Enzyme and Microbial Technology最新文献

{"title":"Tailoring of levansucrase product size by a comparative molecular dynamics approach.","authors":"Zhiwei Li, Tong Bao, Kaiwen Chen, Chao Hu, Xinyu Zhang, Xueqin Hu, Jingwen Yang, Hongbin Zhang","doi":"10.1016/j.enzmictec.2024.110577","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110577","url":null,"abstract":"<p><p>Levan is widely used as food additives. Its utilization is significantly influenced by its molecular weight. Bacillus subtilis levansucrase (Bs-SacB) and Priestia megaterium levansucrase (Pm-SacB) yield levan of different weights. To delve deeper into the molecular underpinnings of the molecular weight disparity between the products of these two enzymes, we conducted a focused study on the eight loops surrounding the active sites of Bs-SacB and Pm-SacB and identified Loop3 and loop4 as critical determinants in changing the molecular weight of Bs-SacB 's products. Subsequently, leveraging mutation energy analysis and non-homologous substitution strategies, we crafted tailored modifications in loop3 and loop4, yielding a spectrum of mutant enzymes that exhibit diverse molecular weight profiles including F182Y (3698 Da), CYTI (3093 Da), 3-Pbl (2776 Da), 4-Bml (1845 Da), and F182K (1571 Da). This research provide a novel comparative molecular dynamics approach to change product molecular weight and it is successfully applied in the modification of levansucrase.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110577"},"PeriodicalIF":3.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892981","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}

{"title":"Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis.","authors":"Jingya Wu, Huan Zeng, Xinyan Zhong, Xi Chen, Peng Zhang, Zeyuan Deng","doi":"10.1016/j.enzmictec.2024.110575","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110575","url":null,"abstract":"<p><p>The exorbitant production costs associated with natural tannases pose a significant challenge to their widespread industrial utilization. Microbial expression systems provide a cost-effective method for enzyme production. In this study, a putative gene encoding the subtype B tannase (Gt-Tan) was cloned from Galactobacillus timonensis and expressed heterologously in Escherichia coli BL21 (DE3) cells. The Gt-Tan was purified using metal affinity chromatography and exhibited a monomeric structure with a molecular weight of 55 kDa. Gt-Tan showed optimal activity at a temperature of 50 ℃ and a pH of 6.0. It was also quite thermostable, with approximately 68.3 % and 54.7 % of its maximal activity retained after incubation at 45 ℃ for 2 h and 40 ℃ for 48 h respectively. Addition of Mn²⁺, Zn²⁺, Al³⁺, urea, n-butanol, and dimethylsulfoxide at a low concentration slightly enhanced the activity of Gt-Tan, whereas Cu²⁺, Fe³⁺, Fe²⁺, Co²⁺, SDS, cetyltrimethylammonium bromide, DTT, Tween 80, and β-mercaptoethanol significantly inhibited its activity. K_m and k_cat/K_m values were estimated to be 0.83 mM and 19.7 s¹ mM¹ for methyl gallate, 0.67 mM and 65.4 s¹ mM¹ for propyl gallate, and 0.22 mM and 240.8 s¹ mM¹ for tannic acid. These results enhanced our understanding of tannase and provided potential sources for applications in the chemical, feed, and food industries.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110575"},"PeriodicalIF":3.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142902714","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}

{"title":"Structural and functional insights into recombinant β-glucosidase from Thermothelomyces thermophilus: Cello-oligosaccharide hydrolysis and thermostability.","authors":"Ana Luiza da Rocha Fortes Saraiva, Gabriela Leila Berto, Bianca Oliva, Paula Macedo Cunha, Lucas Ramos, Leandro Cristante de Oliveira, Fernando Segato","doi":"10.1016/j.enzmictec.2024.110572","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110572","url":null,"abstract":"<p><p>β-glucosidases (BGLs) are key enzymes in the depolymerization of cellulosic biomass, catalyzing the conversion of cello-oligosaccharides into glucose. This conversion is pivotal for enhancing the production of second-generation ethanol or other value-added products in biorefineries. However, the process is often cost-prohibitive due to the high enzyme loadings required. Therefore, the discovery of new highly efficient BGLs represents a significant advancement. In this study, a BGL from the glycoside hydrolase family 3 (GH3) of the thermophilic fungus Thermothelomyces thermophilus (TthBgl3A) was heterologously expressed in Aspergillus nidulans. The recombinant enzyme exhibited optimal activity at pH 5.0 and 55 °C, with noteworthy stability for up to 160 h. A distinctive, extensive loop within the catalytic cavity of TthBgl3A facilitates hydrophobic interactions that enhance the binding and hydrolysis of long cello-oligosaccharides. Consequently, TthBgl3A has proven to be an efficient enzyme for the hydrolysis lignocellulosic biomass. These findings are significant for expanding the repertoire of enzymes produced by T. thermophilus and provide new insights into the potential application of TthBgl3A in the degradation of cellulosic materials and the production of valuable compounds.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110572"},"PeriodicalIF":3.4,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142881501","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}

