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Whole-cell bioconversion using non-Leloir transglycosylation reactions: a review
Inonge Noni Siziya,정종현,서명지,임민철,서동호 한국식품과학회 2023 Food Science and Biotechnology Vol.32 No.6
Microbial biocatalysts are evolving technological tools for glycosylation research in food, feed and pharmaceuticals. Advances in bioengineered Leloir and non-Leloir carbohydrate-active enzymes allow for whole-cell biocatalysts to curtail production costs of purified enzymes while enhancing glucan synthesis through continued enzyme expression. Unlike sugar nucleotide-dependent Leloir glycosyltransferases, non-Leloir enzymes require inexpensive sugar donors and can be designed to match the high value, yield and selectivity of the former. This review addresses the current state of bacterial cell-based production of glucans and glycoconjugates via transglycosylation, and describes how alterations made to microbial hosts to surpass purified enzymes as the preferred mode of catalysis are steadily being acquired through genetic engineering, rational design and process optimization. A comprehensive exploration of relevant literature has been summarized to describe whole-cell biocatalysis in non-Leloir glycosylation reactions with various donors and acceptors, and the characterization, application and latest developments in the optimization of their use.
Siziya Inonge Noni,Yoon Deok Jun,Kim Mibang,서명지 한국미생물·생명공학회 2022 Journal of microbiology and biotechnology Vol.32 No.7
The rising demand for carotenoids can be met by microbial biosynthesis as a promising alternative to chemical synthesis and plant extraction. Several species of lactic acid bacteria (LAB) specifically produce C30 carotenoids and offer the added probiotic benefit of improved gut health and protection against chronic conditions. In this study, the recently characterized Lactiplantibacillus plantarum subsp. plantarum KCCP11226T produced the rare C30 carotenoid, 4,4'-diaponeurosporene, and its yield was optimized for industrial production. The one-factor-at-a-time (OFAT) method was used to screen carbon and nitrogen sources, while the abiotic stresses of temperature, pH, and salinity, were evaluated for their effects on 4,4'-diaponeurosporene production. Lactose and beef extract were ideal for optimal carotenoid production at 25°C incubation in pH 7.0 medium with no salt. The main factors influencing 4,4'-diaponeurosporene yields, namely lactose level, beef extract concentration and initial pH, were enhanced using the Box-Behnken design under response surface methodology (RSM). Compared to commercial MRS medium, there was a 3.3-fold increase in carotenoid production in the optimized conditions of 15% lactose, 8.3% beef extract and initial pH of 6.9, producing a 4,4'-diaponeurosporene concentration of 0.033 A470/ml. To substantiate upscaling for industrial application, the optimal aeration rate in a 5 L fermentor was 0.3 vvm. This resulted in a further 3.8-fold increase in 4,4'-diaponeurosporene production, with a concentration of 0.042 A470/ml, compared to the flask-scale cultivation in commercial MRS medium. The present work confirms the optimization and scale-up feasibility of enhanced 4,4'-diaponeurosporene production by L. plantarum subsp. plantarum KCCP11226T.
홍승표,Inonge Noni Siziya,서명지,박천석,서동호 한국미생물·생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.9
Amylosucrase (ASase, E.C. 2.4.1.4) is capable of efficient glucose transfer from sucrose, acting as the sole donor molecule, to various functional acceptor compounds, such as polyphenols and flavonoids. An ASase variant from Deinococcus geothermalis, in which the 226th alanine is replaced with asparagine (DgAS-A226N), shows increased polymerization activity due to changes in the flexibility of the loop near the active site. In this study, we further investigated how the mutation modulates the enzymatic activity of DgAS using molecular dynamics and docking simulations to evaluate interactions between the enzyme and phenolic compounds. The computational analysis revealed that the A226N mutation could induce and stabilize structural changes near the substratebinding site to increase glucose transfer efficiency to phenolic compounds. Kinetic parameters of DgAS-A226N and WT DgAS were determined with sucrose and 4-methylumbelliferone (MU) as donor and acceptor molecules, respectively. The kcat/Km value of DgAS-A226N with MU (6.352 mM-1min-1) was significantly higher than that of DgAS (5.296 mM-1min-1). The enzymatic activity was tested with a small phenolic compound, hydroquinone, and there was a 1.4-fold increase in α-arbutin production. From the results of the study, it was concluded that DgAS-A226N has improved acceptor specificity toward small phenolic compounds by way of stabilizing the active conformation of these compounds.
김예진,Inonge Noni Siziya,홍승표,이길용,서명지,김영록,유상호,박천석,서동호 한국식품과학회 2021 Food Science and Biotechnology Vol.30 No.2
Amylosucrase (ASase, E.C. 2.4.1.4) is a powerful transglycosylation enzyme that can transfer glucose from sucrose to the hydroxyl (-OH) group of various compounds. In this study, recombinant ASases from Deinococcus geothermalis (DgAS) and Bifidobacterium thermophilum (BtAS) were used to synthesize biosurfactants based on the computational analysis of predicted docking simulations. Successful predictions of the binding affinities, conformations, and three-dimensional structures of three surfactants were computed from receptor-ligand binding modes. DgAS and BtAS were effective in the synthesis of biosurfactants from glyceryl caprylate, glyceryl caprate, and polyglyceryl-2 caprate. The results of the transglycosylation reaction were consistent for both ASases, with glyceryl caprylate acceptor showing the highest concentration, as confirmed by thin layer chromatography. Furthermore, the transglycosylation reactions of DgAS were more effective than those of BtAS. Among the three substrates, glyceryl caprylate glycoside and glyceryl caprate glycoside were successfully purified by liquid chromatography–mass spectrometry (LC–MS) with the corresponding molecular weights.