CRYOLETTERS
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2023
·Vol. 44
·期 5
·280-290
·
IF 1
Wu Jiawei,Liu Yeyu,Zhang Jinping,Wang Xiaohui
BACKGROUND: Chitin is the second largest carbon source on the earth, and chitosan oligosaccharides produced by its degradation have good application prospects in medicine, cosmetics, and agricultural production. OBJECTIVE: The discovery of a chitinase with high efficiency,
high stability and clear degradation mechanism is of great help to promote the research of chitin derivatives and the development of the industrial chain. MATERIALS AND METHODS: In this experiment, a lowtemperature chitinase-producing strain Photobacterium sp. LG-29 was isolated
from deep-sea mud in the Bohai Sea, and studied by means of molecular biology, biochemistry and bioinformatics. RESULTS: Purification of chitinase yielded an enzyme solution with a concentration of 0.918 mg/mL and a specific activity of 21.036 U/mg. The optimum action temperature is
35°C, and it is still active at 4°C, showing low-temperature enzymatic activity, and also has certain thermal stability. The optimum pH is 8.0, and it maintains more than 70% of the enzyme activity at pH 11, which is very stable in an alkaline environment. Mn2+, Ca2+,
and Mg2+ are the main activators of enzymes, while Fe2+, Zn2+, etc. have extremely significant inhibitory effects on enzymes. The Km and Kcat of chitinase were determined to be 269.05 μ mol/L and 0.49 min-1, respectively. Chitinase PbCHI5 has
both endonuclease and exonuclease activity. The theoretical pI of the enzyme is 4.16, which is a stable hydrophilic protein. CONCLUSION: This experiment laid a theoretical foundation for the development and utilization of new low-temperature chitinases.
研究领域:
分子生物学
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生物化学
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生物信息学
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酶学
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海洋微生物学
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DOI
PubMed
高影响因子
JOURNAL OF BIOTECHNOLOGY
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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
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蛋白质工程
·
酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2020
·
IF 3.475
Hong-Yan Wang,Yu-Li Xie,Xin Shi,Hong-Ling Shi,Jian-He Xu,Cun-Duo Tang,Lun-Guang Yao,Yun-Chao Kan
Halogenated mandelate and its derivatives are important scaffolds in chemical industry. However, the natural D -mandelate dehydrogenase (DMDH) showed relatively low catalytic activity toward D -o-chloromandelic acid, which largely limits the efficiency of dehydrogenation processes. To improve catalytic activity of Lactobacillus brevis D -mandelate dehydrogenase ( Lb DMDH) toward halogenated substrate, a D -mandelate dehydrogenase mutant Lb DMDH N253S with higher catalytic activity toward D -o-chloromandelic acid was successfully obtained by directed evolution, which was about 29 times of Lb DMDH. In addition, the temperature optimum of Lb DMDH N253S is 50 °C, and closer to the optimal growth temperature for Escherichia coli , indicating that it may be more advantageous in whole cell catalysis using living cells as catalysts. Meanwhile, the K cat value toward D -o-chloromandelic acid of Lb DMDH N253S is 2.08 s −1 , which is significantly higher than the one (0.09 s −1 ) of Lb DMDH, indicating that Lb DMDH N253S displayed higher catalytic efficiency toward D -o-chloromandelic acid. Then, we preliminarily explained the molecular basis for its catalytic performance by in silico design methods. All of these results established a solid foundation for molecular modification of DMDHs and the other industrial enzymes.
研究领域:
生物化学
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酶工程
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分子生物学
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产品关联
DOI
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
·
2021
·
IF 6.291
Zhijian Ke,Qian Zhu,Wankui Jiang,Yidong Zhou,Mingliang Zhang,Mingli Jiang,Qing Hong
Carbaryl is the representative of carbamate insecticide. As an acetylcholinesterase inhibitor, it poses potential threat to humans and other non-target organisms. Agrobacterium sp. XWY-2, which could grow with carbaryl as the sole carbon source, was isolated and characterized. The carH gene, encoding a carbaryl hydrolase, was cloned from strain XWY-2 and expressed in Escherichia coli BL21 (DE3). CarH was able to hydrolyze carbamate pesticides including carbaryl, carbofuran, isoprocarb, propoxur and fenobucarb efficiently, while it hydrolyzed oxamyl and aldicarb poorly. The optimal pH of CarH was 8.0 and the optimal temperature was 30 ℃. The apparent K m and k cat values of CarH for carbaryl were 38.01 ± 2.81 μM and 0.33 ± 0.01 s −1 , respectively. The point mutation experiment demonstrated that His341, His343, His346, His416 and D437 are the key sites for CarH to hydrolyze carbaryl.
