Process Biochemistry最新文献

英文中文

Enhancing thermostability of lysine hydroxylase via a semi-rational design

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.005

Chengjuan Hu , Zhijie Zheng , Yue Zhang , Feifei Chen , Alei Zhang , Kequan Chen , Peicheng Luo

(2S,4 R)-4-Hydroxylysine (4-OH-Lys), a derivative of L-lysine, possesses a unique chemical structure that makes it a crucial precursor for the synthesis of pharmaceutical molecules, with extensive applications in the pharmaceutical and biochemical industries. Lysine hydroxylase (K4H) catalyzes the conversion of L-lysine to 4-OH-Lys, offering advantages such as mild reaction conditions, straightforward reaction steps, good regioselectivity, and high catalytic efficiency compared to chemical synthesis and natural extraction methods. However, the low thermostability of K4H hinders its application in large-scale production. In this study, we employed a semi-rational design approach, guided by ΔΔG folding free energy calculations and message-passing neural networks to enhance the thermostability of K4H. After two rounds of evolution, we identified two beneficial mutants: M25 (S101P/Q257M) and M32 (Q257M/V298I). Thermostability assessments revealed that the half-lives (t_1/2) of M25 and M32 at 40 °C were 23.9-fold and 13.3-fold higher than that of the wild-type (WT), with melting temperatures (T_m) exceeding those of WT by 4.2 °C and 8.3 °C, respectively. Molecular dynamics simulations illuminated the mechanisms underlying this enhanced thermostability. This work provides valuable insights into the thermostability of K4H and yields key mutants that are promising candidates for practical production of 4-OH-Lys.

{"title":"Enhancing thermostability of lysine hydroxylase via a semi-rational design","authors":"Chengjuan Hu , Zhijie Zheng , Yue Zhang , Feifei Chen , Alei Zhang , Kequan Chen , Peicheng Luo","doi":"10.1016/j.procbio.2024.12.005","DOIUrl":"10.1016/j.procbio.2024.12.005","url":null,"abstract":"<div><div>(2<em>S</em>,4 <em>R</em>)-4-Hydroxylysine (4-OH-Lys), a derivative of L-lysine, possesses a unique chemical structure that makes it a crucial precursor for the synthesis of pharmaceutical molecules, with extensive applications in the pharmaceutical and biochemical industries. Lysine hydroxylase (K4H) catalyzes the conversion of L-lysine to 4-OH-Lys, offering advantages such as mild reaction conditions, straightforward reaction steps, good regioselectivity, and high catalytic efficiency compared to chemical synthesis and natural extraction methods. However, the low thermostability of K4H hinders its application in large-scale production. In this study, we employed a semi-rational design approach, guided by ΔΔG folding free energy calculations and message-passing neural networks to enhance the thermostability of K4H. After two rounds of evolution, we identified two beneficial mutants: M25 (S101P/Q257M) and M32 (Q257M/V298I). Thermostability assessments revealed that the half-lives (t<sub>1/2</sub>) of M25 and M32 at 40 °C were 23.9-fold and 13.3-fold higher than that of the wild-type (WT), with melting temperatures (T<sub>m</sub>) exceeding those of WT by 4.2 °C and 8.3 °C, respectively. Molecular dynamics simulations illuminated the mechanisms underlying this enhanced thermostability. This work provides valuable insights into the thermostability of K4H and yields key mutants that are promising candidates for practical production of 4-OH-Lys.</div></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":"149 ","pages":"Pages 111-120"},"PeriodicalIF":3.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143139249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Chitosan functionalized with EDTA as a new support for enzyme immobilization and its application on enzymatic resolution of (R,S)-1-phenylethanol

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.004

Renata R. Magalhães , Paloma G. Abrantes , Poliana G. Abrantes , Israel F. Costa , Ercules E.S. Teotonio , Juliana A. Vale

