site directed mutagenesis
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2022 ◽  
Wenwei Tang ◽  
Xiaoyu Jin ◽  
Yunying Liu ◽  
Mengtian Zhang ◽  
Xiaoxuan Li ◽  

Abstract The microbial manganese removal process is believed to be the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and found to have high manganese oxidation activity in vitro and Cu(II) can significantly enhance its manganese oxidation activity. The gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA and then obtain four mutant strains. The manganese removal efficiency of the four strains was determined to find that H120 is the catalytic active site of the CopA. Protein modification analysis of CopA obtained under different conditions by mass spectrometry revealed that the loss of Cu(Ⅱ) and the mutation of the conserved copper binding site H120 resulted in the loss of modification of ethoxyformyl and quinone, the number of modifications was reduced and the position of modification was changed, eventually causing a decrease in protein activity. It reveals that Cu(II) and H120 play an indispensable role in the manganese oxidation of the multicopper oxidase CopA. The Mn valence state of BioMnOx was analyzed by XPS, finding that both the strain-mediated product and the CopA-mediated product were composed of MnO2 and Mn3O4 and the average valence of Mn is 3.2.

2022 ◽  
Vol 10 (1) ◽  
pp. 132
Bingxue Sun ◽  
Guangxue Zhu ◽  
Xuewen Xie ◽  
Ali Chai ◽  
Lei Li ◽  

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.

2022 ◽  
Vol 12 ◽  
Anja Müller ◽  
Keisuke Sakurai ◽  
Diana Seinige ◽  
Kunihiko Nishino ◽  
Corinna Kehrenberg

The prototype fexA gene confers combined resistance to chloramphenicol and florfenicol. However, fexA variants mediating resistance only to chloramphenicol have been identified, such as in the case of a Staphylococcus aureus isolate recovered from poultry meat illegally imported to Germany. The effects of the individual mutations detected in the fexA sequence of this isolate were investigated in this study. A total of 11 fexA variants, including prototype fexA and variants containing the different previously described mutations either alone or in different combinations, were generated by on-chip gene synthesis and site-directed mutagenesis. The constructs were inserted into a shuttle vector and transformed into three recipient strains (Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium). Subsequently, minimal inhibitory concentrations (MIC) of florfenicol and chloramphenicol were determined. In addition, protein modeling was used to predict the structural effects of the mutations. The lack of florfenicol-resistance mediating properties of the fexA variants could be attributed to the presence of a C110T and/or G98C mutation. Transformants carrying fexA variants containing either of these mutations, or both, showed a reduction of florfenicol MICs compared to those transformants carrying prototype fexA or any of the other variants. The significance of these mutations was supported by the generated protein models, indicating a substitution toward more voluminous amino-acids in the substrate-binding site of FexA. The remaining mutations, A391G and C961A, did not result in lower florfenicol-resistance compared to prototype fexA.

2022 ◽  
Shu-Fang Li ◽  
Shen-Yuan Xu ◽  
Ya-Jun Wang ◽  
Yu-Guo Zheng

Abstract Pullulanase is a well-known debranching enzyme that can specially hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four sites (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase PulAR were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Four beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 ℃, pH 5.0 and 6.0, respectively. In addition, its thermostabilities at 60 ℃ and 65 ℃ were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced via rational engineering by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.

2022 ◽  
Vol 23 (1) ◽  
pp. 544
Shinhui Lee ◽  
Hee-Soo Seol ◽  
Sanung Eom ◽  
Jaeeun Lee ◽  
Chaelin Kim ◽  

