Bioresources and Bioprocessing
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2021
·Vol. 8
·期 1
·1-11
·
IF 4.578
Xia Yu,Wu Zifeng,He Rui,Gao Yahui,Qiu Yangyu,Cheng Qianqian,Ma Xiaoyuan,Wang Zhouping
Aflatoxin B 1 (AFB 1 ) and zearalenone (ZEN) are two predominant mycotoxins ubiquitously found in corn, peanuts, and other grains, which pose a great threat to human health. Therefore, safe and effective methods for detoxification of these mycotoxins are urgently needed. To achieve simultaneous degradation of multiple mycotoxins, a fusion enzyme ZPF1 was constructed by linking zearalenone hydrolase and manganese peroxidase with a linker peptide GGGGS. This fusion enzyme was secretory expressed successfully in the newly constructed food-grade recombinant strain Kluyveromyces lactis GG799(pKLAC1-ZPF1), and was investigated with the mycotoxins degradation efficiency in two reaction systems. Results showed that both AFB 1 and ZEN can be degraded by ZPF1 in reaction system 1 (70.0 mmol/L malonic buffer with 1.0 mmol/L MnSO 4 , 0.1 mmol/L H 2 O 2 , 5.0 µg/mL AFB 1 and ZEN, respectively) with the ratios of 46.46% and 38.76%, respectively. In reaction system 2 (50.0 mmol/L Tris–HCl, with 5.0 µg/mL AFB 1 and ZEN, respectively), AFB 1 cannot be degraded while ZEN can be degraded with the ratio of 35.38%. To improve the degradation efficiency of these mycotoxins, optimization of the induction and degradation conditions were fulfilled subsequently. The degradation ratios of AFB 1 and ZEN by ZPF1 in reaction system 1 reached 64.11% ± 2.93% and 46.21% ± 3.17%, respectively. While in reaction system 2, ZEN was degraded by ZPF1 at a ratio of 41.45% ± 3.34%. The increases of degradation ratios for AFB 1 and ZEN in reaction system 1 were 17.65% and 7.45%, respectively, while that for ZEN in reaction system 2 was 6.07%, compared with the unoptimized results.
研究领域:
食品科学
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生物技术
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微生物学
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毒理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Green Synthesis and Catalysis
·
2022
·
IF 0
Yongxing Li,Pengqian Luan,Lele Dong,Jianqiao Liu,Luying Jiang,Jing Bai,Fufeng Liu,Yanjun Jiang
Asymmetric reduction of the conjugated C C bonds by the old yellow enzymes (OYEs) presents a promising field in the synthesis of chiral chemicals. Nevertheless, few natural OYEs have been applied in large-scale applications due to the requirement of costly NADPH and low operational stability. Herein, a stable and efficient fusion of YqjM from Bacillus subtilis and glucose dehydrogenase (GDH) from Bacillus megaterium was constructed to stereoselectively reduce the conjugated C C bonds in a self-sufficient continuous process. The effects of the enzyme order and different linkers on the fusions were investigated by structural analysis and all-atom molecular dynamics simulation. The best fusion YqjM_G_GDH gave 98% conversion of 100 mmol/L 2-methylcyclopentenone with an excellent ee value (>99%) in 3 h, while the mixture of individual enzymes only obtained 68% conversion after more than 8 h. The improved substrate conversion of YqjM_G_GDH fusion was probably attributed to the increased flexibility of each fused enzyme and the shortening of the diffusion distance of NADPH regenerated. A one-pot process was designed to purify and immobilize the fusion on the Ni 2+ -nitrilotriacetic acid functionalized magnetic mesoporous silica nanoflowers. The resulting immobilized biocatalyst not only catalyzed the asymmetric reduction of various α,β -unsaturated ketones (20 mmol/L) continuously with only 50 μmol/L NADP + to initiate the whole process, but also retained more than 82% of the initial activity after seven cycles, serving as a good candidate for the industrial applications.
研究领域:
生物催化
·
酶工程
·
有机化学
·
材料科学
✓ 已验证
DOI
Frontiers in Chemistry
·
2021
·
IF 5.221
Zhang Xiaodong,Hu Yun,Peng Wei,Gao Chenghua,Xing Qiong,Wang Binju,Li Aitao
Cytochrome P450 enzyme CYP109B1 is a versatile biocatalyst exhibiting hydroxylation activities toward various substrates. However, the regio- and stereoselective steroid hydroxylation by CYP109B1 is far less explored. In this study, the oxidizing activity of CYP109B1 is reconstituted by coupling redox pairs from different sources, or by fusing it to the reductase domain of two self-sufficient P450 enzymes P450RhF and P450BM3 to generate the fused enzyme. The recombinant Escherichia coli expressing necessary proteins are individually constructed and compared in steroid hydroxylation. The ferredoxin reductase (Fdr_0978) and ferredoxin (Fdx_1499) from Synechococcus elongates is found to be the best redox pair for CYP109B1, which gives above 99% conversion with 73% 15β selectivity for testosterone. By contrast, the rest ones and the fused enzymes show much less or negligible activity. With the aid of redox pair of Fdr_0978/Fdx_1499, CYP109B1 is used for hydroxylating different steroids. The results show that CYP109B1 displayed good to excellent activity and selectivity toward four testosterone derivatives, giving all 15β-hydroxylated steroids as main products except for 9 (10)-dehydronandrolone, for which the selectivity is shifted to 16β. While for substrates bearing bulky substitutions at C17 position, the activity is essentially lost. Finally, the origin of activity and selectivity for CYP109B1 catalyzed steroid hydroxylation is revealed by computational analysis, thus providing theoretical basis for directed evolution to further improve its catalytic properties.
研究领域:
生物化学
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生物催化
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酶学
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类固醇化学
✓ 已验证
DOI
PubMed
高影响因子
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