Enzyme and Microbial Technology最新文献

A novel GH12 xyloglucanase from the white rot fungus Abortiporus biennis, synergistically enhances lignocellulose saccharification by commercial cellulases

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-03-12 DOI: 10.1016/j.enzmictec.2025.110628

Despoina Panoraia Bakouli , Elisavet Pedi , Nikolaos Labrou , Evangelos Topakas , Anastasia Zerva

Xyloglucan is a complex, highly substituted plant biomass polysaccharide, which is largely overlooked in the design of enzyme cocktails for lignocellulose saccharification, due to its presence in specific plant tissues only, and its low content. Thus, the microbial mechanisms for its degradation have not been thoroughly studied. However, in the frame of the biorefinery concept, xyloglucan monomers also have to be utilized for the design of efficient bioprocesses. Moreover, in plant tissues, xyloglucan often covers cellulose fibrils, impeding the access of cellulases. In order to shed light on the enzymatic degradation of xyloglucan, a novel GH12 family xyloglucanase was studied, from the basidiomycete Abortiporus biennis. The enzyme was heterologously produced in Pichia pastoris, purified and characterized. AbiXeg12a is a 28 kDa glycoprotein, with relatively strict substrate specificity, since it is only active in xyloglucan and β-glucan. The main hydrolysis products are the oligomers XXXG, XLXG/XXLG, XLLG and the optimum activity conditions are pH 4.5 and 55 °C. The enzyme contributes to the saccharification of corn bran and apple pulp by a commercial cellulase preparation, increasing the release of reducing sugars by up to 39 % and 18 %, respectively, while the addition of AbiXeg12a can minimize the enzyme load of the reaction, at least for apple pulp, without loss in reducing sugar yield. Overall, the importance of xyloglucanases on the saccharification of xyloglucan-containing substrates was demonstrated in this study. The results could contribute to the design of more efficient, tailor-made enzyme cocktails for the saccharification and subsequent valorization of lignocellulose.

{"title":"A novel GH12 xyloglucanase from the white rot fungus Abortiporus biennis, synergistically enhances lignocellulose saccharification by commercial cellulases","authors":"Despoina Panoraia Bakouli , Elisavet Pedi , Nikolaos Labrou , Evangelos Topakas , Anastasia Zerva","doi":"10.1016/j.enzmictec.2025.110628","DOIUrl":"10.1016/j.enzmictec.2025.110628","url":null,"abstract":"<div><div>Xyloglucan is a complex, highly substituted plant biomass polysaccharide, which is largely overlooked in the design of enzyme cocktails for lignocellulose saccharification, due to its presence in specific plant tissues only, and its low content. Thus, the microbial mechanisms for its degradation have not been thoroughly studied. However, in the frame of the biorefinery concept, xyloglucan monomers also have to be utilized for the design of efficient bioprocesses. Moreover, in plant tissues, xyloglucan often covers cellulose fibrils, impeding the access of cellulases. In order to shed light on the enzymatic degradation of xyloglucan, a novel GH12 family xyloglucanase was studied, from the basidiomycete <em>Abortiporus biennis</em>. The enzyme was heterologously produced in <em>Pichia pastoris</em>, purified and characterized. <em>Abi</em>Xeg12a is a 28 kDa glycoprotein, with relatively strict substrate specificity, since it is only active in xyloglucan and β-glucan. The main hydrolysis products are the oligomers XXXG, XLXG/XXLG, XLLG and the optimum activity conditions are pH 4.5 and 55 °C. The enzyme contributes to the saccharification of corn bran and apple pulp by a commercial cellulase preparation, increasing the release of reducing sugars by up to 39 % and 18 %, respectively, while the addition of <em>Abi</em>Xeg12a can minimize the enzyme load of the reaction, at least for apple pulp, without loss in reducing sugar yield. Overall, the importance of xyloglucanases on the saccharification of xyloglucan-containing substrates was demonstrated in this study. The results could contribute to the design of more efficient, tailor-made enzyme cocktails for the saccharification and subsequent valorization of lignocellulose.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110628"},"PeriodicalIF":3.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143642329","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}

引用次数: 0

A metabolic-engineering framework approach via fed-batch fermentation for enhancing glucaric acid production in Komagataella phaffii

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-03-12 DOI: 10.1016/j.enzmictec.2025.110627

Jayachandran Krishna , Kabilan Subash Chandra Bose , Sindhu Varadharaj , Meenakshisundaram Sankaranarayanan

