Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

Chen, Zhenya; Li, Ye; Feng, Yue; Chen, Liang; Yuan, Qipeng

doi:10.1039/c5ra15220h

Cited by 19 publications

(8 citation statements)

References 42 publications

(49 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1). It was reported that furfural of 10 mM (approximately 1 g/L) slightly increased Thermoanaerobacter pseudethanolicus ’s cell yield by 11% [35]. Furfural could replace glycerol as an electron sink and allow less carbon loss to byproducts and more carbon distribution towards biomass synthesis [36].…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Furfural tolerance and detoxification mechanism in Candida tropicalis

Wang

Cheng

Joshua

et al. 2016

Biotechnol Biofuels

Self Cite

View full text Add to dashboard Cite

BackgroundCurrent biomass pretreatment by hydrothermal treatment (including acid hydrolysis, steam explosion, and high-temperature steaming) and ionic liquids generally generate inhibitors to the following fermentation process. Furfural is one of the typical inhibitors generated in hydrothermal treatment of biomass. Furfural could inhibit cell growth rate and decrease biofuel productivity of microbes. Candida tropicalis is a promising microbe for the production of biofuels and value-added chemicals using hemicellulose hydrolysate as carbon source. In this study, C. tropicalis showed a comparable ability of furfural tolerance during fermentation. We investigated the mechanism of C. tropicalis’s robust tolerance to furfural and relevant metabolic responses to obtain more information for metabolic engineering of microbes for efficient lignocellulose fermentation.Results Candida tropicalis showed comparable intrinsic tolerance to furfural and a fast rate of furfural detoxification. C. tropicalis’s half maximal inhibitory concentration for furfural with xylose as the sole carbon source was 3.69 g/L, which was higher than that of most wild-type microbes reported in the literature to our knowledge. Even though furfural prolonged the lag phase of C. tropicalis, the final biomass in the groups treated with 1 g/L furfural was slightly greater than that in the control groups. By real-time PCR analysis, we found that the expression of ADH1 in C. tropicalis (ctADH1) was induced by furfural and repressed by ethanol after furfural depletion. The expression of ctADH1 could be regulated by both furfural and ethanol. After the disruption of gene ctADH1, we found that C. tropicalis’s furfural tolerance was weakened. To further confirm the function of ctADH1 and enhance Escherichia coli’s furfural tolerance, ctADH1 was overexpressed in E. coli BL21 (DE3). The rate of furfural degradation in E. coli BL21 (DE3) with pET-ADH1 (high-copy plasmid) and pCS-ADH1 (medium-copy plasmid) was increased by 1.59-fold and 1.28-fold, respectively.Conclusions Candida tropicalis was a robust strain with intrinsic tolerance to inhibitor furfural. The mechanism of furfural detoxification and metabolic responses were identified by multiple analyses. Alcohol dehydrogenase 1 was confirmed to be responsible for furfural detoxification. C. tropicalis showed a complex regulation system during furfural detoxification to minimize adverse effects caused by furfural. Furthermore, the mechanism we uncovered in this work was successfully applied to enhance E. coli’s furfural tolerance by heterologous expression of ctADH1. The study provides deeper insights into strain modification for biofuel production by efficient lignocellulose fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0668-x) contains supplementary material, which is available to authorized users.

show abstract

Section: Resultsmentioning

confidence: 99%

“…The concentration of furfural was measured by HPLC. The expression of ctADH1 was confirmed by SDS-PAGE analysis [35]. …”

Section: Methodsmentioning

confidence: 99%

Furfural tolerance and detoxification mechanism in Candida tropicalis

Wang

Cheng

Joshua

et al. 2016

Biotechnol Biofuels

Self Cite

View full text Add to dashboard Cite

show abstract

“…The tertiary structure of PobA complexed with 4‐HBA and FAD (PDB code: 1IUV) (Gatti et al, ) was downloaded from the Protein Data Bank (http://www.rcsb.org). Substrate of 1IUV model was substituted with 3,4‐DHBA using Discovery Studio (version 2.5) (Chen et al, ). Hydrogen bonds of complexes were analyzed by PyMOL (version 1.7).…”

Section: Methodsmentioning

confidence: 99%

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Chen

Shen

Wang

et al. 2017

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Gallic acid (GA) is a naturally occurring phytochemical that has strong antioxidant and antibacterial activities. It is also used as a potential platform chemical for the synthesis of diverse high-value compounds. Hydrolytic degradation of tannins by acids, bases or microorganisms serves as a major way for GA production, which however, might cause environmental pollution and low yield and efficiency. Here, we report a novel approach for efficient microbial production of GA. First, structure-based rational engineering of PobA, a p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa, generated a new mutant, Y385F/T294A PobA, which displayed much higher activity toward 3,4-dihydroxybenzoic acid (3,4-DHBA) than the wild-type and any other reported mutants. Remarkably, expression of this mutant in Escherichia coli enabled generation of 1149.59 mg/L GA from 1000 mg/L 4-hydroxybenzoic acid (4-HBA), representing a 93% molar conversion ratio. Based on that, we designed and reconstituted a novel artificial biosynthetic pathway of GA and achieved 440.53 mg/L GA production from simple carbon sources in E. coli. Further enhancement of precursor supply through reinforcing shikimate pathway was able to improve GA de novo production to 1266.39 mg/L in shake flasks. Overall, this study not only led to the development of a highly active PobA variant for hydroxylating 3,4-DHBA into GA via structure-based protein engineering approach, but also demonstrated a promising pathway for bio-based manufacturing of GA and its derived compounds. Biotechnol. Bioeng. 2017;114: 2571-2580. © 2017 Wiley Periodicals, Inc.

show abstract

“…On the other hand, others reported the diverse stability of ChABC vs temperature. 33 In our work, we showed that the activity of wild-type ChABC was higher than in standard conditions (controls) for 7 days at 37°C ( Figure 1B) in sucrose solutions, thus suggesting that exposure of ChABC to sucrose could enhance its thermostability and prolong its activity for days. 19 In particular, after 1 day, the ChABC in Sal lost more than 98% of enzyme activity, confirming the instability of the enzyme at body temperature.…”

Section: Discussionmentioning

confidence: 52%

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury

et al. 2018

View full text Add to dashboard Cite

show abstract

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

Abstract: Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

Cited by 19 publications

References 42 publications

Furfural tolerance and detoxification mechanism in Candida tropicalis

Furfural tolerance and detoxification mechanism in Candida tropicalis

Rational engineering of p‐hydroxybenzoate hydroxylase to enable efficient gallic acid synthesis via a novel artificial biosynthetic pathway

Feasible stabilization of chondroitinase abc enables reduced astrogliosis in a chronic model of spinal cord injury

Contact Info

Product

Resources

About