ACS Sustainable Chemistry & Engineering
·
2025
·
IF 7.3
Ran Wang,Wei Song,Hangyuan Xu,Jian Wen,Wanqing Wei,Zhaoyang Chen,Guipeng Hu,Cong Gao,Xiaomin Li,Jia Liu,Jing Wu
l-Asparagine (l-Asn) is an important amino acid with broad applications in food, medicine, fine chemicals, and environmental protection. However, its industrial production is limited by the high cost of raw materials and the low catalytic efficiency of enzymes. In this study, a three-enzyme cascade pathway (FDN) was designed to produce l-Asn, using fumaric acid as the raw material. Within this pathway, EcAsnA was identified as the rate-limiting enzyme and was subsequently engineered using a product rescue strategy to reduce product inhibition by opening the closed gate. The optimal mutant, L109 K/K58R, exhibited a 6.61-fold reduction in product inhibition and a 4.24-fold improvement in catalytic efficiency. This mutant was then integrated into Escherichia coli along with the other two enzymes to construct the optimal recombinant strain E. coli 17. Using diatomite-glutaraldehyde cross-linking immobilized E. coli 17 as a biocatalyst, 267.74 g of l-Asn (with 5.35 g·L–1·h–1 STY, > 99% ee) was produced across 50 batch feedings in 1 L reaction volume. The purity of l-Asn exceeded 99% after isolation and purification. This study demonstrates the effective integration of cascade reaction design, enzyme engineering, and cell immobilization, providing a promising approach for synthesizing high-value products from cost-effective substrates.
研究领域:
生物化学
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酶学
·
合成生物学
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DOI
高影响因子
NEW JOURNAL OF CHEMISTRY
·
2019
·Vol. 43
·期 8
·3323-3331
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IF 3.069
Xi Wang,Tian Gao,Mian Yang,Jie Zhao,Feng-Lei Jiang,Yi Liu
L-Asparagine, a common amino acid, is a cost-effective and ideal precursor for carbon dots because of its high nitrogen content and its abundance in nature. By using L-asparagine as a biocompatible precursor, fluorescent nitrogen-doped carbon dots (A-CDs) were prepared by a one-pot microwave-assisted method. Then, a reasonable structure for A-CDs was put forward, which confirmed that unconjugated small molecules could transform into carbon dots with the sp2 core, and was helpful in the discussion of the photoluminescence mechanism. It was concluded that the photoluminescence origin of A-CDs was from the graphite-like structure and surface defects. The A-CDs showed high stability in strong ionic solutions and negligible cytotoxicity even when the incubation concentration was up to 800 μg mL−1. Long-time bioimaging tests suggested that A-CDs have great potential for use in a wide variety of biological applications.
研究领域:
材料科学
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生物医学工程
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化学
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DOI
高影响因子
RSC Advances
·
2015
·Vol. 5
·期 91
·74977-74985
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IF 3.84
Xin Xiao,Shunheng Tu,Chunxia Zheng,Huan Zhong,Xiaoxi Zuo,Junmin Nan
Large-scale synthesis of nanosized β-Bi2O3 is a significant challenge due to its metastable state. A facile L-asparagine-assisted reflux–calcination route was successfully developed for the large-scale preparation of β-Bi2O3 micro/nanostructures under mild conditions (low temperature, atmospheric pressure, and wide temperature windows). The composition, phase structure, morphology, surface area, and photoabsorption properties of as-synthesized β-Bi2O3 and its precursor were systematically characterized. The phase transformation conditions and possible formation mechanism of flower-like β-Bi2O3 were discussed. It is found that with a simple reflux process under atmospheric pressure at 100 °C, uniform monodisperse bismuth–asparagine complex microspheres with average diameters of ∼500 nm were produced and flower-like β-Bi2O3 micro/nanostructures were then conveniently obtained after precursor calcination at temperatures ranging from 340 °C to 420 °C. A surface CO32− coordination effect introduced from L-asparagine explained the formation of stabilized β-Bi2O3 at low temperatures (up to 420 °C). The as-synthesized β-Bi2O3 shows excellent photocatalytic activity toward the degradation of 4-phenylphenol under visible-light irradiation, which is 3.7 and 21.4 times faster than the removal rates of β-Bi2O3 nanospheres and a commercial β-Bi2O3, respectively, and allows for the elimination of 93.2% total organic carbon after 60 min of irradiation. In addition, the photogenerated reactive species were identified by radical scavenger experiments and electron paramagnetic resonance spectroscopy, and a possible visible-light-induced photocatalytic mechanism was then proposed.