Glucaric acid (D-saccharic acid) is an organic compound belonging to glucuronic acid derivatives, whose commercial synthesis involves the use of hazardous solvents. Biosynthetic production in Saccharomyces cerevisiae has limitations, such as ethanolic fermentation, redox strategy limitations, and low pH toxicity. Komagataella phaffii (K. phaffii) formly known Pichia pastoris, an alternative and robust engineerable organism, is a promising biotransformation agent for glucaric acid production. However, K. phaffii lacks native biosynthetic pathways for glucaric acid synthesis at the industrial scale. There is no proof-of-concept glucaric acid production system. Therefore, gene expression profiling-based metabolic engineering of glucaric acid producing gene cassette was performed using in-fusion cloning. Product production was enhanced using fed-batch fermentation of the key metabolite, myo-inositol; this improved the yield of glucaric acid. The expression was optimized through cofactor recycling and codon optimization for the UDH gene. Fed-batch fermentation with mixed supplementation (Myo-inositol + Monosodium glutamate) as substrate in engineered K. phaffii (X33-GA) enhanced glucaric acid synthesis to 17.6 g/L. In addition, we present simple HPLC and LC-MS techniques for quantifying glucaric acid and its precursors in the fermentation samples. The proof-of-concept results from both shake flask and bioreactor studies provide a unique perspective on sustainable, cost-effective, and green technological alternatives for glucaric acid synthesis.

{"title":"A metabolic-engineering framework approach via fed-batch fermentation for enhancing glucaric acid production in Komagataella phaffii","authors":"Jayachandran Krishna , Kabilan Subash Chandra Bose , Sindhu Varadharaj , Meenakshisundaram Sankaranarayanan","doi":"10.1016/j.enzmictec.2025.110627","DOIUrl":"10.1016/j.enzmictec.2025.110627","url":null,"abstract":"<div><div>Glucaric acid (D-saccharic acid) is an organic compound belonging to glucuronic acid derivatives, whose commercial synthesis involves the use of hazardous solvents. Biosynthetic production in <em>Saccharomyces cerevisiae</em> has limitations, such as ethanolic fermentation, redox strategy limitations, and low pH toxicity. <em>Komagataella phaffii</em> (<em>K. phaffii</em>) formly known <em>Pichia pastoris</em>, an alternative and robust engineerable organism, is a promising biotransformation agent for glucaric acid production. However, <em>K. phaffii</em> lacks native biosynthetic pathways for glucaric acid synthesis at the industrial scale. There is no proof-of-concept glucaric acid production system. Therefore, gene expression profiling-based metabolic engineering of glucaric acid producing gene cassette was performed using in-fusion cloning. Product production was enhanced using fed-batch fermentation of the key metabolite, myo-inositol; this improved the yield of glucaric acid. The expression was optimized through cofactor recycling and codon optimization for the UDH gene. Fed-batch fermentation with mixed supplementation (Myo-inositol + Monosodium glutamate) as substrate in engineered <em>K. phaffii</em> (X33-GA) enhanced glucaric acid synthesis to 17.6 g/L. In addition, we present simple HPLC and LC-MS techniques for quantifying glucaric acid and its precursors in the fermentation samples. The proof-of-concept results from both shake flask and bioreactor studies provide a unique perspective on sustainable, cost-effective, and green technological alternatives for glucaric acid synthesis.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110627"},"PeriodicalIF":3.4,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143642330","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

Synergy of GH67 and GH115 α-1,2-glucuronidases with Penicillium subrubescens endoxylanases to stimulate xylooligosaccharide production

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-03-11 DOI: 10.1016/j.enzmictec.2025.110629

Xinxin Li , Lanyu Li , Alessia Manassero , Astrid Müller , Sumitha K. Reddy , Mirjam A. Kabel , Ronald P. de Vries , Peicheng Sun

A primary substitution of the plant cell wall hemicellulosic polysaccharide xylan is (4-O-methyl-)d-glucuronic acid, which hinders the endoxylanases (XLNs) degradation of xylan for the production of valuable xylooligosaccharides (XOS). In this context, α-1,2-glucuronidase (AGU) plays a critical role in hydrolyzing the α-(1→2)-glycosidic linkages between 4-O-methyl-d-glucuronic acid and xylosyl residues in xylan, thereby enhancing XOS production by XLNs. However, AGUs have been relatively poorly studied, and insufficient and incomplete data on their biochemical properties, substrate specificity, and product profiling has limited their application. Here, we cloned, heterologously produced, purified and functionally characterized an AGU from Aspergillus niger (AnAguA) and another AGU from Penicillium subrubescens (PsAguB), belonging to Glycoside Hydrolase family 67 (GH67) and 115 (GH115), respectively, in the Carbohydrate-Active enZyme database. Results showed that neither AGU released 4-O-methyl-d-glucuronic acid from polymeric beech wood glucuronoxylan (BeWX). However, we found that from BeWX pre-digested with GH10 or GH11 XLNs from P. subrubescens (PsXlnA and PsXlnF, respectively), AnAguA released 4-O-methyl-d-glucuronic acid only from the non-reducing end of glucuronoxylan oligosaccharide, whereas PsAguB released 4-O-methyl-d-glucuronic acid from glucuronoxylan oligosaccharides regardless of the xylosyl substitution position. Furthermore, we demonstrated that enhancement of XOS release by adding AGUs to various combinations of GH10 (PsXlnA–C) and GH11 (PsXlnD–F, PsXlnH–I) XLNs from P. subrubescens varied based on the AGU-XLN combination. The combination of AnAguA with PsXlnA was the most effective, achieving at least a 3-fold increase in the release of XOS with a degree of polymerization of 5–7 compared to using PsXlnA alone.

