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Chen Wang,Kai Zhang,Chen Zhongjun,Heng Cai,Wan Honggui,Ping-Kai Ouyang 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3
Lysine decarboxylase (LDC) exhibits a significant role in cadaverine (1,5-pentanediamine, diaminopentane) production from lysine. In this study, an error-prone PCR and DNA shuffling were performed to improve the activity of LDC from Hafnia alvei AS1.1009 for cadaverine production. A sensitive high-throughput screening strategy based on a pH indicator was established for directed evolution of LDC. Several improved mutants were obtained from directed evolution and LDCV147F/E583G mutant showed highest activity to catalyze lysine to cadaverine. This mutant showed 1.62-fold high LDC activity when compared to wild-type. Further analysis by site-directed mutagenesis reveled that only the mutant E583G was sufficient for higher catalytic activity. Wild type LDC and mutant LDCE583G were purified by an improved method including hydrophobic chromatography. These purified enzymes were characterized and the kinetic parameters were compared between LDCE583G and WT LDC. Vmax of LDCE583G was 1.32-fold higher than that of WT LDC. Use of LDCE583G mutant showed 1.48-fold improved productivity of cadaverine when compared to wild type. The concentration of cadaverine in E. coli JM109/pTrc99a-ldc2-41 was 63.9 g/L with conversion yield of 93.4% during 5 h. These results indicate that the mutation has positive effects on improving LDC activity and a potential candidate for cadaverine production.
Hui Li,Sha Li,Hong Xu,Xiao-Ye Chen,Ping-Kai Ouyang 한국생물공학회 2013 Biotechnology and Bioprocess Engineering Vol.18 No.2
Batch fermentations of welan gum from Alcaligenes sp. CGMCC2428 at pH values of 5.5 ~ 7.0were studied. Based on the kinetic analysis, a pH control process for improving welan production was developed. By maintaining the culture pH at 7.0, the process significantly improved the maximal welan concentration and productivity to reach 25.1 ± 0.65 g/L and 0.42 ± 0.003 g/L/h, respectively,compared with those in native pH processes where pH value would decrease from 7.0 to 5.1 (18.5 ± 0.72 g/L and 0.28 ± 0.002 g/L/h). This improvement may be due to the increased metabolic flux of glucose-1-phosphate to welan induced by pH control process. Furthermore, scale-up fermentation under controlled pH was implemented using 300-L fermentor. The highest welan concentration of 27.5± 0.97 g/L was obtained. This simplified process proved effective in industry fermentation for high welan production.
Ke-quan Chen,Zhen Wang,Wen Xiao,Alie Zhang,Hanxiao Ying,Ping-Kai Ouyang 한국화학공학회 2016 Korean Journal of Chemical Engineering Vol.33 No.10
Actinobacillus succinogenes NJ113 is capable of microaerobic fermentation, which offers the possibility of a novel type of pyruvic acid production. A dissolved oxygen environment with stirring at 300 rpm was a key factor in the fermentative production of a maximum concentration of pyruvic acid. Potassium carbonate (K2CO3) was found to have a role in promoting pyruvic acid production, influencing the concentration of pyruvic acid and production of the by-product succinic acid. The final titer of pyruvic acid production was 36.8±0.1 g L−1 with an overall yield of 0.639±0.056 g g−1 glucose and 3.12±0.03mmol g−1 dry cell weight h−1. Significance and impact of the study: This study is the first to illustrate the advantage of using Actinobacillus succinogenes NJ113 with no genetic modification under microaerobic conditions for the production of pyruvic acid.
Enzymatic Synthesis of Theanine with L-glutamine-Zn(II) Complexes
Hao-Qi Wang,Zhong Yao,Zhi Zhou,Yun Sun,Ping Wei,Ping-Kai Ouyang 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.6
Theanine, a unique amino acid found in tea plants, has many important physiological functions. Theanine can be enzymatically synthesized via the γ-glutamyltranspeptidation reaction. In this study, we described a new method of theanine synthesis using the L-glutamine-Zn(II)(Zn(Gln)2) complex instead of glutamine as the donor,which successfully reduced the side autotranspeptidation reaction and led to higher yield of theanine. We prepared the Zn(Gln)2 complexes and showed that they are stable in liquid bulk under 9.0 pH. After using the Zn(Gln)2 in the γ-glutamyltranspeptidation reaction, we utilized HPLC and Mass spectrometry analysis to demonstrated that Zn(Gln)2was an more effective γ-glutamyl donor than glutamine. The autotranspeptidation reaction was restrained effectively. As a result, the theanine yield and the conversion rate for glutamine were vastly improved. In a reaction mixture containing 48 mM of Zn(Gln)2, 1.6 M ethylamine, and 0.5 U/mL GGT, the final concentration of theanine obtained was 61.3 mM after incubation at 37oC for three hours. The conversion rate for glutamine was 63.8%, which showed a 16.9% increase as compared to when using glutamine alone as the donor substrate.