Monoamine serotonin is a major neurotransmitter that acts on a wide range of central nervous system and peripheral nervous system functions and is known to have a role in various processes. Recently, it has been found that 5-HT is involved in cognitive and memory functions through interaction with cholinergic pathways. The natural flavonoid kaempferol (KAE) extracted from Cudrania tricuspidata is a secondary metabolite of the plant. Recently studies have confirmed that KAE possesses a neuroprotective effect because of its strong antioxidant activity. It has been confirmed that KAE is involved in the serotonergic pathway through an in vivo test. However, these results need to be confirmed at the molecular level, because the exact mechanism that is involved in such effects of KAE has not yet been elucidated. Therefore, the objective of this study is to confirm the interaction of KAE with 5-HT3A through electrophysiological studies at the molecular level using KAE extracted from Cudrania tricuspidata. This study confirmed the interaction between 5-HT3A and KAE at the molecular level. KAE inhibited 5-HT3A receptors in a concentration-dependent and voltage-independent manner. Site-directed mutagenesis and molecular-docking studies confirmed that the binding sites D177 and F199 are the major binding sites of human 5-HT3A receptors of KAE.

2022 ◽  
Vol 12 (1) ◽  
pp. 464
Yong Meng ◽  
Yin Tang ◽  
Xiuhong Zhang ◽  
Jin Wang ◽  
Zhengfu Zhou

Keratin is a tough fibrous structural protein that is difficult to digest with pepsin and trypsin because of the presence of a large number of disulfide bonds. Keratin is widely found in agricultural waste. In recent years, especially, the development of the poultry industry has resulted in a large accumulation of feather keratin resources, which seriously pollute the environment. Keratinase can specifically attack disulfide bridges in keratin, converting them from complex to simplified forms. The keratinase thermal stability has drawn attention to various biotechnological industries. It is significant to identify keratinases and improve their thermostability from microorganism in extreme environments. In this study, the keratinases DgoKerA was identified in Deinococcus gobiensis I-0 from the Gobi desert. The amino acid sequence analysis revealed that DgoKerA was 58.68% identical to the keratinase MtaKerA from M. thermophila WR-220 and 40.94% identical to the classical BliKerA sequence from B. licheniformis PWD-1. In vitro enzyme activity analysis showed that DgoKerA exhibited an optimum temperature of 60 °C, an optimum pH of 7 and a specific enzyme activity of 51147 U/mg. DgoKerA can degrade intact feathers at 60 °C and has good potential for industrial applications. The molecular modification of DgoKerA was also carried out using site-directed mutagenesis, in which the mutant A350S enzyme activity was increased by nearly 30%, and the results provide a theoretical basis for the development and optimization of keratinase applications.

Hyuna Park ◽  
Doyeong Bak ◽  
Wooyoung Jeon ◽  
Minjung Jang ◽  
Jung-Oh Ahn ◽  

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

2022 ◽  
Vol 23 (1) ◽  
pp. 487
Wen-Yuan Lin ◽  
Yuan-Ju Lee ◽  
Ping-Hung Yu ◽  
Yi-Lin Tsai ◽  
Pin-Yi She ◽  

Bacterial sensing of environmental signals through the two-component system (TCS) plays a key role in modulating virulence. In the search for the host hormone-sensing TCS, we identified a conserved qseEGF locus following glmY, a small RNA (sRNA) gene in uropathogenic Proteus mirabilis. Genes of glmY-qseE-qseG-qseF constitute an operon, and QseF binding sites were found in the glmY promoter region. Deletion of glmY or qseF resulted in reduced swarming motility and swarming-related phenotypes relative to the wild-type and the respective complemented strains. The qseF mutant had decreased glmYqseEGF promoter activity. Both glmY and qseF mutants exhibited decreased flhDC promoter activity and mRNA level, while increased rcsB mRNA level was observed in both mutants. Prediction by TargetRNA2 revealed cheA as the target of GlmY. Then, construction of the translational fusions containing various lengths of cheA 5′UTR for reporter assay and site-directed mutagenesis were performed to investigate the cheA-GlmY interaction in cheA activation. Notably, loss of glmY reduced the cheA mRNA level, and urea could inhibit swarming in a QseF-dependent manner. Altogether, this is the first report elucidating the underlying mechanisms for modulation of swarming motility by a QseEF-regulated sRNA GlmY, involving expression of cheA, rcsB and flhDC in uropathogenic P. mirabilis.

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