{"title":"Synergy of GH67 and GH115 α-1,2-glucuronidases with Penicillium subrubescens endoxylanases to stimulate xylooligosaccharide production","authors":"Xinxin Li , Lanyu Li , Alessia Manassero , Astrid Müller , Sumitha K. Reddy , Mirjam A. Kabel , Ronald P. de Vries , Peicheng Sun","doi":"10.1016/j.enzmictec.2025.110629","DOIUrl":"10.1016/j.enzmictec.2025.110629","url":null,"abstract":"<div><div>A primary substitution of the plant cell wall hemicellulosic polysaccharide xylan is (4-<em>O</em>-methyl-)<span>d</span>-glucuronic acid, which hinders the endoxylanases (XLNs) degradation of xylan for the production of valuable xylooligosaccharides (XOS). In this context, α-1,2-glucuronidase (AGU) plays a critical role in hydrolyzing the α-(1→2)-glycosidic linkages between 4-<em>O</em>-methyl-<span>d</span>-glucuronic acid and xylosyl residues in xylan, thereby enhancing XOS production by XLNs. However, AGUs have been relatively poorly studied, and insufficient and incomplete data on their biochemical properties, substrate specificity, and product profiling has limited their application. Here, we cloned, heterologously produced, purified and functionally characterized an AGU from <em>Aspergillus niger</em> (<em>An</em>AguA) and another AGU from <em>Penicillium subrubescens</em> (<em>Ps</em>AguB), belonging to Glycoside Hydrolase family 67 (GH67) and 115 (GH115), respectively, in the Carbohydrate-Active enZyme database. Results showed that neither AGU released 4-<em>O</em>-methyl-<span>d</span>-glucuronic acid from polymeric beech wood glucuronoxylan (BeWX). However, we found that from BeWX pre-digested with GH10 or GH11 XLNs from <em>P. subrubescens</em> (<em>Ps</em>XlnA and <em>Ps</em>XlnF, respectively), <em>An</em>AguA released 4-<em>O</em>-methyl-<span>d</span>-glucuronic acid only from the non-reducing end of glucuronoxylan oligosaccharide, whereas <em>Ps</em>AguB released 4-<em>O</em>-methyl-<span>d</span>-glucuronic acid from glucuronoxylan oligosaccharides regardless of the xylosyl substitution position. Furthermore, we demonstrated that enhancement of XOS release by adding AGUs to various combinations of GH10 (<em>Ps</em>XlnA–C) and GH11 (<em>Ps</em>XlnD–F, <em>Ps</em>XlnH–I) XLNs from <em>P. subrubescens</em> varied based on the AGU-XLN combination. The combination of <em>An</em>AguA with <em>Ps</em>XlnA was the most effective, achieving at least a 3-fold increase in the release of XOS with a degree of polymerization of 5–7 compared to using <em>Ps</em>XlnA alone.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"187 ","pages":"Article 110629"},"PeriodicalIF":3.4,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143637135","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}

引用次数: 0

Acetic acid production from corn straw via enzymatic degradation using putative acetyl esterase from the metagenome assembled genome

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-03-04 DOI: 10.1016/j.enzmictec.2025.110619

Hao Chen , Zhiwei Zeng , Rup Lal , Jie Wu , Jia Chen , Mei Li , Lulu Cao , Xiqiang Liu , Ruzhe Zhang , Chunjie Gong

Acetic acid production from corn straw by enzyme catalysis shows its application value in food industry. In this study, a gene encoding for a putative acetyl esterase derived from Sphingobacterium soilsilvae Em02 was discovered in metagenome assembled genome. The gene was expressed in Escherichia coli BL21 to obtain enzyme with a molecular mass of 38.8 kDa. P-Nitrophenyl acetate was used as a substrate to determine the enzyme activity. The enzyme demonstrated optimal activity under conditions of 40 °C and a neutral pH of 7.0. Under optimal conditions, 17.58 mg of acetic acid was obtained using the enzyme from 50 mg corn straw pretreated with amylase. The acetyl esterase derived from Sphingobacterium soilsilvae Em02, demonstrates significant potential for biotechnological applications, particularly in biomass degradation.

