Crocins are the most important active ingredient found in Crocus sativus, a well-known "plant gold". The glycosyltransferase-catalyzed glycosylation of crocetin is the last step of biosynthesizing crocins and contributes to their structural diversity. Crocin biosynthesis is now hampered by the lack of efficient glycosyltransferases with activity toward crocetin. In this study, two microbial glycosyltransferases (Bs-GT and Bc-GTA) were successfully mined based on the comprehensive analysis of the PSPG motif and the N-terminal motif of the target plant-derived UGT75L6 and Cs-GT2. Bs-GT from Bacillus subtilis 168, an enzyme with a higher activity of glycosylation toward crocetin than that of Bc-GTA, was characterized. The efficient synthesis of crocins from crocetin catalyzed by microbial GT (Bs-GT) was first reported with a high molecular conversion rate of 81.9%, resulting in the production of 476.8 mg/L of crocins. The glycosylation of crocetin on its carboxyl groups by Bs-GT specifically produced crocin-5 and crocin-3, the important rare crocins.
A rapid and convenient strategy to monitor the productivity of biomanufacturing is essential for the research in optimizing relevant bioprocesses. In this work, we have developed a fluorescein-derived probe (FL-DT) that reacts rapidly with thiol groups via 1,4-Michael addition reaction of the sulfhydryl to unsaturated ketone and releases fluorescence. FL-DT specifically forms fluorescent adduct with two adjacent thiols in a protein of interest (POI), making the probe a reliable tool for protein quantification.The production of xylanase fused with a short di-Cys tag was then successfully monitored and quantified with FL-DT in Escherichia coli system under different protein expression conditions, providing useful information for optimizing the bioprocess.Our work provides a convenient and efficient strategy for POI labeling and monitoring bioproduction.
Conventional
enzyme immobilization approaches can only
immobilize
certain specific enzymes with poor generality. Attempts to improve
the universality of enzyme types tend to impart them with more enzymatic
catalysis applications. Here, inspired by mussel adhesive proteins,
we present a novel eco-friendly surface carrier that was 3D printed
and modified by electro-oxidation for enzyme immobilization. The carrier
was fabricated through 3D printing by transforming acrylonitrile butadiene
styrene (ABS) material into a suitable structure (3DABS). Then, electro-oxidative
modification was performed on the surface to form a polydopamine (PDA)
coating (3DABS-PDA). The desired structures for the enzyme immobilization
carriers were obtained through 3D printing technology, while electro-oxidation
modification of the surface provided numerous and firmly covalent
binding sites. Based on these features, we have demonstrated that
3D printed and electro-oxidation-modified carriers could be applied
to immobilize different types of enzymes. The loading capacity of
all immobilized enzymes (galV, EG5C-1, XynLK9, and kdcA) exceeded
25 mg·g–1 (37.7 mg·g–1 for galV), and after 10 reuse cycles, the substrate conversion rate
of 3DABS-PDA@galV was still over 85%. The carriers can be reused after
simple processing. These results indicate that 3DABS-PDA provides
an efficient, sustainable, and versatile approach for enzyme immobilization
and exhibits excellent value in various enzymatic catalysis applications.
A rapid and convenient strategy to monitor the productivity of
biomanufacturing is essential for the research in optimizing relevant
bioprocesses. In this work, we have developed a fluorescein-derived
probe (FL-DT) that reacts rapidly with thiol groups via 1, 4-Micheal
addition reaction of the sulfhydryl to unsaturated ketone and releases
fluorescence. FL-DT specifically forms fluorescent adduct with two
adjacent thiols in a protein of interest (POI), making the probe a
reliable tool for protein quantification. The production of xylanase
fused with a short di-Cys tag was then successfully monitored and
quantified with FL-DT in E. coli system under different protein
expression conditions, providing useful information for optimizing the
bioprocess. Our work provides a convenient and efficient strategy for
POI labeling and monitoring bioproduction.
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