Pub Date : 2024-10-31DOI: 10.1016/j.enzmictec.2024.110543
Xin Wang, Jie Gao, Jiahui Wu, Xuan Li, Junxun Li, Haihong Li, Songlin Wang
A high polyglutamic acid (γ-PGA) producing strain of Bacillus natto UV-40-50 was screened by ultraviolet mutagenesis treatment and identified as still belonging to the Bacillus specie. The optimal fermentation medium composition for solid state fermentation (SSF) of B. natto strain UV-40-50 strain was determined by one-way analysis of variance, under which the yield of γ-PGA was 55.19 g/kg, and the presence and molecular weight of γ-PGA in the γ-PGA-purified samples were determined by a series of characterizations. The purification ability of the unseparated solid fermentation product (SFP) on ammonia nitrogen and nitrite in the water column, as well as its effect on soil water retention, germination rate and seedling length of lettuce and cabbage were further investigated. The results showed that the addition of 1 g/m3 SFP could effectively remove more than 60 % of ammonia nitrogen and more than 40 % of nitrite in the water body; the addition of 0.01 % SFP could increase the water retention capacity of cabbage soil by 2.13 times, and increase the water retention capacity of lettuce soil by 12 %; at the same time, the SFP could also significantly increase the germination rate and seedling length of both cabbage and lettuce.
{"title":"Poly-γ-glutamic production by solid-state fermentation of Bacillus natto in ammonia nitrogen movement and soil water retention processes.","authors":"Xin Wang, Jie Gao, Jiahui Wu, Xuan Li, Junxun Li, Haihong Li, Songlin Wang","doi":"10.1016/j.enzmictec.2024.110543","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110543","url":null,"abstract":"<p><p>A high polyglutamic acid (γ-PGA) producing strain of Bacillus natto UV-40-50 was screened by ultraviolet mutagenesis treatment and identified as still belonging to the Bacillus specie. The optimal fermentation medium composition for solid state fermentation (SSF) of B. natto strain UV-40-50 strain was determined by one-way analysis of variance, under which the yield of γ-PGA was 55.19 g/kg, and the presence and molecular weight of γ-PGA in the γ-PGA-purified samples were determined by a series of characterizations. The purification ability of the unseparated solid fermentation product (SFP) on ammonia nitrogen and nitrite in the water column, as well as its effect on soil water retention, germination rate and seedling length of lettuce and cabbage were further investigated. The results showed that the addition of 1 g/m3 SFP could effectively remove more than 60 % of ammonia nitrogen and more than 40 % of nitrite in the water body; the addition of 0.01 % SFP could increase the water retention capacity of cabbage soil by 2.13 times, and increase the water retention capacity of lettuce soil by 12 %; at the same time, the SFP could also significantly increase the germination rate and seedling length of both cabbage and lettuce.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142603884","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}
Pub Date : 2024-10-30DOI: 10.1016/j.enzmictec.2024.110542
Ruofan Wu, Jiahui Yang, Yujia Jiang, Fengxue Xin
Lactic acid is a versatile building block that can be produced via microbial fermentation. Owing to the high optical purity, approximately 90 % of lactic acid is produced by microbes. Recently, the biosynthesis of lactic acid from lignocellulose has concerned much attentions. However, the cost-effective process faces several obstacles because of the complex structure of lignocellulose. This review will comprehensively summarize the state-of-the-art lactic acid production from lignocellulose, including the commonly used lactate-producing microorganisms, the co-utilization of glucose and xylose for the lactic acid production, as well as the lactic acid production from lignocellulose hydrolysate. Furthermore, the strategies regarding the lignocellulosic lactic acid production via consolidated bioprocessing will be also discussed, which can greatly reduce the complexity of the fermentation process.