利用酶催化从玉米秸秆中生产乙酸显示了其在食品工业中的应用价值。本研究在元基因组中发现了一个编码推测乙酰酯酶的基因，该基因来源于Sphingobacterium soilsilvae Em02。将该基因在大肠杆菌 BL21 中表达，获得了分子质量为 38.8 kDa 的酶。以对硝基苯乙酸酯为底物测定酶的活性。该酶在 40 °C、中性 pH 值为 7.0 的条件下表现出最佳活性。在最佳条件下，从 50 毫克用淀粉酶预处理的玉米秸秆中使用该酶可获得 17.58 毫克乙酸。从 Sphingobacterium soilsilvae Em02 中提取的乙酰酯酶具有很大的生物技术应用潜力，特别是在生物质降解方面。

引用次数: 0

Recombinant alginate lyases and mannitol dehydrogenase enhance hydrolysis of macroalgal carbohydrates

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-26 DOI: 10.1016/j.enzmictec.2025.110618

Dominique F. Rocher, Marthinus W. Myburgh, Marinda Viljoen-Bloom, Rosemary A. Cripwell

Brown macroalgae are a promising source for bioethanol production, primarily due to their high carbohydrate, low lignin and high moisture content. Bioconversion of macroalgae to ethanol requires a yeast, such as Saccharomyces cerevisiae, that can hydrolyse the macroalgal carbohydrates, namely laminarin, mannitol and alginate. In this study, the mannitol dehydrogenase (MDH) genes from Aspergillus fumigatus (AfMDH) and Talaromyces islandicus (TiMDH), and the alginate lyase (AL) genes from Sphingomonas sp. (SpxAL and SpeAL) and Talaromyces emersonii (TeeAL) were expressed in the laboratory strain, S. cerevisiae Y294. Co-cultures of a laminarinase-producing yeast, Y294[Relam1/Tvlam1] and yeasts expressing mannitol dehydrogenases and alginate lyases were evaluated for the consolidated bioprocessing of the major carbohydrates in brown macroalgae. Laminarin and mannitol were targeted for ethanol production, while alginate was depolymerised to expose mannitol. A co-culture of S. cerevisiae Y294[Relam1/Tvlam1], [AfMDH] and [TeeAL/SpxAL] strains produced 10.30 g/L ethanol from Ecklonia maxima, representing a 98 % carbon conversion (based on the laminarin and mannitol content). A strain expressing both endo- and exo-alginate lyase improved the ethanol yield by 42.28 % compared to strains expressing only laminarinase- and mannitol dehydrogenase. Scanning electron microscopy further revealed that co-cultures containing laminarinase, MDH, and AL enzymes promoted significant physical degradation and increased porosity in macroalgal substrates, suggesting enhanced alginate hydrolysis and improved enzyme accessibility. This is the first report on the simultaneous hydrolysis of mannitol, alginate and laminarin with recombinant enzymes during macroalgal fermentation. The results demonstrate significant progress towards exploiting brown macroalgae for bioconversion to ethanol and high-value products.

{"title":"Recombinant alginate lyases and mannitol dehydrogenase enhance hydrolysis of macroalgal carbohydrates","authors":"Dominique F. Rocher, Marthinus W. Myburgh, Marinda Viljoen-Bloom, Rosemary A. Cripwell","doi":"10.1016/j.enzmictec.2025.110618","DOIUrl":"10.1016/j.enzmictec.2025.110618","url":null,"abstract":"<div><div>Brown macroalgae are a promising source for bioethanol production, primarily due to their high carbohydrate, low lignin and high moisture content. Bioconversion of macroalgae to ethanol requires a yeast, such as <em>Saccharomyces cerevisiae</em>, that can hydrolyse the macroalgal carbohydrates, namely laminarin, mannitol and alginate. In this study, the mannitol dehydrogenase (MDH) genes from <em>Aspergillus fumigatus</em> (<em>AfMDH</em>) and <em>Talaromyces islandicus</em> (<em>TiMDH</em>), and the alginate lyase (AL) genes from <em>Sphingomonas</em> sp. (<em>SpxAL</em> and <em>SpeAL</em>) and <em>Talaromyces emersonii</em> (<em>TeeAL</em>) were expressed in the laboratory strain, <em>S. cerevisiae</em> Y294. Co-cultures of a laminarinase-producing yeast, Y294[Relam1/Tvlam1] and yeasts expressing mannitol dehydrogenases and alginate lyases were evaluated for the consolidated bioprocessing of the major carbohydrates in brown macroalgae. Laminarin and mannitol were targeted for ethanol production, while alginate was depolymerised to expose mannitol. A co-culture of <em>S. cerevisiae</em> Y294[Relam1/Tvlam1], [AfMDH] and [TeeAL/SpxAL] strains produced 10.30 g/L ethanol from <em>Ecklonia maxima</em>, representing a 98 % carbon conversion (based on the laminarin and mannitol content). A strain expressing both endo- and exo-alginate lyase improved the ethanol yield by 42.28 % compared to strains expressing only laminarinase- and mannitol dehydrogenase. Scanning electron microscopy further revealed that co-cultures containing laminarinase, MDH, and AL enzymes promoted significant physical degradation and increased porosity in macroalgal substrates, suggesting enhanced alginate hydrolysis and improved enzyme accessibility. This is the first report on the simultaneous hydrolysis of mannitol, alginate and laminarin with recombinant enzymes during macroalgal fermentation. The results demonstrate significant progress towards exploiting brown macroalgae for bioconversion to ethanol and high-value products.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"186 ","pages":"Article 110618"},"PeriodicalIF":3.4,"publicationDate":"2025-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143551515","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}