研究领域:
环境科学
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微生物学
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生物化学
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酶学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Food Bioscience
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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
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生物技术
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酶学
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微生物学
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DOI
高影响因子
BIOORGANIC CHEMISTRY
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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
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生物催化
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化学生物学
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药物化学
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DOI
PubMed
高影响因子
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2020
·
IF 3.232
Wen Zheng,Jiahui Liu,Danyang Yi,Yadi Pan,Yijuan Long,Huzhi Zheng
Ficin has been reported to possess peroxidase activity, but its applications in some respects have been limited because of its relatively low activity. Herein, a mesoporous metal-organic framework, PCN-333(Fe), was synthesized, which was selected to encapsulate ficin to form [email protected] (Fe). Compared with ficin, the peroxidase-like activity of [email protected] (Fe) toward 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation was about 3 times increase in the presence of H 2 O 2 , and followed classical Michaelis-Menten model. The kinetic parameters showed that stronger affinity and higher catalytic constant ( K cat ) of [email protected] (Fe) to both TMB and H 2 O 2 compared with ficin, and K cat of [email protected] (Fe) was increased by 3.65 folds and 3.59 folds for TMB and H 2 O 2 , respectively. Taking advantages of higher catalytic property of [email protected] (Fe), we developed a colorimetric method with high sensitivity and selectivity to detect glucose, which displayed a good linear response toward glucose in the range of 0.5–180 μM with a limit of detection of 97 nM. Furthermore, [email protected] (Fe) has been proven to successfully detect glucose in human serum, implying its great potentialities and wide applications as peroxidase mimics.
研究领域:
生物化学
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分析化学
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材料科学
✓ 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
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微生物学
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生物技术
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酶学
✓ 已验证
产品关联
DOI
高影响因子
TALANTA
·
2019
·
IF 4.916
Wen Zheng,Dongjun Shen,Yadi Pan,Danyang Yi,Yijuan Long,Huzhi Zheng
The peroxidase-like activity of ficin is relatively low, which limits its application. It was found that thiol groups of ficin could inhibit its peroxidase-like activity. So, two procedures, i.e., direct blocking with N -ethylmaleimide (NEM), or using tris (2-carboxyethyl) phosphine hydrochloride (TCEP) to interrupt disulfide bonds then blocking thiol groups with NEM, were applied to block thiol groups of ficin, ficin-NEM (ficin-N) and ficin-TCEP-NEM (ficin-TN) were produced, respectively. The blocking of thiol groups accelerated the peroxidase activity dramatically. The peroxidase catalytic activity of ficin-N and ficin-TN toward the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation by H 2 O 2 was about 2.5-fold and 5-fold increase compared with ficin, respectively, which accompanied a color change from colorless to blue and followed classic Michaelis-Menten model. The kinetic parameters indicated that higher affinity of ficin-N ( K m = 0.31) and ficin-TN ( K m = 0.39) to H 2 O 2 compared with ficin ( K m = 0.58), and ficin-TN had the highest K cat which increased by 6.5 times and 4.5 times for TMB and H 2 O 2 , respectively. According to these findings, a colorimetric method with high sensitivity for the detection of biothiols was developed due to sulfhydryl compounds inhibited the peroxidase activity of ficin. Comparing with ficin and ficin-N, ficin-TN had the widest detection range (0.01–16 μM) and the lowest detection limit (3 nM). The practical applications of ficin-TN for biothiol determination in human serum samples have been demonstrated with satisfactory results. Ficin-N and ficin-TN are promising to apply to the bioanalysis.
研究领域:
生物化学
·
分析化学
·
生物技术
✓ 已验证
DOI
PubMed
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）