{"title":"Advances and prospects for lactic acid production from lignocellulose","authors":"Ruofan Wu, Jiahui Yang, Yujia Jiang, Fengxue Xin","doi":"10.1016/j.enzmictec.2024.110542","DOIUrl":"10.1016/j.enzmictec.2024.110542","url":null,"abstract":"<div><div>Lactic acid is a versatile building block that can be produced via microbial fermentation. Owing to the high optical purity, approximately 90 % of lactic acid is produced by microbes. Recently, the biosynthesis of lactic acid from lignocellulose has concerned much attentions. However, the cost-effective process faces several obstacles because of the complex structure of lignocellulose. This review will comprehensively summarize the state-of-the-art lactic acid production from lignocellulose, including the commonly used lactate-producing microorganisms, the co-utilization of glucose and xylose for the lactic acid production, as well as the lactic acid production from lignocellulose hydrolysate. Furthermore, the strategies regarding the lignocellulosic lactic acid production via consolidated bioprocessing will be also discussed, which can greatly reduce the complexity of the fermentation process.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567122","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}
Pub Date : 2024-10-28DOI: 10.1016/j.enzmictec.2024.110532
Shuqi Xing , Wei Xie , Guangli Hu , Chaocheng Luo , Hong Zhu , Laping He , Cuiqin Li , Xiao Wang , Xuefeng Zeng
GDSL-type esterases are promising biocatalysts for the food and pharmaceutical industries. Here, a GDSL-type esterase from Aspergillus niger CCTCC No. M2012538 (INANE1) was expressed and purified in Pichia pastoris GS115, and its catalytic performances were evaluated, including the synthesis of cinnamyl acetate and deacetyl-7-aminocephalosporanic acid (D-7-ACA). In addition, molecular docking and molecular dynamics simulations analyzed INANE1's substrate specificity. The substrate specificity profile indicated the recombinant esterase (rINANE1) was an acetylesterase with high specificity for p-nitrophenyl acetate (p-NPA). The rINANE1 exhibited maximum activity at pH 8.0 and 35 °C, where Km and Vmax were calculated as 0.13±0.03 mM and 22.56 ± 0.32 μmoL/min/mg, respectively. The yield of cinnamyl acetate of about 85 % was achieved in 24 h. The conversion rate of 7-aminocephalosporanic acid (7-ACA) could reach 92.71 ± 1.78 % at 25 °C and 2.5 h. Moreover, the INANE1 structure model, molecular docking, and molecular dynamics simulation demonstrated that the pocket of the catalytic triad Ser34, Asn267, and His270 could only accommodate p-NPA. INANE1 may be the first fungi esterase with cinnamyl acetate synthetic activity and 7-ACA hydrolysis activity. Therefore, INANE1 would be a promising enzyme with industrial values.
{"title":"The synthesis of cinnamyl acetate and deacetyl-7-aminocephalosporanic acid by a GDSL-type esterase and its substrate specificity analysis","authors":"Shuqi Xing , Wei Xie , Guangli Hu , Chaocheng Luo , Hong Zhu , Laping He , Cuiqin Li , Xiao Wang , Xuefeng Zeng","doi":"10.1016/j.enzmictec.2024.110532","DOIUrl":"10.1016/j.enzmictec.2024.110532","url":null,"abstract":"<div><div>GDSL-type esterases are promising biocatalysts for the food and pharmaceutical industries. Here, a GDSL-type esterase from <em>Aspergillus niger</em> CCTCC No. M2012538 (INANE1) was expressed and purified in <em>Pichia pastoris</em> GS115, and its catalytic performances were evaluated, including the synthesis of cinnamyl acetate and deacetyl-7-aminocephalosporanic acid (D-7-ACA). In addition, molecular docking and molecular dynamics simulations analyzed INANE1's substrate specificity. The substrate specificity profile indicated the recombinant esterase (rINANE1) was an acetylesterase with high specificity for <em>p</em>-nitrophenyl acetate (<em>p</em>-NPA). The rINANE1 exhibited maximum activity at pH 8.0 and 35 °C, where K<sub>m</sub> and V<sub>max</sub> were calculated as 0.13±0.03 mM and 22.56 ± 0.32 μmoL/min/mg, respectively. The yield of cinnamyl acetate of about 85 % was achieved in 24 h. The conversion rate of 7-aminocephalosporanic acid (7-ACA) could reach 92.71 ± 1.78 % at 25 °C and 2.5 h. Moreover, the INANE1 structure model, molecular docking, and molecular dynamics simulation demonstrated that the pocket of the catalytic triad Ser34, Asn267, and His270 could only accommodate <em>p</em>-NPA. INANE1 may be the first fungi esterase with cinnamyl acetate synthetic activity and 7-ACA hydrolysis activity. Therefore, INANE1 would be a promising enzyme with industrial values.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142544459","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}
Pub Date : 2024-10-28DOI: 10.1016/j.enzmictec.2024.110531
Hiram Y. Guerrero-Elias , M. Angeles Camacho-Ruiz , Ruben Espinosa-Salgado , Juan Carlos Mateos-Díaz , Rosa María Camacho-Ruiz , Ali Asaff-Torres , Jorge A. Rodríguez
Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), hold notable significance due to their pharmaceutical relevance. Obtaining PUFAs from diverse sources like vegetables, fish oils, and algae poses challenges due to the mixed fatty acid (FA) composition. Therefore, focusing on particular FAs necessitates purification and resolution processes. To address this, we propose a continuous assay for screening lipases selective for ethyl EPA (E-EPA) or ethyl DHA (E-DHA). Utilizing microplate spectrophotometry, the method enables quantification of liberated fatty acids from ethyl esters (E-EPA or E-DHA). This involves assessing enzyme selectivity by measuring the release of FAs through p-nitrophenolate protonation, either separately for each substrate or in competition with a reference substrate, resorufin acetate. Ten lipases underwent screening, revealing Burkholderia cepacia lipase's (BCL) preference for ethyl DHA hydrolysis (E-EPA/E-DHA = 0.82 ± 0.07 and the lipase selectivity ratio (S) for E-EPA/E-DHA = 0.13 ± 0.04) and Candida antarctica lipase B's (CALB) high specific activity towards both E-EPA and E-DHA (531.14 ± 37.76 and 281.79 ± 2.79 U/mg, respectively) and E-EPA preference (E-EPA/E-DHA = 1.86 ± 0.15 and S E-EPA/E-DHA = 2.59±0.15). Candida rugosa recombinant isoform 4 (rCRLip4) and commercial Candida rugosa lipase (CRL) exhibited significant preference for E-EPA hydrolysis (E-EPA/E-DHA = 2.18 ±0.51 and 2.26 ±0.36, respectively; and S E-EPA/E-DHA = 7.59 ± 1.59 and 7.88 ± 2.13, respectively). Docking analyses of rCRLip4, BCL, and CALB demonstrated no statistically significant differences in activation energies or distances to the catalytic serine; however, they agreed with the experimental results. These findings suggest potential mutagenesis or directed evolution strategies for CALB to enhance E-EPA selectivity, with rCRLip4 emerging as a promising candidate for further investigation. This assay offers a valuable tool for identifying lipases with desired substrate selectivity, with broad implications for pharmaceutical and biotechnological applications.
{"title":"Spectrophotometric assay for the screening of selective enzymes towards DHA and EPA ethyl esters hydrolysis","authors":"Hiram Y. Guerrero-Elias , M. Angeles Camacho-Ruiz , Ruben Espinosa-Salgado , Juan Carlos Mateos-Díaz , Rosa María Camacho-Ruiz , Ali Asaff-Torres , Jorge A. Rodríguez","doi":"10.1016/j.enzmictec.2024.110531","DOIUrl":"10.1016/j.enzmictec.2024.110531","url":null,"abstract":"<div><div>Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), hold notable significance due to their pharmaceutical relevance. Obtaining PUFAs from diverse sources like vegetables, fish oils, and algae poses challenges due to the mixed fatty acid (FA) composition. Therefore, focusing on particular FAs necessitates purification and resolution processes. To address this, we propose a continuous assay for screening lipases selective for ethyl EPA (E-EPA) or ethyl DHA (E-DHA). Utilizing microplate spectrophotometry, the method enables quantification of liberated fatty acids from ethyl esters (E-EPA or E-DHA). This involves assessing enzyme selectivity by measuring the release of FAs through <em>p</em>-nitrophenolate protonation, either separately for each substrate or in competition with a reference substrate, resorufin acetate. Ten lipases underwent screening, revealing <em>Burkholderia cepacia</em> lipase's (BCL) preference for ethyl DHA hydrolysis (E-EPA/E-DHA = 0.82 ± 0.07 and the lipase selectivity ratio (S) for E-EPA/E-DHA = 0.13 ± 0.04) and <em>Candida antarctica</em> lipase B's (CALB) high specific activity towards both E-EPA and E-DHA (531.14 ± 37.76 and 281.79 ± 2.79 U/mg, respectively) and E-EPA preference (E-EPA/E-DHA = 1.86 ± 0.15 and S E-EPA/E-DHA = 2.59±0.15). <em>Candida rugosa</em> recombinant isoform 4 (rCRLip4) and commercial <em>Candida rugosa</em> lipase (CRL) exhibited significant preference for E-EPA hydrolysis (E-EPA/E-DHA = 2.18 ±0.51 and 2.26 ±0.36, respectively; and S E-EPA/E-DHA = 7.59 ± 1.59 and 7.88 ± 2.13, respectively). Docking analyses of rCRLip4, BCL, and CALB demonstrated no statistically significant differences in activation energies or distances to the catalytic serine; however, they agreed with the experimental results. These findings suggest potential mutagenesis or directed evolution strategies for CALB to enhance E-EPA selectivity, with rCRLip4 emerging as a promising candidate for further investigation. This assay offers a valuable tool for identifying lipases with desired substrate selectivity, with broad implications for pharmaceutical and biotechnological applications.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142561420","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}
Pub Date : 2024-10-20DOI: 10.1016/j.enzmictec.2024.110526
Chengfeng Xue, Wan-Wen Ting, Jiun-Jang Juo, I-Son Ng
The probiotic Escherichia coli Nissle 1917 (EcN), known for its superior acid resistance (AR), serves as a promising chassis for live therapeutics due to the effective colonization capabilities. However, the enzymatic activity regarding AR in EcN remains poorly understood. First, we investigated the AR systems of EcN by measuring cell growth under acidic stress and exploring the relationship of mutations to their corresponding enzymatic activities. As a result, the catalytic activity of inducible decarboxylases of GadB, AdiA and CadA, responsible for metabolizing glutamate, arginine, and lysine, exhibited an average 2-fold increase in EcN compared to the reference strain MG1655. Furthermore, we discovered that the glutamate-dependent AR2 system in EcN was meticulously regulated by specific regulons such as GadW. This study not only revealed the physiology of EcN under acidic conditions, but also highlighted that the mutated core enzymes in the AR system of EcN exhibit improved activities.