引用次数: 0

Synergy between processive cellulases in Ruminoccocus albus

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-18 DOI: 10.1016/j.enzmictec.2025.110610

Alem Storani , Alberto A. Iglesias, Sergio A. Guerrero

Endoglucanases (EGs), cellobiohydrolases (CBHs), and β-glucosidases are essential components in enzymatic degradation of cellulose. We analyzed the glycosyl hydrolases from families GH5 and GH48 from Ruminococcus albus 8 (RalCel5G and RalCel48A). Both enzymes feature a catalytic motif and a carbohydrate binding domain from family 37 (CBM37). RalCel5G also exhibited a second CBM37 with lower similarity. As a result, RalCel5G showed higher binding affinity toward insoluble substrates and broader recognition capacity. Kinetic characterization using different cellulosic substrates and reaction product analysis confirmed RalCel5G as a processive EG while RalCel48A is a CBH. Interestingly, we found a synergistic effect on their activity at a low EG to CBH ratio, despite the processive activity of RalCel5G. Furthermore, the lignocellulose degradation capacity was improved by supplementing the cellulases with hemicellulase RalXyn10A. These results provide valuable information about the interaction between processive EG and conventional CBH, necessary for the rational design of enzyme cocktails for optimized biomass processing.

{"title":"Synergy between processive cellulases in Ruminoccocus albus","authors":"Alem Storani , Alberto A. Iglesias, Sergio A. Guerrero","doi":"10.1016/j.enzmictec.2025.110610","DOIUrl":"10.1016/j.enzmictec.2025.110610","url":null,"abstract":"<div><div>Endoglucanases (EGs), cellobiohydrolases (CBHs), and β-glucosidases are essential components in enzymatic degradation of cellulose. We analyzed the glycosyl hydrolases from families GH5 and GH48 from <em>Ruminococcus albus</em> 8 (<em>Ral</em>Cel5G and <em>Ral</em>Cel48A). Both enzymes feature a catalytic motif and a carbohydrate binding domain from family 37 (CBM37). <em>Ral</em>Cel5G also exhibited a second CBM37 with lower similarity. As a result, <em>Ral</em>Cel5G showed higher binding affinity toward insoluble substrates and broader recognition capacity. Kinetic characterization using different cellulosic substrates and reaction product analysis confirmed <em>Ral</em>Cel5G as a processive EG while <em>Ral</em>Cel48A is a CBH. Interestingly, we found a synergistic effect on their activity at a low EG to CBH ratio, despite the processive activity of <em>Ral</em>Cel5G. Furthermore, the lignocellulose degradation capacity was improved by supplementing the cellulases with hemicellulase <em>Ral</em>Xyn10A. These results provide valuable information about the interaction between processive EG and conventional CBH, necessary for the rational design of enzyme cocktails for optimized biomass processing.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"186 ","pages":"Article 110610"},"PeriodicalIF":3.4,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143455005","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

Characterization of keratinase from Chryseobacterium camelliae Dolsongi-HT1 and efficacy on skin exfoliation

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-17 DOI: 10.1016/j.enzmictec.2025.110605