This research demonstrates the immobilization of lipases from Burkholderia cepacia (BCL), Candida rugosa (CRL), and Aspergillus niger (ANL) on chitosan functionalized with EDTA (CHT-EDTA) as support, and its employment as a biocatalyst. The adsorption capacity of lipase onto the CHT-EDTA surface was observed to increase with contact time and an optimal pH value (pH 6,5). Enzymatic activity and stability of lipases before and after conjugation to the support were evaluated in different conditions (pH, temperature, and organic solvents) and then compared with those of the free one. Conformational changes were investigated using Fourier Transform Infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and thermogravimetric analysis (TGA). The results revealed that 112 mg/g for BCL/CHT-EDTA was the most significant amount of adsorbed lipase. The immobilized enzymes demonstrated enzymatic activity of 1.539 U/g for BCL/CHT-EDTA, followed by CRL/ and ANL/CHT-EDTA with 1.409 and 1.063 U/g, respectively. BCL/CHT-EDTA was then chosen to study the resolution of R,S-1-phenylethanol, achieving 99.9 % of

{ee}_{P}

, 82 % of

{ee}_{S}

, and 45 % of conversion (E > 200).

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引用次数: 0

Cellulase immobilization within zeolitic imidazolate frameworks by in situ encapsulation

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.001

Shanshan Tong , Chaozhong Xu , Kanghong Wang, Jia Ouyang, Xiaoli Gu

The industrial applications of cellulase are often restricted by its high cost and poor stability under extreme conditions. In this study, cellulase was immobilized using zeolitic imidazolate framework-8 (ZIF-8) through a one-pot encapsulation method, resulting in the formation of cellulase@ZIF-8 composite designed to address these challenges. The effects of varying Zn²⁺/2-methylimidazole molar ratios and different amounts of cellulase on the properties of cellulase@ZIF-8 were systematically investigated. Cellulase was found to act as a nucleation site, accelerating the formation of cellulase@ZIF-8 while promoting controlled crystal growth. At lower Zn²⁺/2-methylimidazole ratios, cross-shaped cellulase@ZIF-8 crystals with moderate enzymatic performance were obtained. Conversely, at higher Zn²⁺/2-methylimidazole ratios, the resulting spindle-shaped cellulase@ZIF-8 crystals exhibited superior enzyme activity of 327.8 U/g, and a relative activity of 88.6 %. Furthermore, this composite demonstrates excellent thermal and storage stability. The immobilized enzyme retained 92.8 % of its activity at a temperature of 70 °C. Additionally, it maintained 69.8 % of its relative enzymatic activity after undergoing five cycles. These findings have significant implications for the future application of cellulase@ZIF-8 composites in efficient and cost-effective lignocellulosic bioconversion.

{"title":"Cellulase immobilization within zeolitic imidazolate frameworks by in situ encapsulation","authors":"Shanshan Tong , Chaozhong Xu , Kanghong Wang, Jia Ouyang, Xiaoli Gu","doi":"10.1016/j.procbio.2024.12.001","DOIUrl":"10.1016/j.procbio.2024.12.001","url":null,"abstract":"<div><div>The industrial applications of cellulase are often restricted by its high cost and poor stability under extreme conditions. In this study, cellulase was immobilized using zeolitic imidazolate framework-8 (ZIF-8) through a one-pot encapsulation method, resulting in the formation of cellulase@ZIF-8 composite designed to address these challenges. The effects of varying Zn²⁺/2-methylimidazole molar ratios and different amounts of cellulase on the properties of cellulase@ZIF-8 were systematically investigated. Cellulase was found to act as a nucleation site, accelerating the formation of cellulase@ZIF-8 while promoting controlled crystal growth. At lower Zn²⁺/2-methylimidazole ratios, cross-shaped cellulase@ZIF-8 crystals with moderate enzymatic performance were obtained. Conversely, at higher Zn²⁺/2-methylimidazole ratios, the resulting spindle-shaped cellulase@ZIF-8 crystals exhibited superior enzyme activity of 327.8 U/g, and a relative activity of 88.6 %. Furthermore, this composite demonstrates excellent thermal and storage stability. The immobilized enzyme retained 92.8 % of its activity at a temperature of 70 °C. Additionally, it maintained 69.8 % of its relative enzymatic activity after undergoing five cycles. These findings have significant implications for the future application of cellulase@ZIF-8 composites in efficient and cost-effective lignocellulosic bioconversion.</div></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":"149 ","pages":"Pages 65-73"},"PeriodicalIF":3.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143139251","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A renewable natural resource for ferulic acid; An efficient precursor in biotechnological production of vanillin and strategies to enhance the yield of bio-vanillin from ferulic acid - Review

