobilized recombinant lipase indicated that the lipase exhibited optimal catalytic activity with lauric acid and methanol as substrates, a reaction temperature of 35 °C, and n-hexane as the preferred solvent medium; its highest conversion rate can reach at 72.3%.
研究领域:
微生物学
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生物技术
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酶学
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分子生物学
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DOI
PubMed
高影响因子
BIOTECHNOLOGY LETTERS
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2024
·
IF 2
Zhang Fei,Wang Xue,Pan Lixia,Wang Zhao,Zheng Jianyong
The α- l -rhamnosidase ( rha 1) gene was homologously expressed in Aspergillus niger strains CCTCC 206047 and CCTCC 206047Δ pyr G, using hygromycin B and auxotrophic as selection markers. The engineered A. niger strains RHA001-1 and RHA003-1 were screened, yielding α- l -rhamnosidase activities of 20.81 ± 0.56 U/mL and 15.35 ± 0.87 U/mL, respectively. The copy numbers of the rha 1 gene in strains RHA001-1 and RHA003-1 were found to be 18 and 14, respectively. Correlation analysis between copy number and enzyme activity in the A. niger strains revealed that α- l -rhamnosidase activity increased with the copy number of the rha 1 gene. Recombinant α- l -rhamnosidase was utilized for the enzymatic debittering of Ougan juice, and its process conditions were optimized. Furthermore, the primary bitter substance neohesperidin (2.22 g/L) in Ougan juice was converted into hesperetin 7-O-glucoside (1.47 g/L) and hesperidin (0.143 g/L). This study presents a novel approach for the production of food-grade α- l -rhamnosidase and establishes a technical foundation for its application in the beverage industry.
研究领域:
生物技术
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食品科学
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酶学
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DOI
PubMed
高影响因子
Fermentation-Basel
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2023
·Vol. 9
·期 10
·880
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IF 3.7
Siqi Zheng,Mingyue Fang,Jiaxin Huang,Yanbin Li,Yuxia Mei
Antrodia cinnamomea(AC), a rare fungus endemic to Taiwan, contains high levels of various secondary metabolites, notably triterpenoids, having useful medicinal and pharmacological properties. Techniques for increasing the production of AC triterpenoids (ACT) for medicinal purposes are a high research priority. We measured and compared the biomass and ACT content of AC mycelia under various liquid fermentation culture conditions. Relative gene expression levels of ten enzymes involved in the mevalonate (MVA) pathway and “subsequent group modification pathway” were determined, and correlation analysis was performed to evaluate the roles of these enzyme genes in ACT synthesis. Two representative genes encoding the enzymes lanosterol synthase (AcLSS) and sterol C-24 reductase (AcERG4), whose activity is closely associated with ACT content, were selected for homologous expression.AcLSSandAcERG4were separately linked to plasmid pCT74, and transformed into prepared AC protoplasts to obtain two recombinant strains, termed RpLSS and RpERG4, by polyethylene glycol (PEG)-CaCl2-mediated protoplast transformation. Upregulated expression levels ofAcLSSandAcERG4(1.78- and 1.41-fold, respectively) were associated with significantly higher (1.82- and 1.37-fold, respectively) ACT content in the recombinant strains in comparison with the wild-type. Our findings provide a theoretical and practical basis for the enhancement of ACT production using homologous expression techniques.Keywords:Antrodia cinnamomea;triterpenoids;homologous expression;synthetic pathway
研究领域:
真菌学
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生物技术
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药理学
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分子生物学
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DOI
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2022
·Vol. 194
·期 8
·3453-3467
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IF 3.094
Ye Hangyu,Li Xiaojun,Li Luyuan,Zhang Yinjun,Zheng Jianyong
Aspergillus niger has been used for homologous and heterologous expressions of many protein products. In this study, the α-L-rhamnosidase from A. niger (Rha-N1, GenBank XP_001389086.1) was homologously expressed in A. niger 3.350 by Agrobacterium tumefaciens -mediated transformation. The enzyme activity of Rha-N1 was 0.658 U/mL, which was obtained by cultivation of engineered A. niger in a 5-L bioreactor. Rha-N1 was purified by affinity chromatography and characterized. The optimum temperature and optimum pH for Rha-N1 were 60 °C and 4.5, respectively. Enzyme activity was promoted by Al 3+ , Li + , Mg 2+ , and Ba 2+ and was inhibited by Mn 2+ , Fe 3+ , Ca 2+ , Cu 2+ , and organic solvents. The result indicated that rutin was the most suitable substrate for Rha-N1 by comparison with the other two flavonoid substrates hesperidin and naringin. The transformed products of isoquercitrin, hesperetin-7-O-glucoside, and prunin were identified by LC–MS and 1 H-NMR.
研究领域:
生物化学
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分子生物学
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生物技术
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DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）