Enzyme and Microbial Technology最新文献_第3页

Engineering glycolytic pathway for improved Lacto-N-neotetraose production in pichia pastoris 改良毕赤酵母生产乳酸-n -新四糖的工程糖酵解途径。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-25 DOI: 10.1016/j.enzmictec.2024.110576

Jiao Yang , Nitesh Kumar Mund , Lirong Yang , Hao Fang

Lacto-N-neotetraose (LNnT) is a primary solid component of human milk oligosaccharides (HMOs) with various promising health effects for infants. LNnT production by GRAS (generally recognized as safe) microorganisms has attracted considerable attention. However, few studies have emphasized Pichia Pastoris as a cell factory for LNnT’s production. Here, we have reported the first-ever synthesis of LNnT employing P. pastoris as the host. Initially, LNnT biosynthetic pathway genes β-1,3-N-acetylglucosaminyltransferase (lgtA) and β-1,4-galactostltransferase (lgtB) along with lactose permease (lac12) and galactose epimerase (gal10) were integrated into the genome of P. pastoris, but only 0.139 g/L LNnT was obtained. Second, the titer of LNnT was improved to 0.162 g/L via up-regulating genes to strengthen the supply of precursors, UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) and UDP-Gal (Uridine diphosphate galactose), for LNnT biosynthesis. Third, by knocking out critical mediator pfk (6-phosphofructokinase) genes in glycolysis, the major glucose metabolic flux was rewired to the LNnT biosynthesis pathway. As a result, the strain accumulated 0.867 g/L LNnT in YPG medium supplemented with glucose and lactose. Finally, LNnT production was increased to 1.24 g/L in a 3 L bioreactor. The work aimed to explore the potential of P. pastoris as a for LNnT production.

乳-n -新四糖（LNnT）是人乳寡糖（HMOs）的主要固体成分，对婴儿的健康具有多种前景。GRAS（通常被认为是安全的）微生物生产LNnT引起了相当大的关注。然而，很少有研究强调毕赤酵母是生产LNnT的细胞工厂。在这里，我们报道了首次以pastoris为宿主合成LNnT。最初将LNnT生物合成途径基因β-1,3- n -乙酰氨基葡萄糖转移酶（lgtA）和β-1,4-半乳糖转移酶（lgtB）以及乳糖渗透酶（lac12）和半乳糖外聚酶（gal10）整合到P. pastoris基因组中，但LNnT仅为0.139 g/L。其次，通过上调基因将LNnT的滴度提高到0.162 g/L，以加强LNnT生物合成的前体UDP-GlcNAc（二磷酸尿苷n -乙酰氨基葡萄糖）和UDP-Gal（二磷酸尿苷半乳糖）的供应。第三，通过敲除糖酵解中的关键介质pfk（6-磷酸果糖激酶）基因，将主要的葡萄糖代谢通量重新连接到LNnT生物合成途径。结果表明，菌株在添加葡萄糖和乳糖的YPG培养基中积累了0.867 g/L LNnT。最后，在3 L的生物反应器中，LNnT产量提高到1.24 g/L。这项工作旨在探索P. pastoris作为LNnT生产的潜力。

{"title":"Engineering glycolytic pathway for improved Lacto-N-neotetraose production in pichia pastoris","authors":"Jiao Yang , Nitesh Kumar Mund , Lirong Yang , Hao Fang","doi":"10.1016/j.enzmictec.2024.110576","DOIUrl":"10.1016/j.enzmictec.2024.110576","url":null,"abstract":"<div><div>Lacto-N-neotetraose (LNnT) is a primary solid component of human milk oligosaccharides (HMOs) with various promising health effects for infants. LNnT production by GRAS (generally recognized as safe) microorganisms has attracted considerable attention. However, few studies have emphasized <em>Pichia Pastoris</em> as a cell factory for LNnT’s production. Here, we have reported the first-ever synthesis of LNnT employing <em>P. pastoris</em> as the host. Initially, LNnT biosynthetic pathway genes β-1,3-N-acetylglucosaminyltransferase (<em>lgtA</em>) and β-1,4-galactostltransferase (<em>lgtB</em>) along with lactose permease (<em>lac12</em>) and galactose epimerase (<em>gal10</em>) were integrated into the genome of <em>P. pastoris</em>, but only 0.139 g/L LNnT was obtained. Second, the titer of LNnT was improved to 0.162 g/L via up-regulating genes to strengthen the supply of precursors, UDP-GlcNAc (Uridine diphosphate N-acetylglucosamine) and UDP-Gal (Uridine diphosphate galactose), for LNnT biosynthesis. Third, by knocking out critical mediator <em>pfk</em> (6-phosphofructokinase) genes in glycolysis, the major glucose metabolic flux was rewired to the LNnT biosynthesis pathway. As a result, the strain accumulated 0.867 g/L LNnT in YPG medium supplemented with glucose and lactose. Finally, LNnT production was increased to 1.24 g/L in a 3 L bioreactor. The work aimed to explore the potential of <em>P. pastoris</em> as a for LNnT production.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110576"},"PeriodicalIF":3.4,"publicationDate":"2024-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142913974","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