{"title":"Potential of a newly isolated lytic bacteriophage to control Pseudomonas coronafaciens pv. garcae in coffee plants: Molecular characterization with in vitro and ex vivo experiments.","authors":"Luan C Mota, Erica C Silva, Carlos A Quinde, Basilio Cieza, Aakash Basu, Lucas M R Rodrigues, Marta M D C Vila, Victor M Balcão","doi":"10.1016/j.enzmictec.2024.110573","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110573","url":null,"abstract":"<p><p>Traditionally, control of coffee plant bacterial halo blight (BHB) caused by the phytopathogen Pseudomonas coronafaciens pv. garcae (Pcg) involves frequent spraying of coffee plantations with non-environmentally friendly and potentially bacterial resistance-promoting copper products or with kasugamycin hydrochloride. In this study we report a leap forward in the quest for a new ecofriendly approach, characterizing (both physicochemically and biologically) and testing both in vitro and ex vivo a new lytic phage for Pcg. An in-depth molecular (genomic and DNA structural features) characterization of the phage was also undertaken. Phage PcgS01F belongs to the class Caudoviricetes, Drexlerviridae family and genus Guelphvirus, and presents a siphovirus-like morphotype. Phage PcgS01F showed a latency period of 40 min and a burst size of 46 PFU/host cell, allowing to conclude that it replicates well in Pcg IBSBF-158. At Multiplicity Of Infection (MOI, or the ratio of phage to bacteria) 1000, the performance of phage PcgS01F was much better than at MOI 10, promoting increasing bacterial reductions until the end of the in vitro inactivation assays, stabilizing at a significant 82 % bacterial load reduction. Phage PcgS01F infected and killed Pcg cells ex vivo in coffee plant leaves artificially contaminated, with a maximum of Pcg inactivation of 7.66 log CFU/mL at MOI 1000 after 36 h of incubation. This study provides evidence that the isolated phage is a promising candidate against the causative agent of BHB in coffee plants.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110573"},"PeriodicalIF":3.4,"publicationDate":"2024-12-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142863471","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}

{"title":"Improving the anti-autolytic ability of alkaline protease from Bacillus alcalophilus by a rationally combined strategy.","authors":"Mian Wu, Lin Cao, Wei Tang, Zhemin Liu, Su Feng","doi":"10.1016/j.enzmictec.2024.110561","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110561","url":null,"abstract":"<p><p>Detergent enzymes have been extensively developed as eco-friendly alternatives to harmful chemicals, with alkaline protease representing a significant portion of detergent enzyme sales. However, the self-cleavage function of alkaline protease impacts its activity and overall application. Therefore, a new rational combinatorial strategy is proposed based on self-molecular docking (Self-ZDOCK) and molecular dynamics (MD) simulations. Self-ZDOCK is a computational method for predicting the binding mode of proteins to themselves, which is crucial for understanding the self-cleavage mechanism of proteases. On the other hand, MD simulation is a powerful tool to gain insight into the dynamic behaviour of proteins over time, and thus to analyse the structural stability and flexibility of BpAP under various conditions. Experiments verified this strategy is an effective way to improve the anti-autolytic ability of BpAP. Among the 28 mutants of BpAP, 5 mutants showed increases in thermal stability, pH stability, and storage stability in detergent, indicating a significant enhancement in their anti-autolytic capacity. Structural analysis and MD simulations confirmed that the enhanced stability characteristic of BpAP is attributed to improved anti-autolytic ability rather than increased structural stability. The three points combined mutant (MT5) showed the best increases in autolytic ability, as well as advanced catalytic efficiency. The low rate of inactive mutants and the high rate of positive mutants indicated that newly introduced screening factors (distance from catalytic residues, Gibbs free energy term, molecular simulation, and visual inspections) greatly enhance the design of anti-autolytic alkaline protease. Additionally, these findings enhance the industrial use of alkaline protease in detergents and similar applications.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110561"},"PeriodicalIF":3.4,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142817351","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}