Luping Zhou,Lulu Chen,Yaqin Wang,Jie Huang,Guo Ping Yang,Zhi-Rong Tang,Yicheng Wang,Jianwei Liao,Gan Zhou,Kai-hua Wei,Zhenyu Li,Dongsheng Ouyang 고려인삼학회 2019 Journal of Ginseng Research Vol.43 No.3
Background: Ginsenoside compound K (CK) is a promising drug candidate for rheumatoid arthritis. Thisstudy examined the impact of polymorphisms in NR1I2, adenosine triphosphateebinding cassette (ABC)transporter genes on the pharmacokinetics of CK in healthy Chinese individuals. Methods: Forty-two targeted variants in seven genes were genotyped in 54 participants using SequenomMassARRAY system to investigate their association with major pharmacokinetic parameters of CK and itsmetabolite 20(S)-protopanaxadiol (PPD). Subsequently, molecular docking was simulated using theAutoDock Vina program. Results: ABCC4 rs1751034 TT and rs1189437 TT were associated with increased exposure of CK anddecreased exposure of 20(S)-PPD, whereas CFTR rs4148688 heterozygous carriers had the lowestmaximum concentration (Cmax) of CK. The area under the curve from zero to the time of the lastquantifiable concentration (AUClast) of CK was decreased in NR1I2 rs1464602 and rs2472682 homozygouscarriers, while Cmax was significantly reduced only in rs2472682. ABCC4 rs1151471 and CFTR rs2283054influenced the pharmacokinetics of 20(S)-PPD. In addition, several variations in ABCC2, ABCC4, CFTR, andNR1I2 had minor effects on the pharmacokinetics of CK. Quality of the best homology model of multidrugresistance protein 4 (MRP4) was assessed, and the ligand interaction plot showed the mode of interactionof CK with different MRP4 residues. Conlusion: ABCC4 rs1751034 and rs1189437 affected the pharmacokinetics of both CK and 20(S)-PPD. NR1I2 rs1464602 and rs2472682 were only associated with the pharmacokinetics of CK. Thus, thesehereditary variances could partly explain the interindividual differences in the pharmacokinetics of CK.
Plasma treatment of multi-walled carbon nanotubes for lipase immobilization
Xun Cao,Rui Zhang,Wei-min Tan,Ce Wei,Jing Wang,Ze-meng Liu,Ke-quan Chen,Ping-Kai Ouyang 한국화학공학회 2016 Korean Journal of Chemical Engineering Vol.33 No.5
Plasma-modified multiwalled carbon nanotubes (MWNTs) were used as a support to immobilize lipase. The effects of vacuum plasma treatment power, vacuum plasma treatment time, immobilization temperature, immobilization time, and initial protein concentration of the lipase on the amount of lipase immobilized and on the subsequent activity of the immobilized lipase were investigated. The results showed that the adsorption capacity of the plasma-modified MWNTs could reach 0.15 g/g and that the maximal enzyme activity of the immobilized lipase was 520U/g under optimized conditions. Fourier transform infrared (FTIR) analysis and transmission electron microscopy (TEM) were used to characterize the properties of the plasma-modified MWNTs and plasma-modified MWNTslipase, and the results showed that the lipase was successfully immobilized on the plasma-modified MWNTs. Also, the MWNTs-lipase produced an esterification rate of approximately 47% in the synthesis of polyethylene glycol (PEG)-aliphatic esters.
( Gui Zi Ye ),( Min Jiang ),( Jian Li ),( Ke Quan Chen ),( Yong Lan Xi ),( Shu Wen Liu ),( Ping Wei ),( Ping Kai Ouyang ) 한국미생물 · 생명공학회 2010 Journal of microbiology and biotechnology Vol.20 No.8
Actinobacillus succinogenes, a representative succinic-acid-producing microorganism, is seriously inhibited by ammonium ions, thereby hampering the industrial use of A. succinogenes with ammonium-ion-based materials as the pH controller. Therefore, this study isolated an ammonium-ion-tolerant mutant of A. succinogenes using a continuous-culture technique in which all the environmental factors, besides the stress (ammonium ions), were kept constant. Instead of operating the mutant-generating system as a nutrient-limited chemostat, it was used as a nutrientunlimited system, allowing the cells to be continuously cultured at the maximum specific growth rate. The mutants were isolated on agar plates containing the acid-base indicator bromothymol blue and a high level of ammonium ions that would normally kill the parent strain by 100%. When cultured in anaerobic bottles with an ammonium ion concentration of 354 mmol/l, the mutant YZ0819 produced 40.21 g/l of succinic acid with a yield of 80.4%, whereas the parent strain NJ113 was unable to grow. When using NH4OH to buffer the culture pH in a 3.0 l stirred bioreactor, YZ0819 produced 35.15 g/l of succinic acid with a yield of 70.3%, which was 155% higher than that produced by NJ113. In addition, the morphology of YZ0819 changed in the fermentation broth, as the cells were aggregated from the beginning to the end of the fermentation. Therefore, these results indicate that YZ0819 can efficiently produce succinic acid when using NH4OH as the pH controller, and the formation of aggregates can be useful for transferring the cells from a cultivation medium for various industrial applications.