Synthesis of the rare disaccharide nigerose by structure-based design of a phosphorylase mutant with altered regioselectivity

M. Kraus; J. Görl; M. Timm; J. Seibel

doi:10.1039/c6cc00934d

Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-α-glucosylglycerol

Microbial Cell Factories ◽

10.1186/s12934-021-01569-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Katharina N. Schwaiger ◽

Monika Cserjan-Puschmann ◽

Gerald Striedner ◽

Bernd Nidetzky

Keyword(s):

Specific Activity ◽

Dry Mass ◽

High Yield ◽

Sucrose Phosphorylase ◽

Bifidobacterium Adolescentis ◽

Enzymatic Production ◽

Intracellular Enzyme ◽

Whole Cell ◽

Freeze Thaw ◽

Thaw Cycle

Abstract Background Glucosylglycerol (2-O-α-d-glucosyl-sn-glycerol; GG) is a natural osmolyte from bacteria and plants. It has promising applications as cosmetic and food-and-feed ingredient. Due to its natural scarcity, GG must be prepared through dedicated synthesis, and an industrial bioprocess for GG production has been implemented. This process uses sucrose phosphorylase (SucP)-catalyzed glycosylation of glycerol from sucrose, applying the isolated enzyme in immobilized form. A whole cell-based enzyme formulation might constitute an advanced catalyst for GG production. Here, recombinant production in Escherichia coli BL21(DE3) was compared systematically for the SucPs from Leuconostoc mesenteroides (LmSucP) and Bifidobacterium adolescentis (BaSucP) with the purpose of whole cell catalyst development. Results Expression from pQE30 and pET21 plasmids in E. coli BL21(DE3) gave recombinant protein at 40–50% share of total intracellular protein, with the monomeric LmSucP mostly soluble (≥ 80%) and the homodimeric BaSucP more prominently insoluble (~ 40%). The cell lysate specific activity of LmSucP was 2.8-fold (pET21; 70 ± 24 U/mg; N = 5) and 1.4-fold (pQE30; 54 ± 9 U/mg, N = 5) higher than that of BaSucP. Synthesis reactions revealed LmSucP was more regio-selective for glycerol glycosylation (~ 88%; position O2 compared to O1) than BaSucP (~ 66%), thus identifying LmSucP as the enzyme of choice for GG production. Fed-batch bioreactor cultivations at controlled low specific growth rate (µ = 0.05 h−1; 28 °C) for LmSucP production (pET21) yielded ~ 40 g cell dry mass (CDM)/L with an activity of 2.0 × 104 U/g CDM, corresponding to 39 U/mg protein. The same production from the pQE30 plasmid gave a lower yield of 6.5 × 103 U/g CDM, equivalent to 13 U/mg. A single freeze–thaw cycle exposed ~ 70% of the intracellular enzyme activity for GG production (~ 65 g/L, ~ 90% yield from sucrose), without releasing it from the cells during the reaction. Conclusions Compared to BaSucP, LmSucP is preferred for regio-selective GG production. Expression from pET21 and pQE30 plasmids enables high-yield bioreactor production of the enzyme as a whole cell catalyst. The freeze–thaw treated cells represent a highly active, solid formulation of the LmSucP for practical synthesis.

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Mapping the acceptor site of sucrose phosphorylase from Bifidobacterium adolescentis by alanine scanning

Journal of Molecular Catalysis B Enzymatic ◽

10.1016/j.molcatb.2013.06.014 ◽

2013 ◽

Vol 96 ◽

pp. 81-88 ◽

Cited By ~ 13

Author(s):

Tom Verhaeghe ◽

Margo Diricks ◽

Dirk Aerts ◽

Wim Soetaert ◽

Tom Desmet

Keyword(s):

Acceptor Site ◽

Sucrose Phosphorylase ◽

Bifidobacterium Adolescentis ◽

Alanine Scanning

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Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083

Applied Microbiology and Biotechnology ◽

10.1007/s00253-003-1534-x ◽

2004 ◽

Vol 65 (2) ◽

pp. 219-227 ◽

Cited By ~ 53

Author(s):

L. A. M. van den Broek ◽

E. L. van Boxtel ◽

R. P. Kievit ◽

R. Verhoef ◽

G. Beldman ◽

...

Keyword(s):

Sucrose Phosphorylase ◽

Bifidobacterium Adolescentis ◽

Physico Chemical

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Continuous process technology for glucoside production from sucrose using a whole cell-derived solid catalyst of sucrose phosphorylase

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11411-x ◽

2021 ◽

Author(s):

Andreas Kruschitz ◽

Linda Peinsipp ◽

Martin Pfeiffer ◽

Bernd Nidetzky

Keyword(s):