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.012

Dhesiga Krishnan, Manian Rameshpathy

Vanillin is a primary flavoring aromatic component derived from Vanilla orchids and has extensive applications in the food, cosmetics, and pharmaceutical industries. Biotechnological approaches have emerged as a worthwhile alternative to natural vanillin's rising demand and cost. Ferulic acid is a suitable precursor for vanillin among various substrates (lignin, isoeugenol, eugenol). Agro-industrial wastes like rice bran, corn cob, and wheat bran are rich in ferulic acid and serve as a sustainable feedstock for vanillin production, concurrently supporting the concept of efficient waste disposal. The crucial steps to improve vanillin production from ferulic acid have been listed, including the usage of immobilized cells/enzymes in the bioconversion process, and their advantages over free cells. This review mainly delves into the natural sources of ferulic acid, and the microbes involved in bio-conversion, providing a comprehensive overview of advancements in biotechnological vanillin production.

引用次数: 0

Cofactor immobilization for efficient dehydrogenase driven upgrading of xylose

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.11.028

Karolina Bachosz , Agnieszka Rybarczyk , Adam Piasecki , Jakub Zdarta , Anne S. Meyer , Teofil Jesionowski

In this study, the coupled immobilization on nanosilica of xylose dehydrogenase and alcohol dehydrogenase was accomplished with high efficiency, which was 90 %. Moreover, immobilization of the cofactors oxidized and reduced nicotinamide adenine dinucleotide, i.e., NAD⁺ and NADH, on silica material was examined and the impact on the effectiveness of the process was determined. The highest efficiency of NAD⁺ immobilization was found to be 56 %, which was obtained after 24 h of immobilization at 30 °C, pH 7 For NADH, the best immobilization efficiency was 53 % which was achieved after 24 h at 25 °C, pH 7. The K_M and V_max values were determined for various configurations of the biocatalytic systems showing, as expected, that immobilization of the enzymes decreased the catalytic rate (V_max) and slightly increased the K_M, but verifying that the immobilization of the cofactors did not significantly affect the kinetics, but would enable high conversion, and potentially continued enzymatic reaction. The use of the system configuration with co-immobilized enzymes, immobilized NAD⁺ and immobilized NADH thus allowed for obtaining over 90 % efficiency of xylose conversion in one batch, which was significantly higher than the systems with single free or only one immobilized cofactor. Using UV-Vis measurements, it was confirmed that effective cofactor regeneration occurred in the systems with immobilized components thus allowing for sustained enzyme catalyzed upgrading of xylose to xylonic acid.

{"title":"Cofactor immobilization for efficient dehydrogenase driven upgrading of xylose","authors":"Karolina Bachosz , Agnieszka Rybarczyk , Adam Piasecki , Jakub Zdarta , Anne S. Meyer , Teofil Jesionowski","doi":"10.1016/j.procbio.2024.11.028","DOIUrl":"10.1016/j.procbio.2024.11.028","url":null,"abstract":"<div><div>In this study, the coupled immobilization on nanosilica of xylose dehydrogenase and alcohol dehydrogenase was accomplished with high efficiency, which was 90 %. Moreover, immobilization of the cofactors oxidized and reduced nicotinamide adenine dinucleotide, i.e., NAD<sup>+</sup> and NADH, on silica material was examined and the impact on the effectiveness of the process was determined. The highest efficiency of NAD<sup>+</sup> immobilization was found to be 56 %, which was obtained after 24 h of immobilization at 30 °C, pH 7 For NADH, the best immobilization efficiency was 53 % which was achieved after 24 h at 25 °C, pH 7. The <em>K</em><sub>M</sub> and <em>V</em><sub>max</sub> values were determined for various configurations of the biocatalytic systems showing, as expected, that immobilization of the enzymes decreased the catalytic rate (<em>V</em><sub>max</sub>) and slightly increased the <em>K</em><sub>M</sub>, but verifying that the immobilization of the cofactors did not significantly affect the kinetics, but would enable high conversion, and potentially continued enzymatic reaction. The use of the system configuration with co-immobilized enzymes, immobilized NAD<sup>+</sup> and immobilized NADH thus allowed for obtaining over 90 % efficiency of xylose conversion in one batch, which was significantly higher than the systems with single free or only one immobilized cofactor. Using UV-Vis measurements, it was confirmed that effective cofactor regeneration occurred in the systems with immobilized components thus allowing for sustained enzyme catalyzed upgrading of xylose to xylonic acid.</div></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":"149 ","pages":"Pages 36-44"},"PeriodicalIF":3.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143139876","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Metal-organic frameworks for nutraceutical delivery: A futuristic perspective

