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Ulvans, complex polysaccharides found in the ulvales (green seaweed) cell wall, contain predominantly 3-sulfated rhamnose (Rha3S) linked to either d-glucuronic acid, l-iduronic acid or d-xylose.

The versatile synthetic intermediate (2S,4R)-4-hydroxylysine can be produced using L-lysine hydroxylase. However, the wild-type enzyme cannot effectively catalyze the C4 hydroxylation of L-lysine to form the product. To overcome this bottleneck, we modified the L-lysine hydroxylase from Niastella koreensis (NkLH4), using the semirational combinatorial active-site saturation test (CAST). We obtained a highly active mutant MT3 (Q161N/T162A/F178Y/E260D) with a 24.97-fold increase of k cat /K m , compared with the wild-type enzyme (791.33 mM −1 s −1 vs 31.69 mM −1 s −1 ). Further analysis of the structure−activity relationship via molecular dynamics (MD) simulations suggested that MT3 had a more flexible conformation, as well as an enlarged substratebinding pocket with decreased steric hindrance and increased binding energy in substrate recognition. Our study provides a highly active NkLH4 mutant for potential commercial use in the production of enantiomerically pure (2S,4R)-4-hydroxylysine.

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BackgroundBiosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor.ResultsRecombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively).ConclusionsThe successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity. Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0981-0) contains supplementary material, which is available to authorized users.

The protein engineering and metabolic engineering strategies are performed to solve rate-limiting steps in the biosynthesis of 5-HLeu.

According to abrupt diagnosis of runoff, two methods, that is, moving approximate entropy and moving permutation entropy, are used to analyse the abrupt year of the daily river runoff from 1961 to 2006 at Lintong station of Wei River in Loess Plateau. The runoff series are divided into 4 stages. With the analysis of hydrological characters of different stages, we find that there are abrupt changes at the three years 1972, 1983, and 2002. The result shows that moving approximate entropy and moving permutation entropy methods are useful tools for abrupt diagnosis of runoff. The attribution of abrupt change at the Lintong runoff series is primarily due to the reduced precipitation, increased water conservancy project, increased water consumption of industry and agriculture, significantly decreased groundwater table, and increased evaporation.

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