Continuous Production ◽

Polymer Materials ◽

Packed Bed Reactor ◽

Solid Catalyst ◽

Scale Production ◽

Process Technology ◽

Sucrose Phosphorylase ◽

Bifidobacterium Adolescentis ◽

Whole Cell ◽

Highly Active

Abstract Advanced biotransformation processes typically involve the upstream processing part performed continuously and interlinked tightly with the product isolation. Key in their development is a catalyst that is highly active, operationally robust, conveniently produced, and recyclable. A promising strategy to obtain such catalyst is to encapsulate enzymes as permeabilized whole cells in porous polymer materials. Here, we show immobilization of the sucrose phosphorylase from Bifidobacterium adolescentis (P134Q-variant) by encapsulating the corresponding E. coli cells into polyacrylamide. Applying the solid catalyst, we demonstrate continuous production of the commercial extremolyte 2-α-d-glucosyl-glycerol (2-GG) from sucrose and glycerol. The solid catalyst exhibited similar activity (≥70%) as the cell-free extract (~800 U g−1 cell wet weight) and showed excellent in-operando stability (40 °C) over 6 weeks in a packed-bed reactor. Systematic study of immobilization parameters related to catalyst activity led to the identification of cell loading and catalyst particle size as important factors of process optimization. Using glycerol in excess (1.8 M), we analyzed sucrose conversion dependent on space velocity (0.075–0.750 h−1) and revealed conditions for full conversion of up to 900 mM sucrose. The maximum 2-GG space-time yield reached was 45 g L−1 h−1 for a product concentration of 120 g L−1. Collectively, our study establishes a step-economic route towards a practical whole cell-derived solid catalyst of sucrose phosphorylase, enabling continuous production of glucosides from sucrose. This strengthens the current biomanufacturing of 2-GG, but also has significant replication potential for other sucrose-derived glucosides, promoting their industrial scale production using sucrose phosphorylase. Key points • Cells of sucrose phosphorylase fixed in polyacrylamide were highly active and stable. • Solid catalyst was integrated with continuous flow to reach high process efficiency. • Generic process technology to efficiently produce glucosides from sucrose is shown. Graphical abstract

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Prebiotics from energy cane bagasse

Sugar Industry ◽

10.36961/si24679 ◽

2020 ◽

pp. 488-494

Author(s):

Giovanna M. Aita ◽

Young Hwan Moon

Keyword(s):

Biological Effects ◽

Caloric Content ◽

Gut Health ◽

Activated Carbon Adsorption ◽

Bifidobacterium Adolescentis ◽

Great Opportunity ◽

Energy Cane ◽

Carbon Adsorption ◽

Combined Use ◽

Crude Sample

Xylooligosaccharides (XOS) is a group of emerging prebiotics that selectively stimulate the growth of advantageous gastrointestinal bacteria benefitting the host’s gut health and functionality. XOS can achieve positive biological effects at low daily doses and low caloric content, properties that are the same or more desirable than the already established prebiotics. XOS are present in plants in very low amounts so there is a great opportunity to isolate XOS with varying degrees of polymerization from the hemicellulose (xylan) fraction of lignocellulosic materials (e.g., bagasse), a source that offers both economic and environmental advantages. In this study, the recovery of XOS by the combined use of activated carbon adsorption, water washing and ethanol desorption from diluted acid pretreated energy cane bagasse hydrolysates was evaluated. The recovered XOS was tested for its prebiotic activity on Bifidobacterium adolescentis ATCC 15703. The final product of extracted XOS from energy cane bagasse (XOS EC Bagasse crude sample) had a purity of 93%, which was comparable to the purities observed with two commercially available XOS prebiotics, CPA (89%) and CPB (93%). XOS EC Bagasse crude sample exhibited prebiotic properties by stimulating the growth of B. adolescentis ATCC 15703 and by producing lactic acid, which were comparable to those observed with the commercial prebiotics.

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LC-MS/MS Method Development and Validation for the Determination of Ilexsaponin A1 and Its Application in Intestinal Bacterial Metabolic Study

Current Pharmaceutical Analysis ◽

10.2174/1573412915666190304141416 ◽

2020 ◽

Vol 16 (6) ◽

pp. 774-781

Author(s):

Liang Wu ◽

An Kang ◽

Yujie Lin ◽

Chenxiao Shan ◽

Zhu Zhou ◽

...

Keyword(s):

Method Development ◽

Ischemia Reperfusion ◽

Scientific Evidence ◽

Potential Effect ◽

Bacterial Strains ◽

Metabolic Potential ◽

Bifidobacterium Adolescentis ◽

Metabolic Behavior ◽

And Behavior

Background: Ilexsaponin A1, one of the most representative triterpene saponin components in the roots of I. pubescens, showed its effects in anticoagulation and antithrombosis, attenuating ischemia-reperfusion-induced myocardial, angiogenesis and inhibiting phosphodiesterase. Objective: Reveal the key intestinal bacterial strains responsible for ilexsaponin A1 metabolism, and clarify their metabolic behavior. Methods: An accurate and sensitive LC-MS/MS method for the determination of “ilexsaponin A1 in General Anaerobic Medium (GAM) broth” was established and systematically validated. Then it was applied to screen and study the metabolic potential of the intestinal bacterial strains in an anaerobic incubation system. Results: Quantitation of ilexsaponin A1 could be performed within an analytical run time of 14.5 min, in the linear range of 2 - 2000 ng/ml. Enterobacter sakazakii, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium angulatum were identified to have a potential effect to metabolize ilexsaponin A1 to different extents; and further bacterial metabolic studies were performed to clarify their metabolic capacity and behavior. Conclusion: This paper contributes to a better understanding of the intestinal bacterial metabolism of ilexsaponin A1 and provides scientific evidence for its clinical application. Additionally, the importance of intestinal bacterial strains in the disposition of natural products was also highlighted.

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