Dataset Information

Efficient biotransformation of polysialogangliosides for preparation of GM1 by Cellulosimicrobium sp. 21.

ABSTRACT: A new ganglioside transformed strain isolated from soil was identified as Cellulosimicrobium sp. 21. It produced a sialidase which transformed polysialo-gangliosides GD1 and GT1 into a monosialoterahexosylganglioside, i.e., ganglioside GM1. The sialidase had both NeuAc-?-2,3- and NeuAc-?-2,8-sialidase activity without producing asiolo-GM1. The optimum conditions were evaluated and it was found that the transformation was optimally performed at 30 °C and pH 7.0. The substrate should be added at the beginning of the reaction and the concentration of substrate was 3% (w/v). Under these optimum conditions, Cellulosimicrobium sp. 21 converted GD1 and GT1 into GM1 in inorganic medium in a 5 L bioreactor with the recovery rate of 69.3%. The product contained 50.3% GM1 and was purified on silica to give the product with 95% of GM1 with a recovery rate of 30.5%. Therefore, Cellulosimicrobium sp. 21 has potential to be applied in the production of GM1 in the pharmaceutical industry.

SUBMITTER: Zheng Y

PROVIDER: S-EPMC6271061 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Efficient biotransformation of polysialogangliosides for preparation of GM1 by Cellulosimicrobium sp. 21.

Zheng Yan Y Ji Li L Leng Jiayi J Yuan Ye Y Chen Honglei H Gou Dongxia D Gao Yufei Y Zhou Yifa Y

Molecules (Basel, Switzerland) 20141008 10

A new ganglioside transformed strain isolated from soil was identified as Cellulosimicrobium sp. 21. It produced a sialidase which transformed polysialo-gangliosides GD1 and GT1 into a monosialoterahexosylganglioside, i.e., ganglioside GM1. The sialidase had both NeuAc-α-2,3- and NeuAc-α-2,8-sialidase activity without producing asiolo-GM1. The optimum conditions were evaluated and it was found that the transformation was optimally performed at 30 °C and pH 7.0. The substrate should be added at t ...[more]

PMID: 25299818

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Project description:Background21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity.ResultsIn this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known.ConclusionsHeterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.

Dataset Information

Efficient biotransformation of polysialogangliosides for preparation of GM1 by Cellulosimicrobium sp. 21.

Publications

Efficient biotransformation of polysialogangliosides for preparation of GM1 by Cellulosimicrobium sp. 21.

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