Fine-Tuning Enzyme Activity Through Saturation Mutagenesis

2010 ◽  
pp. 271-283 ◽  
Author(s):  
Holly H. Hogrefe
Keyword(s):  
Enzyme Activity ◽  
Fine Tuning ◽  
Saturation Mutagenesis

scholarly journals Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Junyan Ma ◽  
Qian Li ◽  
Haidong Tan ◽  
Hao Jiang ◽  
Kuikui Li ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Specific Activity ◽  
Jerusalem Artichoke ◽  
Fine Tuning ◽  
Substrate Affinity ◽  
Carbohydrate Binding ◽  
Secondary Structure Analysis ◽  
C Terminus ◽  
Glycosylation Sites ◽  
Carbohydrate Binding Modules

Abstract Background Inulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity. Results Five N-glycosylation sites with variable high mannose-type oligosaccharides (Man3–9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in Tm) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity. Conclusion The increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

scholarly journals Saturation mutagenesis at Ser196 in BaCsn46A from Bacillus Amyloliquefaciens Enhances Enzyme Activity and Thermostability

2021 ◽  
Author(s):  
Wang Yi ◽  
Gao Wenjun ◽  
Wang Hailong ◽  
Xu Kepan ◽  
Luo Wen ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Secondary Structure ◽  
Industrial Production ◽  
Bacillus Amyloliquefaciens ◽  
Three Dimensional ◽  
Saturation Mutagenesis ◽  
Specific Enzyme ◽  
Mutation Site ◽  
Excellent Thermal Stability ◽  
Specific Enzyme Activity

Abstract The chitosanase (BaCsn46A) was extracted from Bacillus amyloliquefaciens (GenBank: QEK97559.1) and synthesized after codon optimization. The saturation mutation site was determined by analyzing the sequence and three-dimensional protein model. WT and mutant chitosanase genes were cloned and expressed in E. coli BL21 (DE3). The enzymatic properties of WT and mutants were compared, including the optimal reaction pH, temperature and thermostability. Three mutants S196F, S196Y and S196A with the highest specific enzyme activity were selected for further study. Compared with WT, the specific enzyme activity of S196Y increased by 144.76% (more than other two mutants), and the thermostability was not significantly improved. While the specific enzyme activity of S196A increased by 118.79%, and the thermostability of S196A was much higher than WT. From the perspective of industrial production, S196A is more in line with the requirements of industrial production because of its excellent thermal stability at 60°C. From the results of circular dichroism spectrum, the mutation of chitosanase at Ser196 did not change the secondary protein structure. In addition, CD analysis showed that the secondary structure of WT and mutants did not change significantly, indicating that the improvement of thermostability of S196A was not related to the secondary structure.

scholarly journals Evolved β-Galactosidases from Geobacillus stearothermophilus with Improved Transgalactosylation Yield for Galacto-Oligosaccharide Production

2009 ◽  
Vol 75 (19) ◽  
pp. 6312-6321 ◽  
Author(s):  
Gaël Placier ◽  
Hildegard Watzlawick ◽  
Claude Rabiller ◽  
Ralf Mattes
Keyword(s):  
Enzyme Activity ◽  
Screening Strategy ◽  
Saturation Mutagenesis ◽  
Geobacillus Stearothermophilus ◽  
Wild Type ◽  
Transglycosylation Reaction ◽  
Lactose Concentration ◽  
Hydrolysis Of ◽  
Simple Screening

ABSTRACT A mutagenesis approach was applied to the β-galactosidase BgaB from Geobacillus stearothermophilus KVE39 in order to improve its enzymatic transglycosylation of lactose into oligosaccharides. A simple screening strategy, which was based on the reduction of the hydrolysis of a potential transglycosylation product (lactosucrose), provided mutant enzymes possessing improved synthetic properties for the autocondensation product from nitrophenyl-galactoside and galacto-oligosaccharides (GOS) from lactose. The effects of the mutations on enzyme activity and kinetics were determined. An change of one arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved β-galactosidases, a major trisaccharide was formed. Its structure was characterized as β-d-galactopyranosyl-(1→3)-β-d-galactopyranosyl-(1→4)-d-glucopyranoside (3′-galactosyl-lactose). At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3′-galactosyl-lactose production of 23%.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR).Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS.Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An Automated Workflow to Screen Alkene Reductases Using High-throughput Thin Layer Chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the extent of epoxidation and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at the Leu377 site of SaGGR. We show this TLC-based screen can distinguish between fourfold differences in enzyme activity for select mutants and validated those results by GC–MS. Conclusions With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

scholarly journals An automated workflow to screen alkene reductases using high-throughput thin layer chromatography

