JOURNAL OF BIOTECHNOLOGY
·
2024
·
IF 4.1
Kehao Yuan,Zongchao Huo,Ya`ning Zhang,Zuran Guo,Yucan Chang,Yunming Jin,Lining Gao,Tong Zhang,Yanwei Li,Qinyuan Ma,Xiuzhen Gao
During the deamination and amination processes of meso -diaminopimelate dehydrogenase ( meso -DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso -DAP during deamination . Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso -DAP. However, some H154 variants showed enhanced k cat / K m values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved k cat / K m values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.
研究领域:
生物化学
·
蛋白质工程
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Bioscience
·
2021
·
IF 4.24
Jiao Yuan,Yuvaraj Ravikumar,Guoyan Zhang,Junhua Yun,Yufei Zhang,Hossain M. Zabed,Xianghui Qi
l -arabinose isomerases (L-AIS) are enzymes that mediate the conversion of l -arabinose into l -ribulose and D-galactose into D-tagatose. The potential of L-AI from Lactobacillus parabuchneri CICC 6004 (LPAI) in D-galactose biotransformation using E. coli whole-cells was studied. The parameters investigated in this study were 1) the optimum pH, temperature, and cofactor specificity for maximum enzyme activity ; 2) the D-tagatose biosynthesis from D-galactose using whole-cells containing LPAI. The results showed that with D-galactose, the purified LPAI showed the highest activity, 15.52 U/mg at pH 6.7, 45 °C, and necessitates 3 mM Ca 2+ as its cofactor. The K m , V max and K cat /K m values of LPAI for D-galactose were 5.4 mM, 35.5 U/mg and 2.8 mM −1 min −1 , respectively, while the respective values for l -arabinose were found to be 4.3 mM, 63.1 U/mg and 5.9 mM −1 min −1 .The applicability of LPAI for D-tagatose synthesis was shown by using whole-cell biocatalyst approach. Under the optimum conditions, during one-stage biotransformation of D-galactose by E. coli whole cells, 39.0 g/L of D-tagatose was produced from 140 g/L of D-galactose after 50 h, which corresponded to the yield and productivity of 0.28 g/g and 0.78 g/L/h, respectively. Further addition of new cells into the reaction mixture in the second stage produced 54.0 g/L of D-tagatose after 100 h. The D-tagatose total yield and productivity were 0.39 g/g and 0.54 g/L/h, respectively. The results attained in this study showed LPAI as a potential candidate for the industrial production of D-tagatose.
研究领域:
生物化学
·
生物技术
·
酶学
·
微生物学
✓ 已验证
DOI
高影响因子
BIOORGANIC CHEMISTRY
·
2022
·
IF 5.307
Hua-Tao Liu,Chun-Yue Weng,Shen-Yuan Xu,Shu-Fang Li,Ya-Jun Wang,Yu-Guo Zheng
Traditional screening methods of enzyme engineering often require building large mutant libraries to screen for potentially beneficial sites, which are often time-consuming and labor-intensive with low mining efficiency. In this study, a novel enzyme engineering strategy was established to modify carbonyl reductase Ls CR for the synthesis of (1 S )-2-chloro-1-(3,4-difluorophenyl) ethanol (( S )-CFPL), which is a key intermediate of anticoagulant drug ticagrelor. The strategy was developed by combining HotSpot, FireProt and multiple sequence alignment, resulting in the construction of a “small and smart” mutant library including 10 mutations. Among them, 5 mutations were positive, resulting in a 50% mining accuracy of beneficial sites. Finally, a highly active mutant Ls CR M3 (N101D/A117G/F147L) was obtained by further screening through saturation mutation and iterative mutation. Compared with wild type (WT) Ls CR, the catalytic activity of Ls CR M3 was increased by 4.7 times, the catalytic efficiency k cat / K M value was increased by 2.9 times, and the half-life t 1/2 at 40 °C was increased by 1.3 times. Due to the low aqueous solubility of the substrate 2-chloro-1-(3,4-difluorophenyl) ethanone (CFPO), isopropanol was used as not only the co-substrate but also co-solvent. In the presence of 40% ( v / v ) isopropanol, Ls CR M3 completely reduced 400 g/L CFPO to enantiomerically pure CFPL (99.9%, e.e. ) in 11 h with a space–time yield (STY) as high as 809 g/L∙d.
研究领域:
酶工程
·
生物催化
·
化学生物学
·
药物化学
✓ 已验证
DOI
PubMed
高影响因子
PROCESS BIOCHEMISTRY
·
2023
·
IF 4.4
Xiaoliang Wang,Yibo Li,Yan Liu,Qian Wu,Zunxi Huang,Shukun Tang,Junmei Ding
The newly semi-synthetic β -lactam antibiotics play critical roles in prevention and treatment of β -lactam antibiotic-resistant bacteria. Acetyl xylan esterases belong to carbohydrate esterase 7 family can specifically hydrolyze 7-aminocephalosporanic acid (7-ACA) to produce deacetyl-7-aminocephalosporanic acid (D-7ACA) which is one of the most important starting materials to synthesize newly semi-synthetic β -lactam antibiotics. Here, an effective acetyl xylan esterase, ThAXE, from Thermobifida halotolerans YIM 90462 T , was firstly identified and characterized. ThAXE exhibited its highest catalytic activity against p -nitrophenyl acetate ( p -NPC 2 ) at pH 8.5 and 50 °C, with K m and K cat / K m were 1.0 ± 0.1 mM and 2262.9 mM −1 s −1 , respectively. Additionally, ThAXE can hydrolyze the acetyl group of 7-ACA at its C3' position with the specific activity of 7.4 U mg −1 . Furthermore, molecular docking combined with site-directed mutagenesis validation demonstrated that the pocket of catalytic residues Ser186, Asp272, and His301 could accommodate p -NPC 2 and 7-ACA ligands. This is the first report of a novel acetyl xylan esterase from T. halotolerans that has considerable potential biotechnological values.
研究领域:
生物化学
·
微生物学
·
生物技术
·
酶学
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2021
·
IF 5.279
Juan Han,Hui Feng,Jiacong Wu,Yuanyuan Li,Yang Zhou,Lei Wang,Peng Luo,Yun Wang
Today, we are seeking an efficient biotransformation of cellulosic material into sustainable biochemical products to meet the increasing global energy demand. Herein, we report the fabrication of multienzyme hybrid nanoflowers (ECG-NFs) by co-immobilizing three recombinant enzymes (cellobiohydrolase (CBH), endo-glucanase (EG), and β-glucosidase (BG)) integrating a binary tag composed of elastin-like polypeptide (ELP) and His-tag to act as a tri-enzyme biocatalyst, which catalyzes the hydrolysis of cellulose into glucose. The prepared ECG-NFs exhibited excellent performance in terms of pH stability, thermal stability, storage stability, and catalytic efficiency compared to free multienzyme system. Notably, ECG-NFs could be recycled for up to eight consecutive runs. The Km and kcat/Km values for ECG-NFs were 9.33 g L–1 and 0.0051 L min–1 g–1, respectively, which were better than those of the free multienzyme system, indicating a better substrate affinity. Finally, the overall enzyme activity of ECG-NFs increased by 1.12 times and the degradation efficiency of ECG-NFs was superior to the free multienzyme system, which revealed that ECG-NFs could facilitate an effective one-pot hydrolysis of cellulose into glucose.
研究领域:
生物化学
·
生物技术
·
材料科学
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）