{"title":"Enhanced degradation activity of PET plastics by fusion protein of anchor peptide LCI and Thermobifida fusca cutinase.","authors":"Yidi Liu, Zhanzhi Liu, Xuehong Guo, Ke Tong, Yueheng Niu, Zhiyu Shen, Hanzhi Weng, Fengshan Zhang, Jing Wu","doi":"10.1016/j.enzmictec.2024.110562","DOIUrl":"https://doi.org/10.1016/j.enzmictec.2024.110562","url":null,"abstract":"<p><p>The substantial accumulation of polyethylene terephthalate (PET) plastic waste in the environment has exacerbated the issue of plastic pollution. The biodegradation of PET plastics using biological enzymes has garnered considerable attention due to its efficiency and environmentally friendly nature. Nevertheless, the low binding affinity of PET plastics presents a significant limitation to the application of biocatalysts in their degradation. This study endeavors to engineer a fusion protein comprising the anchor peptide LCI, derived from Bacillus subtilis A014, and a thermally stabilized variant of Thermobifida fusca cutinase, D204C/E253C (Tfuc2), with the objective of augmenting its polyethylene terephthalate (PET) degradation efficacy. The findings demonstrate that LCI exhibits a high binding affinity for PET, and the hydrolytic efficiency of the LCI-containing fusion protein is enhanced by a factor of 1.8-34.5 compared to the free Tfuc2 enzyme. The enzymatic characteristics and molecular dynamics simulation outcomes indicate that the improved hydrolytic efficiency of PET may originate from the flexible oscillatory behavior of LCI, which exhibits a high binding affinity for PET. This study presents a novel methodology for the enzymatic degradation of PET plastic waste.</p>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"110562"},"PeriodicalIF":3.4,"publicationDate":"2024-12-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142799958","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}

{"title":"Semi-rational engineering of glucosamine-6-phosphate deaminase for catalytic synthesis of glucosamine from D-fructose","authors":"Zi-Hao Zhang,&nbsp;Yun-Xing Liao,&nbsp;Xue-Ting Deng,&nbsp;Zheng-Bing Guan","doi":"10.1016/j.enzmictec.2024.110552","DOIUrl":"10.1016/j.enzmictec.2024.110552","url":null,"abstract":"<div><div>Glucosamine (GlcN), as one of the important derivatives of D-glucose, is formed by the substitution of the hydroxyl group at position 2 of glucose with an amino group. As a bioactive amino monosaccharide, GlcN is known for its various biological effects, including immune enhancement, antioxidant, anti-inflammatory, hepatoprotective, joint pain relief, and alleviation of osteoporosis. These properties highlight the broad applications of GlcN and its derivatives in pharmaceuticals, cosmetics, food production, and other fields, underscoring their promising prospects. Thus, the efficient industrial production of GlcN is gaining increasing attention as well. Here, we report a novel biosynthetic method for GlcN, utilizing engineered <em>Escherichia coli</em> expressing glucosamine-6-phosphate deaminase (GlmD) to directly convert D-fructose into GlcN. The best mutant screened using the Morgan-Elson colorimetric method is the triple mutant G42S/G43C/G136T (designated as GlmD-ZH11), which exhibits approximately 21 times higher catalytic activity towards D-fructose compared to the wild type. Using the purified enzyme of GlmD-ZH11 in shaken flask fermentation for six hours, we achieved a conversion rate of 72.11 % from D-fructose to GlcN. To further elucidate the mechanism behind the enhanced activity of the GlmD-ZH11 mutant, we conducted hydrogen bond network analysis to investigate the hydrogen bond interactions between the mutant and fructose. Additionally, we performed molecular dynamics simulations to study the RMSD and RMSF curves of the mutant. The results indicate that the protein structure of the mutant ZH11 is more stable and binds more tightly to the substrate. Calculations of the solvent-accessible surface area and binding free energy suggested that Thr41, Ser42, Asp72, Gly137, and Ala145 may be key amino acid residues in the catalytic process of ZH11. Finally, based on these findings and the catalytic mechanism of the wild type, we hypothesized a potential catalytic reaction mechanism for the ZH11 mutant.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110552"},"PeriodicalIF":3.4,"publicationDate":"2024-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142746425","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}