研究领域:
材料科学
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环境科学
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DOI
高影响因子
ACS Applied Materials & Interfaces
·
2019
·
IF 8.456
Juan Qiao,Junfang Jiang,Lili Liu,Ji Shen,Li Qi
The challenge for polymeric enzyme reactors at present is to selectively control the enzymolysis rate in complex conditions. Additionally, the fabrication methodology is hindered by complex processes, especially for achieving diverse stimuli responsiveness and functions. Here, we reported a kind of pH-sensitive polymer, poly(styrene-co-maleic anhydride-acrylic acid) (PS-MAn-AA)-based hybrid enzyme reactor. It comprised magnetic nanoparticles and a pH-sensitive PS-MAn-AA porous polymer membrane made by breath figure method. The enzyme l-asparaginase (l-ASNase) could covalently bond on the surface of the pH-sensitive porous polymer membrane (pH-PPM), and the resultant enzyme reactor was characterized by Fourier transform infrared spectroscopy and vibrating sample magnetometer. The apparent Michaelis–Menten constants (Km and Vmax) of the l-ASNase enzyme reactor at different pH values were determined by a chiral ligand-exchange capillary electrophoresis method with l-asparagine as the substrate. The Vmax value of the l-ASNase enzyme reactor (0.67 mM/min) was almost 3-fold of that of the free l-ASNase (0.23 mM/min) at pH 8.2. Its ability to precisely control the enzymolysis rate in complex conditions is triggered primarily by the pH of the buffer solution, allowing controlled enzymatic reactions and displaying excellent stability and reusability of the proposed pH-PPM. This strategy for porous polymer membrane enzyme reactor fabrication has established a platform for enzyme efficiency adjusting. These valve-like distinguished features highlight the outstanding potential of stimuli-responsive enzyme reactor applied for enzyme immobilization and enzyme-related disease treatment.
研究领域:
生物化学
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材料科学
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酶工程
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
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2022
·Vol. 23
·期 17
·9663
·
IF 6.208
Yawen Zhou,Linshu Jiao,Juan Shen,Huibing Chi,Zhaoxin Lu,Huawei Liu,Fengxia Lu,Ping Zhu
Low catalytic activity is a key factor limiting the widespread application of type II L-asparaginase (ASNase) in the food and pharmaceutical industries. In this study, smart libraries were constructed by semi-rational design to improve the catalytic activity of type II ASNase fromBacillus licheniformis. Mutants with greatly enhanced catalytic efficiency were screened by saturation mutations and combinatorial mutations. A quintuple mutant ILRAC was ultimately obtained with specific activity of 841.62 IU/mg andkcat/Kmof 537.15 min−1·mM−1, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity andkcat/Kmwere firstly reported in type II ASNase fromBacillus licheniformis. Additionally, enhanced pH stability and superior thermostability were both achieved in mutant ILRAC. Meanwhile, structural alignment and molecular dynamic simulation demonstrated that high structure stability and strong substrate binding were beneficial for the improved thermal stability and enzymatic activity of mutant ILRAC. This is the first time that enzymatic activity of type II ASNase fromBacillus licheniformishas been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.Keywords:L-asparaginase;protein engineering;saturation mutagenesis;catalytic efficiency;molecular dynamics simulation
研究领域:
生物技术
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酶学
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蛋白质工程
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产品关联
DOI
PubMed
高影响因子
ELECTROPHORESIS
·
2012
·Vol. 34
·期 3
·409-416
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IF 3.303
Juan Qiao,Li Qi,Huijuan Yan,Yaping Li,Xiaoyu Mu
l -Asparaginase ( l -Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, l -glutamine ( l -Gln) and l -asparagine ( l -Asn). To study the cytotoxic effect and the secondary complications of l -Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of l -Asnase for the hydrolysis of l -Gln, employing the enzyme-gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)-LIF was established for the separation of l -amino acids ( l -Gln and l -glutamic acid) and studying the hydrolysis of l -Gln by using l -Asnase enzyme reactor. Then, using l -Gln as target analyte, the enzyme kinetics of l -Asnase in free solution, enzyme-gold nanoparticle conjugates (E-GNP), and the enzyme-gold nanoparticle conjugates immobilized in capillary (E-GNP-C) were investigated in detail with the proposed MCE-LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of l -Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E-GNP-C enzyme reactor exhibited the best performance for the hydrolysis of l -Gln.