Eun-Mi Kim, Soojung Oh, Hyeongwon Choi, Won-Seok Park

Keratin is the outermost layer that protects our skin and has an appropriate turnover cycle. With age, the keratin turnover cycle begins to dysfunction. To overcome this issue, we artificially remove dead skin cells. In this study, we attempted to screen enzymes that could be useful in the cosmetics industry to develop enzymes suitable for the enzyme-based method, a mild exfoliation method that does not damage the skin. Chryseobacterium camelliae Dolsongi-HT1 with keratinolytic activity was isolated from green tea leaves (sourced from the Dolsongi tea garden, Jeju Island). The keratinolytic activity of C. camelliae Dolsongi-HT1 was detected in the culture media, indicating that the target keratinolytic enzyme is a secreted protein. Keratinolytic activity was demonstrated using forearm skin keratin and reconstituted human skin models. The enzyme from C. camelliae Dolsng-HT1 (HT1) could efficiently decompose human skin keratin. Moreover, experiments using the reconstituted human skin model demonstrated that HT1 is efficient in exfoliating the outermost stratum corneum. Compared with the popularly used chemical exfoliation method, enzymatic exfoliation using HT1 was less abrasive and did not damage the epidermal layer. Keratinolytic enzyme was identified using protein purification and mass spectrometry. The identified enzyme (iHT1) was expressed in the Bacillus subtilis RIK 1285 secretory protein expression system. The iHT1 enzyme showed high activity over a wide temperature range (30–60 °C), with the highest activity at 30 °C. The optimum pH for the activity of iHT was pH8.

{"title":"Characterization of keratinase from Chryseobacterium camelliae Dolsongi-HT1 and efficacy on skin exfoliation","authors":"Eun-Mi Kim, Soojung Oh, Hyeongwon Choi, Won-Seok Park","doi":"10.1016/j.enzmictec.2025.110605","DOIUrl":"10.1016/j.enzmictec.2025.110605","url":null,"abstract":"<div><div>Keratin is the outermost layer that protects our skin and has an appropriate turnover cycle. With age, the keratin turnover cycle begins to dysfunction. To overcome this issue, we artificially remove dead skin cells. In this study, we attempted to screen enzymes that could be useful in the cosmetics industry to develop enzymes suitable for the enzyme-based method, a mild exfoliation method that does not damage the skin. <em>Chryseobacterium camelliae</em> Dolsongi-HT1 with keratinolytic activity was isolated from green tea leaves (sourced from the Dolsongi tea garden, Jeju Island). The keratinolytic activity of <em>C. camelliae</em> Dolsongi-HT1 was detected in the culture media, indicating that the target keratinolytic enzyme is a secreted protein. Keratinolytic activity was demonstrated using forearm skin keratin and reconstituted human skin models. The enzyme from <em>C. camelliae</em> Dolsng-HT1 (HT1) could efficiently decompose human skin keratin. Moreover, experiments using the reconstituted human skin model demonstrated that HT1 is efficient in exfoliating the outermost stratum corneum. Compared with the popularly used chemical exfoliation method, enzymatic exfoliation using HT1 was less abrasive and did not damage the epidermal layer. Keratinolytic enzyme was identified using protein purification and mass spectrometry. The identified enzyme (iHT1) was expressed in the <em>Bacillus subtilis</em> RIK 1285 secretory protein expression system. The iHT1 enzyme showed high activity over a wide temperature range (30–60 °C), with the highest activity at 30 °C. The optimum pH for the activity of iHT was pH8.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"186 ","pages":"Article 110605"},"PeriodicalIF":3.4,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143429904","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

Enzymatic production of chitooligosaccharides with high degree of polymerisations and their potential application to soy sauce preservation 高聚合度壳寡糖的酶法生产及其在酱油保鲜中的潜在应用

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-17 DOI: 10.1016/j.enzmictec.2025.110608

Yihao Liu , Guangru Sun , Yimeng Lou , Peng Cheng , Qian Song , Wen Lv , Chunling Wang

Chitooligosaccharides (COSs) with a high degree of polymerisation (DP 5–10) have been reported to possess diverse bioactivities. Thus, the development of novel methods for the acquisition of high-DP COSs has become increasingly significant. In the study, a novel GH family 46 chitosanase gene (ThCsn46) was expressed and characterized. ThCsn46 was further applied to COSs production, and the highest yield of 95.7 % (143.6 g/L) was obtained using 15 % (w/v) of chitosan as the substrate. The proportion of high-DP COSs occupied 40.6 % of the total COSs. Moreover, the high (GlcN)₆ content was achieved. The total viable count (TVC) and amino acid nitrogen (AAN) of soy sauce incorporated with 0.1 % (w/v) of COSs were better than that of the negative control. The potential of ThCsn46 for application in the production of COSs and the preservation of soy sauce is significant. The green and efficient bioproduction process represents a promising way for further research.