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.11.034

P. Santhoshkumar, J.A. Moses

Metal-organic frameworks (MOFs) in conjunction with nutraceuticals exemplify the interdisciplinary nature of modern research, where materials science converges with the nutritional and health sciences to create innovative solutions. While MOFs find extensive applications in diverse industries, their utilization in the food industry, particularly the nutraceutical segment, is relatively naive. Recent efforts have focused on synthesizing novel MOFs utilizing biological materials, particularly cyclodextrin-based MOFs; imparting them with renewable, non-toxic, and edible properties. However, MOFs show promising potential, particularly in targeted delivery applications in pharmaceutical and nutraceuticals. Accordingly, this review explores the related design, synthesis, and structural aspects, further introducing the range of emerging applications in nutraceutical delivery. Their unique capabilities are highlighted, and challenges that require attention are detailed as the research landscape progresses. Having demonstrated merits in the field of drug delivery, their scope with nutraceuticals, as detailed in this review article, is untapped yet promising. Overall, a significant number of scientific documents focus on cyclodextrin; however, further research is necessary, majorly focusing on material identification, synthesis and applications.

{"title":"Metal-organic frameworks for nutraceutical delivery: A futuristic perspective","authors":"P. Santhoshkumar, J.A. Moses","doi":"10.1016/j.procbio.2024.11.034","DOIUrl":"10.1016/j.procbio.2024.11.034","url":null,"abstract":"<div><div>Metal-organic frameworks (MOFs) in conjunction with nutraceuticals exemplify the interdisciplinary nature of modern research, where materials science converges with the nutritional and health sciences to create innovative solutions. While MOFs find extensive applications in diverse industries, their utilization in the food industry, particularly the nutraceutical segment, is relatively naive. Recent efforts have focused on synthesizing novel MOFs utilizing biological materials, particularly cyclodextrin-based MOFs; imparting them with renewable, non-toxic, and edible properties. However, MOFs show promising potential, particularly in targeted delivery applications in pharmaceutical and nutraceuticals. Accordingly, this review explores the related design, synthesis, and structural aspects, further introducing the range of emerging applications in nutraceutical delivery. Their unique capabilities are highlighted, and challenges that require attention are detailed as the research landscape progresses. Having demonstrated merits in the field of drug delivery, their scope with nutraceuticals, as detailed in this review article, is untapped yet promising. Overall, a significant number of scientific documents focus on cyclodextrin; however, further research is necessary, majorly focusing on material identification, synthesis and applications.</div></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":"149 ","pages":"Pages 85-98"},"PeriodicalIF":3.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143140075","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

An electrochemical enzymatic biosensor based on Xylaria sp. laccase isolated from cassava waste is applied to quantify dopamine

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.002

Mariana Pontes Vieira , Patrícia Alessandra Bersanetti , Vitor Stabile Garcia , Gerhard Ett , Andreia de Araújo Morandim-Giannetti

Cassava waste (leaves and stems) was used to obtain endophytic fungi to assess laccase production. All the fungi (twenty-three) underwent an enzymatic extraction process to evaluate laccase production. The results revealed that six fungi from the stems and one from the leaves performed best. The fungus Xylaria sp. stood out for its greater productivity, and the growth parameters were optimized for this fungus (11 days, pH 6.70, and temperature 29°C). The enzyme extract enriched with laccase was then used to produce an electrochemical enzymatic biosensor immobilized in chitosan crosslinked with STMP. This biosensor was applied to quantify dopamine, with analysis conditions optimized for detecting low concentrations of the compound. The biosensor demonstrated its highest potential when operating at pH 5.60 and a temperature of 38.64°C, allowing the quantification of dopamine at concentrations from 0.17 to 492.83 µmol. L⁻¹ with a detection limit of 0.17 µmol. L⁻¹ . The biosensor demonstrated high reproducibility (0.4 % error) and stability after seven cycles.