益生菌大肠埃希氏菌 Nissle 1917（EcN）以其卓越的耐酸性（AR）而闻名，由于其有效的定植能力，它是一种很有前景的活体疗法底盘。然而，人们对 EcN 中 AR 的酶活性仍然知之甚少。首先，我们通过测量酸性胁迫下的细胞生长来研究 EcN 的 AR 系统，并探索突变与其相应酶活性的关系。结果发现，与参考菌株 MG1655 相比，EcN 中负责谷氨酸、精氨酸和赖氨酸代谢的诱导性脱羧酶 GadB、AdiA 和 CadA 的催化活性平均增加了 2 倍。此外，我们还发现 EcN 中依赖谷氨酸的 AR2 系统受到 GadW 等特定调控子的严格调控。这项研究不仅揭示了 EcN 在酸性条件下的生理机理，还突出显示了 EcN AR 系统中突变的核心酶表现出更高的活性。
{"title":"New insight into acid-resistant enzymes from natural mutations of Escherichia coli Nissle 1917","authors":"Chengfeng Xue, Wan-Wen Ting, Jiun-Jang Juo, I-Son Ng","doi":"10.1016/j.enzmictec.2024.110526","DOIUrl":"10.1016/j.enzmictec.2024.110526","url":null,"abstract":"<div><div>The probiotic <em>Escherichia coli</em> Nissle 1917 (EcN), known for its superior acid resistance (AR), serves as a promising chassis for live therapeutics due to the effective colonization capabilities. However, the enzymatic activity regarding AR in EcN remains poorly understood. First, we investigated the AR systems of EcN by measuring cell growth under acidic stress and exploring the relationship of mutations to their corresponding enzymatic activities. As a result, the catalytic activity of inducible decarboxylases of GadB, AdiA and CadA, responsible for metabolizing glutamate, arginine, and lysine, exhibited an average 2-fold increase in EcN compared to the reference strain MG1655. Furthermore, we discovered that the glutamate-dependent AR2 system in EcN was meticulously regulated by specific regulons such as GadW. This study not only revealed the physiology of EcN under acidic conditions, but also highlighted that the mutated core enzymes in the AR system of EcN exhibit improved activities.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142497334","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}
Pub Date : 2024-10-20DOI: 10.1016/j.enzmictec.2024.110530
Yinghui He , Hui Zhang , Yeledana Huwati , Na Shu , Wei Hu , Xiwen Jia , Kaili Ding , Xueyan Liang , Luoyang Liu , Lujia Han , Weihua Xiao
Cellulases are essential for the enzymatic saccharification of lignocellulose. They play a crucial role in breaking down the structure of lignocellulose to obtain fermentable sugars. In this study, we conducted on-site cellulase production by Trichoderma reesei RutC-30 through submerged fermentation. The effects of carbon source, nitrogen source, KH2PO4, and mineral elements on cellulase production were evaluated using the hydrolyzed total sugar concentration of ball-milled corn stover as an indicator. The optimal fermentation medium conditions for cellulase production were determined through orthogonal experimental design analysis. Additionally, by optimizing culture conditions, including inoculation, pH, and bottling volume, we achieved a total sugar concentration of 92.25 g/L. After the optimization, the FPA, CMCA, protein, and total sugar concentration increased by 75.49 %, 18.43 %, 89.71 %, and 17.83 %, respectively. Furthermore, corn stover pretreated by different methods was applied to induce cellulase production. Ball-milled and steam-exploded corn stover was identified as suitable incubation carbon sources with total sugar concentration up to 94.31 g/L. Our work exploits the cellulase induced by lignocellulose and then applies it to lignocellulose, enabling the customization and providing a reference for the production of cellulase with corn stover as an inducer.
