Bacterial production of maize and human serine racemases as partially active inclusion bodies for d-serine synthesis

Wang, Ruyue; Li, Jinfeng; Dang, Dongya; Hu, Jiong; Hu, Yafang; Fan, Jun

doi:10.1016/j.enzmictec.2020.109547

Cited by 6 publications

(2 citation statements)

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“…However, it is now clear that at least some proteins can retain certain activity when embedded in these aggregates, turning them into functional sub-micron particles [23,85]. These nanostructures are finding amazing applications in biotechnology [86] and biomedicine [18,19].…”

Section: Discussionmentioning

confidence: 99%

Coiled-coil inspired functional inclusion bodies

2020

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Background Recombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded. Results In order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only α-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins. Conclusions Here, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This α-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of β-sheet-based IBs and renders highly active and homogeneous submicrometric particles.

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Section: Discussionmentioning

confidence: 99%

Coiled-coil inspired functional inclusion bodies

2020

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“…In addition, AtHNL CatIBs displayed a higher stability at low pH values compared to the native enzyme. Dimeric serine racemases from maize and human sources, which are dependent on pyridoxal 5′-phosphate (PLP) as a cofactor, have been recently added to the growing list of POIs that were incorporated within CatIBs . It is worthwhile to note that, in the case of human and maize serine racemase CatIBs, from the two functions of these enzymes, namely, the reversible racemization of l -serine to d -serine, and the dehydration of both enantiomers to produce pyruvate and ammonia, only the first function has been retained, therefore generating CatIBs with altered substrate specificity.…”

Section: Display/entrapment Of Proteins On/within Inclusion Bodies An...mentioning

confidence: 99%

Emerging Solutions for in Vivo Biocatalyst Immobilization: Tailor-Made Catalysts for Industrial Biocatalysis

Ölçücü

Klaus

Jaeger

et al. 2021

ACS Sustainable Chem. Eng.

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In industry, enzymes are often immobilized to generate more stable enzyme preparations that are easier to store, handle, and recycle for repetitive use. Traditionally, enzymes are bound to inorganic carrier materials, which requires caseto-case optimization and incurs additional labor and costs. Therefore, with the advent of rational protein design strategies as part of bottom-up synthetic biology approaches, numerous immobilization methods have been developed that enable the one-step production and immobilization of enzymes onto biogenic carrier materials often directly within the production host, which we here refer to as in vivo immobilization. As a result, nano-to micro-meter-sized functionalized biomaterials, or biologically produced enzyme immobilizates, are obtained that can directly be used for synthetic purposes. In this Perspective, we provide an overview over established and recently emerging in vivo enzyme immobilization methods, with special emphasis on their applicability for (industrial) biocatalysis. For each approach, we present fundamental working principles as well as advantages and limitations guiding future research avenues toward sustainable applications in the bioindustry.

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