{"title":"Enzymatic production of chitooligosaccharides with high degree of polymerisations and their potential application to soy sauce preservation","authors":"Yihao Liu , Guangru Sun , Yimeng Lou , Peng Cheng , Qian Song , Wen Lv , Chunling Wang","doi":"10.1016/j.enzmictec.2025.110608","DOIUrl":"10.1016/j.enzmictec.2025.110608","url":null,"abstract":"<div><div>Chitooligosaccharides (COSs) with a high degree of polymerisation (DP 5–10) have been reported to possess diverse bioactivities. Thus, the development of novel methods for the acquisition of high-DP COSs has become increasingly significant. In the study, a novel GH family 46 chitosanase gene (<em>ThCsn46</em>) was expressed and characterized. ThCsn46 was further applied to COSs production, and the highest yield of 95.7 % (143.6 g/L) was obtained using 15 % (w/v) of chitosan as the substrate. The proportion of high-DP COSs occupied 40.6 % of the total COSs. Moreover, the high (GlcN)<sub>6</sub> content was achieved. The total viable count (TVC) and amino acid nitrogen (AAN) of soy sauce incorporated with 0.1 % (w/v) of COSs were better than that of the negative control. The potential of ThCsn46 for application in the production of COSs and the preservation of soy sauce is significant. The green and efficient bioproduction process represents a promising way for further research.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"186 ","pages":"Article 110608"},"PeriodicalIF":3.4,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143429810","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

Screening and characterization of thermostable xylose isomerase from Rhodothermus marinus for erythrose production from one-carbon source 筛选和鉴定海红藻中的可热稳定木糖异构酶，以利用单碳源生产赤藓糖

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-17 DOI: 10.1016/j.enzmictec.2025.110607

Yanfei Wang , Wenwen Li , Dandan Wang , Yan Zeng , Ming Li , Yuanxia Sun , Jiangang Yang

Four-carbon (C4) sugars, which rarely exist in nature, usually have various biological functions and serve as building units for pharmaceutical agents. For example, erythrose possesses various pharmacological activities. Although several iso/epimerases have a catalytic function on C5/C6 sugars, only a few studies have demonstrated the enzymatic iso/epimerization of C4 sugars. In this work, we presented a xylose isomerase from Rhodothermus marinus possessing isomerization activity toward C4 l-erythrulose. This enzyme showed higher affinity and catalytic efficiency toward l-erythrulose than d-fructose and d-xylulose. Its specific activity reached 24.2 U/mg under optimal reaction conditions, and its half-life was over 8 days at 50 ℃. Demonstration of this enzyme under 833 mM of L-erythrulose gave 130 mM L-erythrose with a conversion yield of 15.6 %. On the basis of this beneficial enzyme, we further designed a multienzyme system and presented a one-pot cascade process for the synthesis of l-erythrose from low-cost, one-carbon formaldehyde (FALD) as the sole feedstock. Given that FALD can be derived from CO₂ electrocatalysis or methanol oxidization, l-erythrose synthesis can be realized from methanol and even CO₂. l-erythrose can further function as feedstock for synthesizing other high-carbon sugars by coupling enzymatic aldol condensation and reduction reactions.

{"title":"Screening and characterization of thermostable xylose isomerase from Rhodothermus marinus for erythrose production from one-carbon source","authors":"Yanfei Wang , Wenwen Li , Dandan Wang , Yan Zeng , Ming Li , Yuanxia Sun , Jiangang Yang","doi":"10.1016/j.enzmictec.2025.110607","DOIUrl":"10.1016/j.enzmictec.2025.110607","url":null,"abstract":"<div><div>Four-carbon (C4) sugars, which rarely exist in nature, usually have various biological functions and serve as building units for pharmaceutical agents. For example, erythrose possesses various pharmacological activities. Although several iso/epimerases have a catalytic function on C5/C6 sugars, only a few studies have demonstrated the enzymatic iso/epimerization of C4 sugars. In this work, we presented a xylose isomerase from <em>Rhodothermus marinus</em> possessing isomerization activity toward C4 <span>l</span>-erythrulose. This enzyme showed higher affinity and catalytic efficiency toward <span>l</span>-erythrulose than <span>d</span>-fructose and <span>d</span>-xylulose. Its specific activity reached 24.2 U/mg under optimal reaction conditions, and its half-life was over 8 days at 50 ℃. Demonstration of this enzyme under 833 mM of L-erythrulose gave 130 mM L-erythrose with a conversion yield of 15.6 %. On the basis of this beneficial enzyme, we further designed a multienzyme system and presented a one-pot cascade process for the synthesis of <span>l</span>-erythrose from low-cost, one-carbon formaldehyde (FALD) as the sole feedstock. Given that FALD can be derived from CO<sub>2</sub> electrocatalysis or methanol oxidization, <span>l</span>-erythrose synthesis can be realized from methanol and even CO<sub>2</sub>. <span>l</span>-erythrose can further function as feedstock for synthesizing other high-carbon sugars by coupling enzymatic aldol condensation and reduction reactions.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"186 ","pages":"Article 110607"},"PeriodicalIF":3.4,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143429811","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