{"title":"On-site cellulase production by Trichoderma reesei RutC-30 to enhance the enzymatic saccharification of ball-milled corn stover","authors":"Yinghui He , Hui Zhang , Yeledana Huwati , Na Shu , Wei Hu , Xiwen Jia , Kaili Ding , Xueyan Liang , Luoyang Liu , Lujia Han , Weihua Xiao","doi":"10.1016/j.enzmictec.2024.110530","DOIUrl":"10.1016/j.enzmictec.2024.110530","url":null,"abstract":"<div><div>Cellulases are essential for the enzymatic saccharification of lignocellulose. They play a crucial role in breaking down the structure of lignocellulose to obtain fermentable sugars. In this study, we conducted on-site cellulase production by <em>Trichoderma reesei</em> RutC-30 through submerged fermentation. The effects of carbon source, nitrogen source, KH<sub>2</sub>PO<sub>4</sub>, and mineral elements on cellulase production were evaluated using the hydrolyzed total sugar concentration of ball-milled corn stover as an indicator. The optimal fermentation medium conditions for cellulase production were determined through orthogonal experimental design analysis. Additionally, by optimizing culture conditions, including inoculation, pH, and bottling volume, we achieved a total sugar concentration of 92.25 g/L. After the optimization, the FPA, CMCA, protein, and total sugar concentration increased by 75.49 %, 18.43 %, 89.71 %, and 17.83 %, respectively. Furthermore, corn stover pretreated by different methods was applied to induce cellulase production. Ball-milled and steam-exploded corn stover was identified as suitable incubation carbon sources with total sugar concentration up to 94.31 g/L. Our work exploits the cellulase induced by lignocellulose and then applies it to lignocellulose, enabling the customization and providing a reference for the production of cellulase with corn stover as an inducer.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142497335","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}
Pub Date : 2024-10-18DOI: 10.1016/j.enzmictec.2024.110529
Hyun Jin Kim , Suwon Kim , Yeda Lee , Yuni Shin , Suhye Choi , Jinok Oh , Jaeho Jeong , HyunA Park , Jungoh Ahn , Jeong Chan Joo , Kwon-Young Choi , Shashi Kant Bhatia , Yung-Hun Yang
Indigo is a unique blue dye that has been used in the textile industry for centuries and is currently mass-produced commercially through chemical synthesis. However, the use of toxic substrates and reducing agents for chemical synthesis is associated with environmental concerns, necessitating the development of eco-friendly alternatives based on microbial production. In this study, a robust industrial strategy for indigo production was developed using Pseudomonas putida KT2440 as the host strain, which is characterized by its excellent ability to degrade aromatic compounds and high resistance to environmental stress. By introducing the genes tryptophanase (tnaA) and Flavin-containing monooxygenase (FMO), a P. putida HI201 strain was constructed to produce indigo from tryptophan. To enhance the indigo yield, culture conditions, including medium composition, temperature, tryptophan concentration, and shaking speed, were optimized. Under optimal conditions such as TB medium, 15 mM tryptophan, 30°C, 200 rpm, P. putida HI201 biosynthesized 1.31 g/L indigo from tryptophan in a fed-batch fermentation system. The introduction of tnaA and FMO genes also enabled the production of indigo in various P. putida species, and the indigo-producing strain had a blue color, which served as a visual indicator. This study presents a strategy for using P. putida as a host for robust and sustainable microbial production of indigo, highlighting the strain's applicability and efficiency in environment friendly dye synthesis.
靛蓝是一种独特的蓝色染料，几个世纪以来一直用于纺织业，目前通过化学合成进行大规模商业化生产。然而，使用有毒底物和还原剂进行化学合成会引起环境问题，因此有必要开发基于微生物生产的生态友好型替代品。该菌株具有降解芳香族化合物能力强、抗环境胁迫能力强等特点。通过引入色氨酸酶（tnaA）和含黄素单氧化酶（FMO）基因，构建了一株利用色氨酸生产靛蓝的假单胞菌 HI201。为提高靛蓝产量，对培养基成分、温度、色氨酸浓度和振荡速度等培养条件进行了优化。在 TB 培养基、15 mM 色氨酸、30°C、200 rpm 等最佳条件下，P. putida HI201 在饲料批量发酵系统中从色氨酸中生物合成了 1.31 g/L 的靛蓝。引入 tnaA 和 FMO 基因也能在不同种类的 P. putida 中产生靛蓝，而且产生靛蓝的菌株呈蓝色，可作为视觉指标。本研究提出了一种以 P. putida 为宿主进行稳健、可持续的靛蓝微生物生产的策略，凸显了该菌株在环境友好型染料合成中的适用性和高效性。