研究领域:
生物化学
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医学
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纳米技术
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DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2023
·Vol. 107
·期 7
·2321-2333
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IF 5
Chi Huibing,Zhu Xiaoyu,Shen Juan,Lu Zhaoxin,Lu Fengxia,Lyu Yunbin,Zhu Ping
Abstract Acrylamide alleviation in food has represented as a critical issue due to its neurotoxic effect on human health. L-Asparaginase (ASNase, EC 3.5.1.1) is considered a potential additive for acrylamide alleviation in food. However, low thermal stability hinders the application of ASNase in thermal food processing. To obtain highly thermal stable ASNase for its industrial application, a consensus-guided approach combined with site-directed saturation mutation (SSM) was firstly reported to engineer the thermostability of Mycobacterium gordonae L-asparaginase (GmASNase). The key residues Gly97, Asn159, and Glu249 were identified for improving thermostability. The combinatorial triple mutant G97T/N159Y/E249Q (TYQ) displayed significantly superior thermostability with half-life values of 61.65 ± 8.69 min at 50 °C and 5.12 ± 1.66 min at 55 °C, whereas the wild-type was completely inactive at these conditions. Moreover, its T m value increased by 8.59 °C from parent wild-type. Interestingly, TYQ still maintained excellent catalytic efficiency and specific activity. Further molecular dynamics and structure analysis revealed that the additional hydrogen bonds, increased hydrophobic interactions, and favorable electrostatic potential were essential for TYQ being in a more rigid state for thermostability enhancement. These results suggested that our strategy was an efficient engineering approach for improving fundamental properties of GmASNase and offering GmASNase as a potential agent for efficient acrylamide mitigation in food industry. Key points
• The thermostability of GmASNase was firstly improved by consensus-guided engineering.
• The half-life and T
m
value of triple mutant TYQ were significantly increased.
• Insight on improved thermostability of TYQ was revealed by MD and structure analysis.
研究领域:
生物化学
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食品科学
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蛋白质工程
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产品关联
DOI
PubMed
高影响因子
Foods
·
2021
·Vol. 10
·期 11
·2819
·
IF 4.35
Huibing Chi,Meirong Chen,Linshu Jiao,Zhaoxin Lu,Xiaomei Bie,Haizhen Zhao,Fengxia Lu
L-asparaginase (E.C.3.5.1.1) is a well-known agent that prevents the formation of acrylamide both in the food industry and against childhood acute lymphoblastic leukemia in clinical settings. The disadvantages of L-asparaginase, which restrict its industrial application, include its narrow range of pH stability and low thermostability. In this study, a novel L-asparaginase fromMycobacterium gordonae(GmASNase) was cloned and expressed inEscherichia coliBL21 (DE3). GmASNase was found to be a tetramer with a monomeric size of 32 kDa, sharing only 32% structural identity withHelicobacter pyloriL-asparaginases in the Protein Data Bank database. The purified GmASNase had the highest specific activity of 486.65 IU mg−1at pH 9.0 and 50 °C. In addition, GmASNase possessed superior properties in terms of stability at a wide pH range of 5.0–11.0 and activity at temperatures below 40 °C. Moreover, GmASNase displayed high substrate specificity towards L-asparagine with Km, kcat, and kcat/Km values of 6.025 mM, 11,864.71 min−1and 1969.25 mM−1min−1, respectively. To evaluate its ability to mitigate acrylamide, GmASNase was used to treat potato chips prior to frying, where the acrylamide content decreased by 65.09% compared with the untreated control. These results suggest that GmASNase is a potential candidate for applications in the food industry.Keywords:L-asparaginase;acrylamide;Mycobacterium gordonae;thermal stability
研究领域:
食品科学
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酶学
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生物技术
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生物化学
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DOI
PubMed
高影响因子
Catalysts
·
2022
·Vol. 12
·期 8
·887
·
IF 4.501
Yu Hao,Shaohua Chen,Xueyin Zhang,Tiehong Chen
Pt1/CeO2nanospheres (Pt/CeO2-NS) were synthesized by the bath oiling method with L-asparagine as a necessary additive. Owing to the morphology control effect and coordination interaction of L-asparagine, CeO2nanospheres can retain their nanosphere structure and show stronger electronic metal-support interaction with highly dispersed Pt. Moreover, the toluene catalytic combustion performance of Pt/CeO2-NS was investigated. The structure-performance relationship is analyzed according to the coordination state of Pt. The Pt/CeO2-NS catalyst exhibited superior catalytic activity than the commercial CeO2-supported Pt catalyst, which is attributed to its higher oxygen vacancy and Pt4+.