2020 ◽  
Author(s):  
Brett M. Garabedian ◽  
Corey W. Meadows ◽  
Florence Mingardon ◽  
Joel M. Guenther ◽  
Tristan de Rond ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reductase Activity ◽  
Enzyme Engineering ◽  
Screening Tools ◽  
Saturation Mutagenesis ◽  
Layer Chromatography

Abstract Background: Synthetic biology efforts often require high-throughput screening tools for enzyme engineering campaigns. While innovations in chromatographic and mass spectrometry-based techniques provide relevant structural information associated with enzyme activity, these approaches can require cost-intensive instrumentation and technical expertise not broadly available. Moreover, complex workflows and analysis time can significantly impact throughput. To this end, we develop an automated, 96-well screening platform based on thin layer chromatography (TLC) and use it to monitor in vitro activity of a geranylgeranyl reductase isolated from Sulfolobus acidocaldarius (SaGGR). Results: Unreduced SaGGR products are oxidized to their corresponding epoxide and applied to thin layer silica plates by acoustic printing. These derivatives are chromatographically separated based on the number of epoxides they possess and are covalently ligated to a chromophore, allowing detection of enzyme variants with unique product distributions or enhanced reductase activity. Herein, we employ this workflow to examine farnesol reduction using a codon-saturation mutagenesis library at site Leu377 of SaGGR. We show this TLC-based screen can distinguish between 4-fold differences in enzyme activity for select mutants and validated those results by GC-MS. Conclusions: With appropriate quantitation methods, this workflow can be used to screen polyprenyl reductase activity and can be readily adapted to analyze broader catalyst libraries whose products are amenable to TLC analysis.

Artificial Transfer Hydrogenases Based on the Biotin−(Strept)avidin Technology:  Fine Tuning the Selectivity by Saturation Mutagenesis of the Host Protein

2006 ◽  
Vol 128 (25) ◽  
pp. 8320-8328 ◽  
Author(s):  
Christophe Letondor ◽  
Anca Pordea ◽  
Nicolas Humbert ◽  
Anita Ivanova ◽  
Sylwester Mazurek ◽  
...  
Keyword(s):  
Fine Tuning ◽  
Host Protein ◽  
Saturation Mutagenesis

Morphological and biochemical analyses of changes in rat hepatocytes related to wine and ethanol consumption

1984 ◽  
Vol 42 ◽  
pp. 232-233
Author(s):  
S.M. Geyer ◽  
C.L. Mendenhall ◽  
J.T. Hung ◽  
E.L. Cardell ◽  
R.L. Drake ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Malic Enzyme ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Mature Male ◽  
Treatment Groups ◽  
Malic Enzyme Activity ◽  
Biochemical Analyses

Thirty-three mature male Holtzman rats were randomly placed in 3 treatment groups: Controls (C); Ethanolics (E); and Wine drinkers (W). The animals were fed synthetic diets (Lieber type) with ethanol or wine substituted isocalorically for carbohydrates in the diet of E and W groups, respectively. W received a volume of wine which provided the same gram quantity of alcohol consumed by E. The animals were sacrificed by decapitation after 6 weeks and the livers processed for quantitative triglycerides (T3), proteins, malic enzyme activity (MEA), light microscopy (LM) and electron microscopy (EM). Morphometric analysis of randomly selected LM and EM micrographs was performed to determine organellar changes in centrilobular (CV) and periportal (PV) regions of the liver. This analysis (Table 1) showed that hepatocytes from E were larger than those in C and W groups. Smooth endoplasmic reticulum decreased in E and increased in W compared to C values.

Fine-Tuning Futures

ASHA Leader ◽  
2017 ◽  
Vol 22 (6) ◽  
Author(s):  
Christi Miller
Keyword(s):  
Fine Tuning
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