Processivity and enzymatic mechanism of a non-modular family 5 endoglucanase from Sporocytophaga sp. CX11 with potential applications in cellulose saccharification

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2025-02-15 DOI: 10.1016/j.enzmictec.2025.110609

Xiaoyi Chen , Zilong Gao , Shang Wang, Fan Yang

In this study, a novel GH5 processive endoglucanase SmCel5A from Sporocytophaga sp. CX11 was functionally expressed in E. coli. It could rapidly decrease the viscosity of carboxymethyl cellulose (CMC-Na) solution by nearly 50 % within the initial 8 min of incubation and exhibited a significantly high specific activity towards CMC-Na of 9940 U/µmol. SmCel5A could also hydrolyze other cellulosic substrates such as RAC, Avicel, filter paper, β-glucan and the chromogenic substrate pNPC. When hydrolyzing filter paper, about 89.1 % of soluble reducing sugars were generated after 180 min of incubation, and the main products were cellobiose followed by cellotriose and glucose. The processive ratio of SmCel5A increased from 2.32 to 11.22 as the reaction time was extended from 5 min to 180 min, which is significantly higher than those of other known processive endoglucanases. Moreover, SmCel5A could hydrolyze cellodextrins with the degree of polymerization (DP) ≥ 3, but it was not active on cellobiose. In combination reaction with β-glucosidase, the maximum substrate conversion rate reached 73.2 %, showing that the synergistic reaction of the two enzymes could efficiently reduce the accumulation of cellobiose and greatly improve the hydrolysis efficiency of cellulose. The three-dimensional structure of SmCel5A was predicted by AlphaFold2 and showed to feature a classic GH5 family (β/α)₈-barrel structure, with a deep substrate-binding cleft formed by the amino acids at the C-terminus. Molecular docking results indicated that when hydrolyzing cellulosic substrates, SmCel5A exhibits a preference for the exo-type mechanism of action over the endo-type mechanism of action.

{"title":"Processivity and enzymatic mechanism of a non-modular family 5 endoglucanase from Sporocytophaga sp. CX11 with potential applications in cellulose saccharification","authors":"Xiaoyi Chen , Zilong Gao , Shang Wang, Fan Yang","doi":"10.1016/j.enzmictec.2025.110609","DOIUrl":"10.1016/j.enzmictec.2025.110609","url":null,"abstract":"<div><div>In this study, a novel GH5 processive endoglucanase <em>Sm</em>Cel5A from <em>Sporocytophaga</em> sp. CX11 was functionally expressed in <em>E. coli.</em> It could rapidly decrease the viscosity of carboxymethyl cellulose (CMC-Na) solution by nearly 50 % within the initial 8 min of incubation and exhibited a significantly high specific activity towards CMC-Na of 9940 U/µmol. <em>Sm</em>Cel5A could also hydrolyze other cellulosic substrates such as RAC, Avicel, filter paper, β-glucan and the chromogenic substrate <em>p</em>NPC. When hydrolyzing filter paper, about 89.1 % of soluble reducing sugars were generated after 180 min of incubation, and the main products were cellobiose followed by cellotriose and glucose. The processive ratio of <em>Sm</em>Cel5A increased from 2.32 to 11.22 as the reaction time was extended from 5 min to 180 min, which is significantly higher than those of other known processive endoglucanases. Moreover, <em>Sm</em>Cel5A could hydrolyze cellodextrins with the degree of polymerization (DP) ≥ 3, but it was not active on cellobiose. In combination reaction with β-glucosidase, the maximum substrate conversion rate reached 73.2 %, showing that the synergistic reaction of the two enzymes could efficiently reduce the accumulation of cellobiose and greatly improve the hydrolysis efficiency of cellulose. The three-dimensional structure of <em>Sm</em>Cel5A was predicted by AlphaFold2 and showed to feature a classic GH5 family (β/α)<sub>8</sub>-barrel structure, with a deep substrate-binding cleft formed by the amino acids at the C-terminus. Molecular docking results indicated that when hydrolyzing cellulosic substrates, <em>Sm</em>Cel5A exhibits a preference for the exo-type mechanism of action over the endo-type mechanism of action.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"185 ","pages":"Article 110609"},"PeriodicalIF":3.4,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143429162","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