研究领域:
催化化学
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材料科学
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环境科学
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DOI
高影响因子
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
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2019
·
IF 2.032
Xiaohong Lu,Juhua Chen,Linshu Jiao,Lei Zhong,Zhaoxin Lu,Chong Zhang,Fengxia Lu
Directed evolution methodologies have been used as promising strategies for improving the catalytic properties of many existing enzymes . In the presented work, this approach was applied to improve the enzyme activity of l -asparaginase I obtained from Bacillus megaterium H-1. After two rounds of error-prone polymerase chain reaction (epPCR) and two generations of sequential DNA shuffling , all of 5 different mutants showed a significant increase in the enzyme activity of l -asparaginase I, ranging from 6.27 to 22.78 IU/mL. Among these mutants, D-9B and DD-12G displayed the relatively high catalytic activity , which were 20.22-fold and 21.33-fold higher than the wild-type enzyme (WT), respectively. Furthermore, the catalytic efficiency ( k cat / K m ) of D-9B and DD-12G were also improved, which were 132.73 min −1 mM −1 and 146.39 min −1 mM −1 , respectively, in comparison to that of WT (3.39 min −1 mM −1 ). In addition, mutant DD-12G showed tolerance toward wider range of pH values and higher temperatures than its WT counterpart. Homology modeling of above two mutants reflected a reduction of hydrogen bonds and an introduction of flexible residues in the loops near the active catalytic site Thr15. These changes contributed to the flexibility of loops, which may lead to further enhancement in catalytic efficiency. Results also showed that approximately 88.5% (0.978 mg/kg) acrylamide could be removed from mutant DD-12G pre-treated fried potato chips. This study clearly shows that directional evolution methods can indeed be utilized to improve the activity of l -asparaginase, which could also provide research basis for future application in food industry.
研究领域:
生物技术
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酶学
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食品科学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2022
·
IF 8.025
Huibing Chi,Bingjie Xia,Juan Shen,Xiaoyu Zhu,Zhaoxin Lu,Fengxia Lu,Ping Zhu
Type II L-asparaginase as a pivotal enzyme agent has been applied to treating for acute lymphoblastic leukemia (ALL) and efficient mitigation of acrylamide formed in fried and baked foods. However, low activity, narrow range of pH stability, as well as undesirable glutaminase activity hinder the applications of this enzyme. In our work, A novel type II L-asparaginase (CgASNase) from Corynebacterium glutamicum with molecular mass of about 35 kDa was chosen to express in E. coli . CgASNase shared only 27 % structural identity with the reported L-asparaginase from Helicobacter pylori . The purified CgASNase showed the highest specific activity of 1979.08 IU mg −1 to L-asparagine, compared with reported type II ASNases in the literature. CgASNase displayed superior stability at a wide pH range from 5.0 to 11.0, and retained about 76 % of its activity at 30 °C for 30 min. The kinetic parameters K m (Michaelis constant), k cat (turnover number), and k cat / K m (catalytic efficiency) values of 4.66 mM, 79,697.40 min −1 , and 17,102.45 mM −1 min −1 , respectively. More importantly, CgASNase exhibited strict substrate specificity towards L-asparagine, no detectable activity to l -glutamine. To explore its ability to catalyze L-asparagine, CgASNase was supplied in frying potato chips, which produced the fries with 84 % less acrylamide content compared with no supply. These findings suggest that CgASNase presents excellent properties for chemotherapy against diseases and great potential in the food processing industry.
研究领域:
生物化学
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酶学
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食品科学
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肿瘤学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2022
·Vol. 23
·期 12
·6588
·
IF 6.208
Jiafeng Niu,Ruxue Yan,Juan Shen,Xiaoyu Zhu,Fanqiang Meng,Zhaoxin Lu,Fengxia Lu
Type I L-asparaginase fromBacillus licheniformisZ-1 (BlAase) was efficiently produced and secreted inBacillus subtilisRIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis inB. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.Keywords:L-asparaginase;Bacillus subtilis;promoter;dual-promoter systems;ribosome binding site;scale-up fermentation;acrylamide
研究领域:
生物技术
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分子生物学
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食品科学
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微生物学
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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）