{"title":"Exploring the complexity of xylitol production in the fungal cell factory Aspergillus niger","authors":"Astrid Müller ,&nbsp;Jiali Meng ,&nbsp;Robin Kuijpers ,&nbsp;Miia R. Mäkelä ,&nbsp;Ronald P. de Vries","doi":"10.1016/j.enzmictec.2024.110550","DOIUrl":"10.1016/j.enzmictec.2024.110550","url":null,"abstract":"<div><div>Production of xylitol from agricultural by-products offers a promising approach for the circular bioeconomy. This study investigates the roles of transcription factors XlnR and CreA in xylitol production from wheat bran in <em>Aspergillus niger</em> by generating strains with a constitutively active XlnR (XlnR_c, V756F mutation) and/or deletion of <em>creA</em>, in a previously generated xylitol-producing strain. The XlnR_c mutation increased the initial rate of xylitol production but lowered the overall accumulation. Deletion of <em>creA</em> in this strain significantly improved both the onset and rate of xylitol production, indicating an inhibitory role of CreA in the PCP. These results demonstrate the complexity of metabolic engineering to generate fungal cell factories for valuable biochemicals, such as xylitol, as not only metabolic but also multiple gene regulation aspects need to be considered.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110550"},"PeriodicalIF":3.4,"publicationDate":"2024-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699659","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}

{"title":"Screening of lipase TiL from Tilletia indica for chemo-enzymatic epoxidation of alkenes","authors":"Jiang Pan ,&nbsp;Nan Yang ,&nbsp;Yuan-Lin Lv ,&nbsp;Zi-Yang Zhang ,&nbsp;Chun-Xiu Li,&nbsp;Jian-He Xu","doi":"10.1016/j.enzmictec.2024.110547","DOIUrl":"10.1016/j.enzmictec.2024.110547","url":null,"abstract":"<div><div>Lipase can mediate the chemo-enzymatic epoxidation of alkenes with the presence of free carboxylic acid and hydrogen peroxide. Four novel lipases with the abilities of chemo-enzymatic epoxidation were mined from the gene database. Lipase <em>Ti</em>L originated from <em>Tilletia indica</em> was identified with significant activity on formation of methyl epoxystearate from methyl oleate. <em>n</em>-Heptanoic acid was determined as the optimal carboxylic acid substrate of <em>Ti</em>L. Methyl oleate and α-pinene were efficiently converted to corresponding epoxy compound in micro-aqueous media and aqueous-organic biphase, respectively. A preparative scale chemo-enzymatic transformation of α-pinene was conduct using the optimized reaction condition, with 30 % yield of α-pinene oxide obtained.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110547"},"PeriodicalIF":3.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699660","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}

{"title":"Improvement of lipid production from glucose/xylose mixed-sugar by the oleaginous yeast Lipomyces starkeyi through ultra-violet mutagenesis","authors":"Sota Kamba,&nbsp;Ryosuke Yamada ,&nbsp;Takuya Matsumoto,&nbsp;Hiroyasu Ogino","doi":"10.1016/j.enzmictec.2024.110551","DOIUrl":"10.1016/j.enzmictec.2024.110551","url":null,"abstract":"<div><div>The oleaginous yeast <em>Lipomyces starkeyi</em> is a promising triacylglycerol (TAG) producer for biodiesel fuel. However, it is necessary to further improve TAG productivity in <em>L. starkeyi</em> from a mixed sugar of glucose and xylose. This study aimed to construct an <em>L. starkeyi</em> mutant with increased TAG productivity from glucose/xylose mixed-sugar and to elucidate the causes underlying increased lipid productivity. Ultra-violet (UV) mutagenesis combined with enrichment culture with ethanol and H₂O₂ and selection of low-density cells was applied to <em>L. starkeyi</em> to obtain the <em>L. starkeyi</em> mutant strain UMP47, which exhibited higher TAG production from glucose/xylose. Transcriptome analysis revealed high expression of genes involved in transporter activity and carbohydrate metabolism, whereas genes involved in DNA replication exhibited lower expression in the mutant strain UMP47 than in the wild-type strain. Altogether, the lipid productivity of <em>L. starkeyi</em> was successfully improved by UV mutagenesis. Transcriptome analysis suggested the importance of previously unidentified genes in TAG production. This study provides information on potential target genes for improving TAG production through the genetic modification of oleaginous yeast.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"183 ","pages":"Article 110551"},"PeriodicalIF":3.4,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142699661","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}