{"title":"An electrochemical enzymatic biosensor based on Xylaria sp. laccase isolated from cassava waste is applied to quantify dopamine","authors":"Mariana Pontes Vieira , Patrícia Alessandra Bersanetti , Vitor Stabile Garcia , Gerhard Ett , Andreia de Araújo Morandim-Giannetti","doi":"10.1016/j.procbio.2024.12.002","DOIUrl":"10.1016/j.procbio.2024.12.002","url":null,"abstract":"<div><div>Cassava waste (leaves and stems) was used to obtain endophytic fungi to assess laccase production. All the fungi (twenty-three) underwent an enzymatic extraction process to evaluate laccase production. The results revealed that six fungi from the stems and one from the leaves performed best. The fungus <em>Xylaria</em> sp. stood out for its greater productivity, and the growth parameters were optimized for this fungus (11 days, pH 6.70, and temperature 29°C). The enzyme extract enriched with laccase was then used to produce an electrochemical enzymatic biosensor immobilized in chitosan crosslinked with STMP. This biosensor was applied to quantify dopamine, with analysis conditions optimized for detecting low concentrations of the compound. The biosensor demonstrated its highest potential when operating at pH 5.60 and a temperature of 38.64°C, allowing the quantification of dopamine at concentrations from 0.17 to 492.83 µmol. L⁻¹ with a detection limit of 0.17 µmol. L⁻¹ . The biosensor demonstrated high reproducibility (0.4 % error) and stability after seven cycles.</div></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":"149 ","pages":"Pages 237-247"},"PeriodicalIF":3.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143139502","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Unlocking the potential of CMP synthesis: Merging enzyme design, host genome editing, and fermentation optimization strategy

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.017

Zhou-Lei Qing , Chuan-Qi Sun , Zong-Lin Li , Zhi-Min Li

Cytidine-5′-monophosphate (CMP) and its derivatives play an important role in the fields of food, medicine and cosmetics. In this study, we used crude enzyme preparations of nucleoside kinase (NK) and polyphosphate kinase (PPK) to synthesize CMP in a synergistic reaction. A variant NK enzyme A220L/A236V/V237A/H252A was developed through a rational channel engineering approach, and its activity was increased by 9.17 times compared with the wild type. However, when cytidine was used as a substrate, the accumulation of a large amount of byproducts hindered the reaction efficiency, resulting in CMP yields that were always less than 80 %. By knocking out the cdd, rihA and rihC genes related to cytidine metabolism in the Escherichia coli host genome, the CMP yield was significantly increased to more than 95 %. Subsequently, we optimized the fermentation process in a 5-L bioreactor to further improve the crude enzyme activity. The optimized crude enzyme coupling reaction successfully generated 291 mM CMP within 20 h with a yield of up to 97 %. This study significantly improved the production efficiency of CMP by integrating key enzyme engineering, host bacteria modification and fermentation process optimization, laying a solid foundation for its industrial-scale production.

{"title":"Unlocking the potential of CMP synthesis: Merging enzyme design, host genome editing, and fermentation optimization strategy","authors":"Zhou-Lei Qing , Chuan-Qi Sun , Zong-Lin Li , Zhi-Min Li","doi":"10.1016/j.procbio.2024.12.017","DOIUrl":"10.1016/j.procbio.2024.12.017","url":null,"abstract":"<div><div>Cytidine-5′-monophosphate (CMP) and its derivatives play an important role in the fields of food, medicine and cosmetics. In this study, we used crude enzyme preparations of nucleoside kinase (NK) and polyphosphate kinase (PPK) to synthesize CMP in a synergistic reaction. A variant NK enzyme A220L/A236V/V237A/H252A was developed through a rational channel engineering approach, and its activity was increased by 9.17 times compared with the wild type. However, when cytidine was used as a substrate, the accumulation of a large amount of byproducts hindered the reaction efficiency, resulting in CMP yields that were always less than 80 %. By knocking out the <em>cdd</em>, <em>rihA</em> and <em>rihC</em> genes related to cytidine metabolism in the <em>Escherichia coli</em> host genome, the CMP yield was significantly increased to more than 95 %. Subsequently, we optimized the fermentation process in a 5-L bioreactor to further improve the crude enzyme activity. The optimized crude enzyme coupling reaction successfully generated 291 mM CMP within 20 h with a yield of up to 97 %. This study significantly improved the production efficiency of CMP by integrating key enzyme engineering, host bacteria modification and fermentation process optimization, laying a solid foundation for its industrial-scale production.</div></div>","PeriodicalId":20811,"journal":{"name":"Process Biochemistry","volume":"149 ","pages":"Pages 204-212"},"PeriodicalIF":3.7,"publicationDate":"2025-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143139248","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

