Content Access Key:.
Enzyme-catalyzed Synthesis and Degradation of Biopolymers
Volume 6 , Issue 1 , Journal Home. Keywords: Biopolymer, polyhydroxyalkanoate PHA , PHA synthase, enzyme evolution, PHB depolymerase, lipase Abstract: The synthesis and degradation of biopolymers are cardinal reactions that take place to maintain homeostasis in biosystems. Close Print this page. Content: Citation Only. Crystal structure analysis of Bgl3 revealed that Lys is part of a network of salt bridges, which suggests that this residue plays a role in the structural stability of the enzyme.
It was therefore suggested that Asn induced a crucial shift in the p K a of this catalytic residue. Again, 10 7 enzyme variants were analyzed, revealing several single mutants with improved thermostability including mutant SC.
Further characterization of this mutant revealed a 5. All synthesized genes were pooled in equimolar amounts and used to transform E. After this selection, DNA was isolated from the surviving cells and sequenced by an Illumina sequencing approach to determine the frequency of occurrence of each mutant. From these maps, amino acid substitutions that led to shifts in substrate specificity either towards kanamycin or towards one of the other five tested aminoglycoside antibiotics were identified.
This remarkable shift in substrate specificity underlines the applicability of mutability landscapes to identification of hotspots for enzyme engineering. This ubiquitination promotes degradation of the substrate protein, which is a crucial process for homeostasis. Libraries with on average two random nucleotide mutations per gene were generated, sequenced, and subsequently displayed on bacteriophages. DNA was isolated from enriched bacteriophages and subsequently sequenced by use of Illumina technology.
Enzyme Engineering: Volume 6
Interestingly, several single mutants with improved activity relative to WT could be identified from this mutability landscape. This illustrates the fact that beneficial mutations can be useful both for the generation of superior enzymes and to provide useful insight into enzyme mechanisms. When using a defined collection of single mutants, the bottleneck lies in the generation of this defined mutant gene collection. Other more recently developed mutagenesis techniques include chemoenzymatic methods e.
Moreover, because of the ever decreasing costs of commercially available synthetic DNA, the most economical way to obtain a defined collection of single mutants of an enzyme might be DNA synthesis. Both of these techniques are rapidly evolving, 36 , 37 , 46 which might facilitate the generation of mutability landscapes by use of deep mutational scanning. The generation of mutability landscapes for several properties of one enzyme e.
Furthermore, mutability landscapes can be used to advance our understanding of sequence—function relationships in enzymes because they provide systematic information on neutral, beneficial, and detrimental amino acid substitutions. Both detrimental and beneficial mutations can be extremely helpful for elucidation of enzyme mechanisms.
Enzyme Engineering Volume 6 : Ichiro Chibata :
Volume 17 , Issue The full text of this article hosted at iucr. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. If the address matches an existing account you will receive an email with instructions to retrieve your username. ChemBioChem Volume 17, Issue Minireview Open Access. Gerrit J. Poelarends Corresponding Author E-mail address: g. Tools Request permission Export citation Add to favorites Track citation. Share Give access Share full text access.
Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article.
Abstract Beneficial, detrimental, or neutral? Figure 1 Open in figure viewer PowerPoint. Scheme 1 Open in figure viewer PowerPoint. Figure 2 Open in figure viewer PowerPoint. Scheme 2 Open in figure viewer PowerPoint. Figure 3 Open in figure viewer PowerPoint. A: activity. E: expression. ES: enantioselectivity. SS: substrate specificity.
References 1 U. Bornscheuer , G. Huisman , R. Kazlauskas , S. Lutz , J.
Most Cited Articles The most cited articles published since , extracted from Scopus. Giovanni Benelli. Line Munk Mogens Larsen Andersen Visit our new webpage. Receiving the prestigious award for his research in light-activated ion-channels. Special Issues. Special issues published in Enzyme and Microbial Technology. Sheldon May Dr. Enrico Marsili. Enrico Marsili Sujoy K Das. Tai Hyun Park.
PlumX Metrics. Below is a recent list of — articles that have had the most social media attention.
The Plum Print next to each article shows the relative activity in each of these categories of metrics: Captures, Mentions, Social Media and Citations. Go here to learn more about PlumX Metrics. Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.
Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8. Baeyer-Villiger monooxygenases from Yarrowia lipolytica catalyze preferentially sulfoxidations. Tweets by ElsevierBiotech. Advertisers Media Information.