{"title":"Production of bio-indigo from engineered Pseudomonas putida KT2440 harboring tryptophanase and flavin-containing monooxygenase","authors":"Hyun Jin Kim , Suwon Kim , Yeda Lee , Yuni Shin , Suhye Choi , Jinok Oh , Jaeho Jeong , HyunA Park , Jungoh Ahn , Jeong Chan Joo , Kwon-Young Choi , Shashi Kant Bhatia , Yung-Hun Yang","doi":"10.1016/j.enzmictec.2024.110529","DOIUrl":"10.1016/j.enzmictec.2024.110529","url":null,"abstract":"<div><div>Indigo is a unique blue dye that has been used in the textile industry for centuries and is currently mass-produced commercially through chemical synthesis. However, the use of toxic substrates and reducing agents for chemical synthesis is associated with environmental concerns, necessitating the development of eco-friendly alternatives based on microbial production. In this study, a robust industrial strategy for indigo production was developed using <em>Pseudomonas putida</em> KT2440 as the host strain, which is characterized by its excellent ability to degrade aromatic compounds and high resistance to environmental stress. By introducing the genes tryptophanase (<em>tnaA</em>) and Flavin-containing monooxygenase <em>(FMO</em>), a <em>P. putida</em> HI201 strain was constructed to produce indigo from tryptophan. To enhance the indigo yield, culture conditions, including medium composition, temperature, tryptophan concentration, and shaking speed, were optimized. Under optimal conditions such as TB medium, 15 mM tryptophan, 30°C, 200 rpm, <em>P. putida</em> HI201 biosynthesized 1.31 g/L indigo from tryptophan in a fed-batch fermentation system. The introduction of <em>tnaA</em> and <em>FMO</em> genes also enabled the production of indigo in various <em>P. putida</em> species, and the indigo-producing strain had a blue color, which served as a visual indicator. This study presents a strategy for using <em>P. putida</em> as a host for robust and sustainable microbial production of indigo, highlighting the strain's applicability and efficiency in environment friendly dye synthesis.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142497336","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}
Pub Date : 2024-10-16DOI: 10.1016/j.enzmictec.2024.110528
Bjorn Thor Adalsteinsson , Hörður Guðmundsson , Andrius Jasilionis , Morten Schiøtt , Maria Dalgaard Mikkelsen , Elísabet Eik Guðmundsdóttir , Pavithra Sivakumar , Annika Malmgren , Tushar Kaushik , Erik Apelqvist , Signe Vangsgaard , Rébecca Leblay , Ólafur Friðjónsson , Anne S. Meyer , Eva Nordberg Karlsson , Guðmundur Óli Hreggviðsson
Seaweeds (macroalgae) are an attractive resource for diverse microbial- and enzymatic production processes. They are abundant, underutilized, cheap, and rich in carbohydrates, and therefore have the potential to be used as a source of mono- or oligosaccharides, and as substrates for industrial fermentation processes. Many seaweed polysaccharides, including the sulfated polysaccharides ulvan and fucoidan, are however complex and heterogenous in structure, and there are currently few enzymes available to modify them, and understanding of their enzymatic depolymerization remains limited. The present study aimed to identify and characterize robust fucoidanases and ulvan lyases. Metagenomes were obtained from microbial enrichments from an intertidal hot-spring, genes identified that expressed putative fucoidanases and ulvan lyases, and following gene cloning and expression, the respective enzymes were screened for enzymatic activity. Consistent with their origin, the identified protein sequences were considerably divergent from previously characterized enzymes, with a 44 % average maximal sequence identity. In total, the study resulted in the characterization of 10 new fucoidanases (GH107 and GH168 families) and 8 new ulvan lyases (PL24, PL25 and PL40 families). Notably, the new fucoidanases appeared to have functional specificity towards fucoidan containing α-1,3 linked L-fucosyl and several functioned at high temperature. The study contributes a metagenomics-based approach to identify new seaweed polysaccharide degrading enzymes and an increased understanding of the diversity of such enzymes, which may have implications for the realization of biotechnology based valorization of seaweed biomass.