In vitro enzymatic production of 3-hydroxypropionic acid from glycerol via CoA-independent pathway

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.023

Hanjoo Jo , Byeongsu Gu , Minkyeong Jeon , Sung In Lim

3-Hydroxypropionic acid (3-HP) is a C3-based versatile platform chemical offering significant industrial and environmental advantages over petrochemical derivatives. Microbial cell factories are commonly employed to produce 3-HP from various carbon sources, with glycerol being a key substrate via CoA-dependent and -independent pathways. However, challenges such as inhibited growth and reduced productivity, often due to substrate inhibition and toxic byproducts like 3-hydroxypropionaldehyde (3-HPA), limit the efficiency of glycerol-based microbial 3-HP production and its industrial scalability. To address these challenges, we propose an alternative in vitro approach for 3-HP production from glycerol through a coupled enzymatic reaction. We optimized the recombinant production of glycerol dehydratase (GDHt) and alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH), two key enzymes from Escherichia coli. After purification, GDHt and KGSADH showed 23.4 ± 4.6 s⁻¹ mM⁻¹ and 28.7 ± 5.0 s⁻¹ mM⁻¹ of k_cat/K_m, respectively. We then systematically varied enzyme concentrations, reaction temperature, and pH to determine the optimal conditions for 3-HP biosynthesis. The chain reaction, conducted with 100 nM of each enzyme, produced 43.5 μg/mL of 3-HP within 20 min under optimal conditions of 45 ºC and pH 9.0. This approach demonstrated the potential for a clean and efficient in vitro system for 3-HP production from renewable sources.

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Novel myco-fabrication of copper and nickel nanoparticles and evaluation of their effects against antibiotic resistance genes in different bacterial strains and anticancer potentials

IF 3.7 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Process Biochemistry

Pub Date : 2025-02-01 DOI: 10.1016/j.procbio.2024.12.008

Aisha Shaban , Zainab Zakaria , Ismail A. Abdelhamid , Heba K.A. Elhakim , El-Sayed R. El-Sayed

Several endophytic fungal species were isolated and tested for their biosynthetic capabilities of Cu-NPs and Ni-NPs, as a potentially green and simple method with low cost. A promising fungus Aspergillus terreus was found to synthesize the two types of NPs. The synthesis of Cu-NPs and Ni-NPs was confirmed by several techniques. The antibacterial activity of the synthesized NPs alone and in combinations with different antibiotics (Benzathine penicillin and Ceftriaxone) against some antibiotic-resistant bacterial strains was assessed. The results showed that the antibiotic resistance genes of the tested bacterial strains were expressed when treated with either antibiotics, Cu-NPs, or Ni-NPs alone. Otherwise, it were down-regulated when treated with a combination of NPs and antibiotics, indicating the synergistic and additive effects of such combinations. Moreover, the cytotoxic activities of Cu-NPs and Ni-NPs were evaluated, and the results confirmed their moderate safety margin against several cancer and normal cell lines. Finally, the synthesis efficiency of both NPs was optimized using the response surface methodology. Under optimal conditions, the synthesis efficiency was significantly enhanced reaching 85.08 (Cu-NPs) and 80.71 % (Ni-NPs). These findings indicate that the fungal strain is a promising biofactory for Cu-NPs and Ni-NPs with promising activities to combat antibiotic-resistant bacteria and cancer.

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