ChemInform Abstract: Directed Evolution of Enzymes for Industrial Biocatalysis

Porter, Joanne L.; Rusli, Rukhairul A.; Ollis, David L.

doi:10.1002/chin.201615268

Cited by 4 publications

(4 citation statements)

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“…The use of enzymes has gained space by presenting high specificity and not generating undesirable parallel products as occurs in chemical synthesis reactions, in addition to the easy Research, Society and Development, v. 9, n. 7, e694974810, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i7.4810 4 regulation of enzymatic activity through variables such as temperature and pH. As a result, enzymatic industrial processes become easy to control, relatively simple, energy efficient and low-cost (Porter, Rusli, & Ollis, 2016).…”

Section: Introductionmentioning

confidence: 99%

Produção e caracterização da amilase obtida de Rhizopus microsporus var. oligosporus

Santos

Mendes

Miranda

et al. 2020

RSD

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Amylases have the ability to catalyze the α-1,4 binding of starch by releasing glucose and dextrin, with prominence in several industrial fields. The research sought to characterize the enzyme amylase obtained from the fungus Rhizopus microsporus var. oligosporus, through the Doehlert methodology, evaluating the behavior of the enzyme in the face of variations in pH and temperature. The production of amylase occurred with the use of starch as an inducer, and the characterization was carried out using the response surface methodology, with pH analysis at 5 levels (3.0, 4.0, 5.0, 6.0 and 7.0) and temperature in 3 levels (30, 50 and 70°C). The evaluation of the amylase thermostability occurred at 60, 70 and 80ºC. The application of the experimental model indicated that the amylase obtained from R. microsporus var. oligosporus shows better catalytic development at temperatures between 40ºC and 55ºC and pH between 2.5 and 3.2. The evaluation of thermostability indicated that the increase in temperature negatively influences the catalytic activity of amylase. The experimental model led to the understanding of the conditions favorable to the production of amylases from R. microsporus var. oligosporus.

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Section: Introductionmentioning

confidence: 99%

Produção e caracterização da amilase obtida de Rhizopus microsporus var. oligosporus

Santos

Mendes

Miranda

et al. 2020

RSD

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“…Protein engineers have been successfully improving enzymes for more than 30 years using a variety of experimental and computational techniques. Tremendous advances have been made including the design and engineering of enzymes with activities not found in nature [1][2][3]. Metabolic engineering and some synthetic biology applications focus on assembling enzymes from different organisms to perform novel multi-step chemical reac-tions, in vitro or in vivo [4][5][6][7].…”

Section: Protein Engineering Scaffold Structuresmentioning

confidence: 99%

Extreme makeover: Engineering the activity of a thermostable alcohol dehydrogenase (AdhD) from Pyrococcus furiosus

Solanki

Abdallah

Banta

2016

Biotechnology Journal

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Alcohol dehydrogenase D (AdhD) is a monomeric thermostable alcohol dehydrogenase from the aldo-keto reductase (AKR) superfamily of proteins. We have been exploring various strategies of engineering the activity of AdhD so that it could be employed in future biotechnology applications. Driven by insights made in other AKRs, we have made mutations in the cofactor-binding pocket of the enzyme and broadened its cofactor specificity. A pre-steady state kinetic analysis yielded new insights into the conformational behavior of this enzyme. The most active mutant enzyme concomitantly gained activity with a non-native cofactor, nicotinamide mononucleotide, NMN(H), and an enzymatic biofuel cell was demonstrated with this enzyme/cofactor pair. Substrate specificity was altered by grafting loop regions near the active site pocket from a mesostable human aldose reductase (hAR) onto the thermostable AdhD. These moves not only transferred the substrate specificity of hAR but also the cofactor specificity of hAR. We have added alpha-helical appendages to AdhD to enable it to self-assemble into a thermostable catalytic proteinaceous hydrogel. As our understanding of the structure/function relationship in AdhD and other AKRs advances, this ubiquitous protein scaffold could be engineered for a variety of catalytic activities that will be useful for many future applications.