{"title":"Targeted metagenomics – Enrichment for enzymes active on sulfated polysaccharides from seaweeds","authors":"Bjorn Thor Adalsteinsson , Hörður Guðmundsson , Andrius Jasilionis , Morten Schiøtt , Maria Dalgaard Mikkelsen , Elísabet Eik Guðmundsdóttir , Pavithra Sivakumar , Annika Malmgren , Tushar Kaushik , Erik Apelqvist , Signe Vangsgaard , Rébecca Leblay , Ólafur Friðjónsson , Anne S. Meyer , Eva Nordberg Karlsson , Guðmundur Óli Hreggviðsson","doi":"10.1016/j.enzmictec.2024.110528","DOIUrl":"10.1016/j.enzmictec.2024.110528","url":null,"abstract":"<div><div>Seaweeds (macroalgae) are an attractive resource for diverse microbial- and enzymatic production processes. They are abundant, underutilized, cheap, and rich in carbohydrates, and therefore have the potential to be used as a source of mono- or oligosaccharides, and as substrates for industrial fermentation processes. Many seaweed polysaccharides, including the sulfated polysaccharides ulvan and fucoidan, are however complex and heterogenous in structure, and there are currently few enzymes available to modify them, and understanding of their enzymatic depolymerization remains limited. The present study aimed to identify and characterize robust fucoidanases and ulvan lyases. Metagenomes were obtained from microbial enrichments from an intertidal hot-spring, genes identified that expressed putative fucoidanases and ulvan lyases, and following gene cloning and expression, the respective enzymes were screened for enzymatic activity. Consistent with their origin, the identified protein sequences were considerably divergent from previously characterized enzymes, with a 44 % average maximal sequence identity. In total, the study resulted in the characterization of 10 new fucoidanases (GH107 and GH168 families) and 8 new ulvan lyases (PL24, PL25 and PL40 families). Notably, the new fucoidanases appeared to have functional specificity towards fucoidan containing α-1,3 linked L-fucosyl and several functioned at high temperature. The study contributes a metagenomics-based approach to identify new seaweed polysaccharide degrading enzymes and an increased understanding of the diversity of such enzymes, which may have implications for the realization of biotechnology based valorization of seaweed biomass.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142578510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-10-16DOI: 10.1016/j.enzmictec.2024.110527
Yi-An Hong , Aye Aye Khine , Yu-Wei Lin , Pei-Yun Lee , Wei-Xiang Hong , Anren Hu , Tzenge-Lien Shih , Hao-Ping Chen
Borneol, a medicinally important bicyclic monoterpene, facilitates drug transport across mucous membranes and the blood-brain barrier. Derivatives of borneol and camphor also have numerous biomedical applications. Borneol is currently industrially synthesized via the conversion of turpentine and α-pinene. However, the major product is racemic isoborneol rather than racemic borneol. Both borneol and isoborneol are degraded by the soil bacterium Pseudomonas via a well-established degradation pathway. Two indigenous Pseudomonas strains were used to convert racemic isoborneol to other optically pure bicyclic monoterpenes here. Our results showed that deletion of the camE2,5 gene alone from the strain TCU-HL1 genome led to the complete loss of borneol and camphor degradation ability. Knockout of both camE2,5 and bdh1 (TCU-HL1Δbdh1ΔcamE2,5) restored the degradation capability as the role of Bdh1 was replaced by that of Bdh2. This mutant converted racemic isoborneol into an optically pure bicyclic monoterpene, 2,5-diketocamphane, with a 45 % recovery yield. RT-qPCR results suggested that camE2,5 expression plays a pivotal role in regulating the borneol/camphor degradation cluster. While (+)-borneol, (–)-borneol and (+)-camphor can be obtained from plants for mass production purposes, (–)-camphor cannot be obtained in the same manner. P. monteilii TCU-CK1 converted racemic isoborneol into (–)-camphor and 3,6-diketocamphane, with 15 % and 10 % recovery yields, respectively. In conclusion, we report the role of camE2,5 in regulating the borneol/camphor degradation operon and biotransformation methods to produce several optically pure bicyclic monoterpenes.
{"title":"Metabolic engineering of the borneol and camphor degradation pathways in Pseudomonas to produce optically pure bicyclic monoterpenes","authors":"Yi-An Hong , Aye Aye Khine , Yu-Wei Lin , Pei-Yun Lee , Wei-Xiang Hong , Anren Hu , Tzenge-Lien Shih , Hao-Ping Chen","doi":"10.1016/j.enzmictec.2024.110527","DOIUrl":"10.1016/j.enzmictec.2024.110527","url":null,"abstract":"<div><div>Borneol, a medicinally important bicyclic monoterpene, facilitates drug transport across mucous membranes and the blood-brain barrier. Derivatives of borneol and camphor also have numerous biomedical applications. Borneol is currently industrially synthesized via the conversion of turpentine and α-pinene. However, the major product is racemic isoborneol rather than racemic borneol. Both borneol and isoborneol are degraded by the soil bacterium <em>Pseudomonas</em> via a well-established degradation pathway. Two indigenous <em>Pseudomonas</em> strains were used to convert racemic isoborneol to other optically pure bicyclic monoterpenes here. Our results showed that deletion of the <em>camE</em><sub><em>2,5</em></sub> gene alone from the strain TCU-HL1 genome led to the complete loss of borneol and camphor