Recent advances in the improvement of enzyme thermostability by structure modification

Xu, Zhe; Cen, Yu-Ke; Zou, Shu-Ping; Xue, Ya‐Ping; Zheng, Yu‐Guo

doi:10.1080/07388551.2019.1682963

Cited by 169 publications

(74 citation statements)

References 127 publications

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“…The glycosylated product has superior performance and can be used in food, pharmaceuticals, cosmetics, and other industrial fields. However, the deficient thermostability limits enzyme’s application in continuous industrial production (Xu et al 2020 ). Take the production of αGG as an example.…”

Section: Discussionmentioning

confidence: 99%

“…In the process of synthesizing αGG, due to the long reaction time, it is usually necessary to keep the temperature at 30 °C (Bolivar et al 2017 ; Goedl et al 2008 ). Therefore, improving the thermostability of enzymes to enhance their usability in the industry has attracted increasing interest, and thus, various auxiliary methods have been developed to help predict potential thermostable mutants (Sun et al 2019 ; Xu et al 2020 ). The semi-rational design combines the benefits of directed evolution and rational design and is suitable for modifying proteins without high-throughput determination methods and structure–function understanding (Chica et al 2005 ).…”

Section: Introductionmentioning

confidence: 99%

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Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Xia

Yang

et al. 2021

Appl Microbiol Biotechnol

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Sucrose phosphorylase (SPase) can specifically catalyze transglycosylation reactions and can be used to enzymatically synthesize α-D-glycosides. However, the low thermostability of SPase has been a bottleneck for its industrial application. In this study, a SPase gene from Leuconostoc mesenteroides ATCC 12,291 (LmSPase) was synthesized with optimized codons and overexpressed successfully in Escherichia coli. A semi-rational design strategy that combined the FireProt (a web server designing thermostable proteins), structure–function analysis, and molecular dynamic simulations was used to improve the thermostability of LmSPase. Finally, one single-point mutation T219L and a combination mutation I31F/T219L/T263L/S360A (Mut4) with improved thermostability were obtained. The half-lives at 50 °C of T219L and Mut4 both increased approximately two-fold compared to that of wild-type LmSPase (WT). Furthermore, the two variants T219L and Mut4 were used to produce α-D-glucosylglycerol (αGG) from sucrose and glycerol by incubating with 40 U/mL crude extracts at 37 °C for 60 h and achieved the product concentration of 193.2 ± 12.9 g/L and 195.8 ± 13.1 g/L, respectively, which were approximately 1.3-fold higher than that of WT (150.4 ± 10.0 g/L). This study provides an effective strategy for improving the thermostability of an industrial enzyme. Key points • Predicted potential hotspot residues directing the thermostability of LmSPase by semi-rational design • Screened two positive variants with higher thermostability and higher activity • Synthesized α-D-glucosylglycerol to a high level by two screened positive variants

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Xia

Yang

et al. 2021

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

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“…This might be explained by a differing influence of intraand intermolecular interactions. For thermal stability, intramolecular interactions such as hydrogen bonds or salt bridges within a molecule are mainly responsible for delaying unfolding under heat [30], while for mechanical stability, in addition to intramolecular interactions, intermolecular interactions, especially at crystal contacts, and the arrangement of molecules in the crystal lattice are important. In addition, the mechanical stability can be strongly influenced by the cross-linking with glutaraldehyde [17].…”

Section: Mechanical Characterization Of Pga Clecsmentioning

confidence: 99%

Structure-Properties Correlation of Cross-Linked Penicillin G Acylase Crystals

et al. 2021

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In biocatalytic processes, the use of free enzymes is often limited due to the lack of long-term stability and reusability. To counteract this, enzymes can be crystallized and then immobilized, generating cross-linked enzyme crystals (CLECs). As mechanical stability and activity of CLECs are crucial, different penicillin G acylases (PGAs) from Gram-positive organisms have proven to be promising candidates for industrial production of new semisynthetic antibiotics, which can be crystallized and cross-linked to characterize the resulting CLECs regarding their mechanical and catalytic properties. The greatest hardness and Young’s modulus determined by indentation with an atomic force microscope were observed for CLECs of Bacillus species FJAT-PGA CLECs (26 MPa/1450 MPa), followed by BmPGA (Priestia megaterium PGA, 23 MPa/1170 MPa) and BtPGA CLECs (Bacillus thermotolerans PGA, 11 MPa/614 MPa). In addition, FJAT- and BtPGA CLECs showed up to 20-fold higher volumetric activities compared to BmPGA CLECs. Correlation to structural characteristics indicated that a high solvent content and low number of cross-linking residues might lead to reduced stability. Furthermore, activity seems to be restricted by small water channels due to severe diffusion limitations. To the best of our knowledge, we show for the first time in this study that the entire process chain for the characterization of diverse industrially relevant enzymes can be performed at the microliter scale to discover the most important relationships and limitations.

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“…However, researchers are now focused on how to improve its efficacy with respect to various aspects by involving various biotechnological tools. Furthermore, scientists are also emphasizing enabling or modifying pectinase so that it could be of application in different bioprocessing industrial sectors (11,12). Pectinolytic enzymes are employed in many industrial applications but are mainly utilized in food industries in specific operations such as the clarification of fruit juices and wines and the extraction of vegetable oils.…”

Section: Introductionmentioning

confidence: 99%

Biochemical Prospects of Various Microbial Pectinase and Pectin: An Approachable Concept in Pharmaceutical Bioprocessing

et al. 2020

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Both pectin and pectinase are vitally imperative biomolecules in the biotechnological sector. These molecules are a feasible non-toxic contrivance of nature with extensive applicative perception. Understanding pectic substances and their structure, unique depolymerization, and biochemical properties such as a catalytic mechanism and the strong interrelationship among these molecules could immensely enhance their applicability in industries. For instance, gaining knowledge with respect to the versatile molecular heterogeneity of the compounds could be considered as the center of concern to resolve the industrial issues from multiple aspects. In the present review, an effort has been made to orchestrate the fundamental information related to structure, depolymerization characteristics, and classification of pectin as well as the types and biochemical properties of pectinase. Furthermore, various production methods related to the optimization of the product and its significant contribution to the pharmaceutical industry (either pectinase or derived pectic substances) are described in this article.

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Recent advances in the improvement of enzyme thermostability by structure modification

Cited by 169 publications

References 127 publications

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Development of thermostable sucrose phosphorylase by semi-rational design for efficient biosynthesis of alpha-D-glucosylglycerol

Structure-Properties Correlation of Cross-Linked Penicillin G Acylase Crystals

Biochemical Prospects of Various Microbial Pectinase and Pectin: An Approachable Concept in Pharmaceutical Bioprocessing

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