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“…Directed evolution enables the selection of protein variants with improved properties as therapeutics and biocatalysts 1, 2, 3 . The generation of genetic variability followed by a screening process are the essential steps of directed evolution 4, 5 .…”

Section: Introductionmentioning

confidence: 99%

In vivodiversification of target genomic sites using processive T7 RNA polymerase-base deaminase fusions blocked by RNA-guided dCas9

Álvarez

Mencı́a

Fernández

2019

Preprint

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32Diversification of specific DNA segments typically involve in vitro generation of large sequence 33 libraries and their introduction in cells for selection. Alternative in vivo mutagenesis systems on 34 cells often show deleterious offsite mutations and restricted capabilities. To overcome these 35 limitations, we have developed an in vivo platform to diversify specific DNA segments based on 36 protein fusions between various base deaminases (BD) and the T7 RNA polymerase (T7RNAP) 37 that recognizes a cognate promoter oriented towards the target sequence. The transcriptional 38 elongation of these fusions generates transitions C to T or A to G on both DNA strands and in 39 long DNA segments. To delimit the boundaries of the diversified DNA, the catalytically dead 40 Cas9 (dCas9) is tethered with custom-designed crRNAs as a "roadblock" for BD-T7RNAP 41 elongation. While the efficiency of this platform is demonstrated in E. coli, the system can be 42 adapted to a variety of bacterial and eukaryotic hosts. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58Directed evolution enables the selection of protein variants with improved properties as 59 therapeutics and biocatalysts 1, 2, 3 . The generation of genetic variability followed by a screening 60 process are the essential steps of directed evolution 4, 5 . In vitro mutagenesis techniques (e.g., 61error-prone PCR) can quickly produce large number of variants of the target gene but their 62 selection requires, in most cases, cloning and transformation into a host cell for expression (e.g., 63E. coli). These steps are time-consuming and labor-intensive, especially when iterative cycles 64 of mutagenesis and selection are needed. Cell-free selection methods are also feasible 6 , but 65 they are technically demanding and functional expression of complex proteins (e.g. membrane 66 proteins, multimeric enzymes) is often difficult to achieve. Hence, in vivo mutagenesis systems 67 are preferred because the generation of genetic variants, their expression and selection can 68 done in a continuous process, which accellerates directed evolution 5, 7 . 70Long-established in vivo mutagenesis methods (e.g., chemical mutagens, radiations) do not 71 target specific genes and are deleterious for the host cell due to accumulation of random 72 mutations in the genome 8 . Similarly, "mutator" host strains do not allow the concentration of the 73 mutagenic activity on the target gene, inducing the accumulation of unwanted mutations in the 74 host genome 9, 10, 11, 12 . A few in vivo mutagenesis systems with targeted specificity have been 75 reported for E. coli and yeast cells, which are the preferred hosts for cloning and expression of 76 gene libraries 5 . For example, an error-prone variant of E. coli DNA polymersase I enables the 77 mutagenesis of cloned genes in ColE1 plasmids, although it concentrates the mutations close 78 to the origin of replication 13 . A different approach involves the transformation of E. coli with 79 mutant oligonucleotide libraries, targeting one or multip...

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ChemInform Abstract: Directed Evolution of Enzymes for Industrial Biocatalysis

Abstract: Review: 106 refs.

Cited by 4 publications

References 78 publications

Produção e caracterização da amilase obtida de Rhizopus microsporus var. oligosporus

Produção e caracterização da amilase obtida de Rhizopus microsporus var. oligosporus

Extreme makeover: Engineering the activity of a thermostable alcohol dehydrogenase (AdhD) from Pyrococcus furiosus

In vivodiversification of target genomic sites using processive T7 RNA polymerase-base deaminase fusions blocked by RNA-guided dCas9

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