degradation ability. Knockout of both <em>camE</em><sub><em>2,5</em></sub> and <em>bdh1</em> (TCU-HL1<em>Δbdh1ΔcamE</em><sub><em>2,5</em></sub>) restored the degradation capability as the role of Bdh1 was replaced by that of Bdh2. This mutant converted racemic isoborneol into an optically pure bicyclic monoterpene, 2,5-diketocamphane, with a 45 % recovery yield. RT-qPCR results suggested that <em>camE</em><sub><em>2,5</em></sub> expression plays a pivotal role in regulating the borneol/camphor degradation cluster. While (+)-borneol, (–)-borneol and (+)-camphor can be obtained from plants for mass production purposes, (–)-camphor cannot be obtained in the same manner. <em>P. monteilii</em> TCU-CK1 converted racemic isoborneol into (–)-camphor and 3,6-diketocamphane, with 15 % and 10 % recovery yields, respectively. In conclusion, we report the role of <em>camE</em><sub><em>2,5</em></sub> in regulating the borneol/camphor degradation operon and biotransformation methods to produce several optically pure bicyclic monoterpenes.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142460872","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}
Pub Date : 2024-10-10DOI: 10.1016/j.enzmictec.2024.110524
Sen-Lin Peng , Yi Ding , Meng-Hai Xiang , Ken Chen , Xiao-Dong Gao , Ning Wang
N-Glycosylation is one of the most important posttranslational modifications of proteins. Nearly the entire surface of cells and almost all secreted proteins in humans are modified with complex-type N-glycans, whose functions are affected by the number of N-glycan branches. N-Acetylglucosaminyltransferase-IVa (GnT-IVa) is a Golgi glycosyltransferase that transfers a GlcNAc to the α-1,3 mannose arm of the biantennary N-glycan GlcNAc2Man3GlcNAc2 to form a β-1,4 GlcNAc branched structure. The soluble expression of mammalian glycosyltransferases in heterologous hosts is often challenging. In the present study, human GnT-IVa (HsGnT-IVa) was cloned as an N-terminal truncated form that was fused with solubility-enhancing tags or signal peptides and overexpressed in Escherichia coli (E. coli). Our results showed that recombinant HsGnT-IVa could be overexpressed in its highest soluble and active form when the first 87 amino acids were removed and was fused with maltose-binding protein (MBP). By optimizing the induction conditions, the expression level of the recombinant protein was increased to yield approximately 540 mg per liter of culture after affinity purification. The purified enzyme exhibited appropriate glycosyltransferase activity, and the Km value of the acceptor substrate was calculated as 1.1 mM. Characterization of the enzyme revealed that it reached its maximum activity with 5 mM Mn2+ at 37 °C in MES/NaOH (pH 7.0). In addition, the effects of key amino acids in the catalytic and lectin domains on enzyme activity were measured. This work offers an efficient approach for the large-scale production of bioactive HsGnT-IVa, which can be used for in vitro synthesis and functional studies of multiantennary complex-type N-glycans.
{"title":"Enhanced soluble expression and characterization of human N-acetylglucosaminyltransferase IVa in Escherichia coli","authors":"Sen-Lin Peng , Yi Ding , Meng-Hai Xiang , Ken Chen , Xiao-Dong Gao , Ning Wang","doi":"10.1016/j.enzmictec.2024.110524","DOIUrl":"10.1016/j.enzmictec.2024.110524","url":null,"abstract":"<div><div><em>N</em>-Glycosylation is one of the most important posttranslational modifications of proteins. Nearly the entire surface of cells and almost all secreted proteins in humans are modified with complex-type <em>N</em>-glycans, whose functions are affected by the number of <em>N</em>-glycan branches. <em>N</em>-Acetylglucosaminyltransferase-IVa (GnT-IVa) is a Golgi glycosyltransferase that transfers a GlcNAc to the α-1,3 mannose arm of the biantennary <em>N</em>-glycan GlcNAc2Man3GlcNAc2 to form a β-1,4 GlcNAc branched structure. The soluble expression of mammalian glycosyltransferases in heterologous hosts is often challenging. In the present study, human GnT-IVa (<em>Hs</em>GnT-IVa) was cloned as an N-terminal truncated form that was fused with solubility-enhancing tags or signal peptides and overexpressed in <em>Escherichia coli</em> (<em>E. coli</em>). Our results showed that recombinant <em>Hs</em>GnT-IVa could be overexpressed in its highest soluble and active form when the first 87 amino acids were removed and was fused with maltose-binding protein (MBP). By optimizing the induction conditions, the expression level of the recombinant protein was increased to yield approximately 540 mg per liter of culture after affinity purification. The purified enzyme exhibited appropriate glycosyltransferase activity, and the <em>K</em><sub><em>m</em></sub> value of the acceptor substrate was calculated as 1.1 mM. Characterization of the enzyme revealed that it reached its maximum activity with 5 mM Mn<sup>2+</sup> at 37 °C in MES/NaOH (pH 7.0). In addition, the effects of key amino acids in the catalytic and lectin domains on enzyme activity were measured. This work offers an efficient approach for the large-scale production of bioactive <em>Hs</em>GnT-IVa, which can be used for in vitro synthesis and functional studies of multiantennary complex-type <em>N</em>-glycans.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142460871","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}
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