Structures and properties of α-amylase and glucoamylase immobilized by ZIF-8 via one-pot preparation 用 ZIF-8 单锅制备固定α-淀粉酶和葡萄糖淀粉酶的结构和特性。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-25 DOI: 10.1016/j.enzmictec.2024.110579

Yuxin Liu, Qinghua Pan, Zesheng Liang, Jingqiao Li, Rulong Wu

The immobilization of α-amylase and glucoamylase using a metal-organic framework (enzyme@ZIF-8) was prepared in situ through a one-pot method. The morphology, crystal structure, and molecular characteristics of the free enzyme and enzyme@ZIF-8 were characterized. The enzyme@ZIF-8 exhibited the rhombic dodecahedron morphology, with a decrease in particle size. Successful immobilization of α-amylase and glucoamylase within ZIF-8 was confirmed, with 30–40 % loading rate. The immobilization process did not significantly alter the crystal structure of ZIF-8. The changes in secondary structure of enzyme after immobilization resulted in modiﬁcation of catalytic activity of enzyme. The melting enthalpy of enzyme @ZIF-8 increased with the increase of enzyme content. The melting peak temperature of the enzyme immobilized by ZIF-8 increased. The activity of free and immobilized enzymes was influenced by the different time, pH and temperature. At pH 5–8 and temperature 60–80 °C, the activity of the immobilized enzyme was significantly greater than that of the free enzyme. The repeatability of enzyme@ZIF-8 was 61.52 % after three cycles. The kinetic parameters of Michaelis-Menten model for enzymatic reaction were determined by fitting the initial rate of reactions and initial substrate concentration data. The Michaelis-Menten constant (K_M) values of immobilized enzyme were lower than that of free enzyme, indicating the greater affinity between the enzyme and the substrate.

用金属有机骨架（enzyme@ZIF-8）原位固定化α-淀粉酶和葡萄糖淀粉酶。对游离酶和enzyme@ZIF-8的形态、晶体结构和分子特性进行了表征。enzyme@ZIF-8呈菱形十二面体形态，粒径减小。证实了α-淀粉酶和葡萄糖淀粉酶在ZIF-8内的固定成功，负载率为30-40 %。固定过程没有显著改变ZIF-8的晶体结构。固定化后酶的二级结构发生变化，导致酶的催化活性发生改变。酶@ZIF-8的熔化焓随酶含量的增加而增大。ZIF-8固定化酶的熔化峰温度升高。游离酶和固定化酶的活性受时间、pH和温度的影响。在pH 5 ~ 8、温度60 ~ 80℃条件下，固定化酶的活性显著高于游离酶。3个循环后enzyme@ZIF-8的重复性为61.52 %。通过拟合初始反应速率和初始底物浓度，确定了Michaelis-Menten模型的动力学参数。固定化酶的Michaelis-Menten常数（KM）值低于游离酶，说明固定化酶与底物的亲和力较强。

{"title":"Structures and properties of α-amylase and glucoamylase immobilized by ZIF-8 via one-pot preparation","authors":"Yuxin Liu, Qinghua Pan, Zesheng Liang, Jingqiao Li, Rulong Wu","doi":"10.1016/j.enzmictec.2024.110579","DOIUrl":"10.1016/j.enzmictec.2024.110579","url":null,"abstract":"<div><div>The immobilization of α-amylase and glucoamylase using a metal-organic framework (enzyme@ZIF-8) was prepared in situ through a one-pot method. The morphology, crystal structure, and molecular characteristics of the free enzyme and enzyme@ZIF-8 were characterized. The enzyme@ZIF-8 exhibited the rhombic dodecahedron morphology, with a decrease in particle size. Successful immobilization of α-amylase and glucoamylase within ZIF-8 was confirmed, with 30–40 % loading rate. The immobilization process did not significantly alter the crystal structure of ZIF-8. The changes in secondary structure of enzyme after immobilization resulted in modiﬁcation of catalytic activity of enzyme. The melting enthalpy of enzyme @ZIF-8 increased with the increase of enzyme content. The melting peak temperature of the enzyme immobilized by ZIF-8 increased. The activity of free and immobilized enzymes was influenced by the different time, pH and temperature. At pH 5–8 and temperature 60–80 °C, the activity of the immobilized enzyme was significantly greater than that of the free enzyme. The repeatability of enzyme@ZIF-8 was 61.52 % after three cycles. The kinetic parameters of Michaelis-Menten model for enzymatic reaction were determined by fitting the initial rate of reactions and initial substrate concentration data. The Michaelis-Menten constant (<em>K</em><sub><em>M</em></sub>) values of immobilized enzyme were lower than that of free enzyme, indicating the greater affinity between the enzyme and the substrate.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110579"},"PeriodicalIF":3.4,"publicationDate":"2024-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142926836","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

Identification of a bacteria P450 enzyme from B. megaterium H-1 with vitamin D3 C-25 hydroxylation capabilities 具有维生素D3 C-25羟基化能力的巨型芽孢杆菌H-1细菌P450酶的鉴定。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-24 DOI: 10.1016/j.enzmictec.2024.110578

Yulin He , Yina Hou , Hui Li , Fan He , Jingyi Zhou , Xiaomei Zhang , Jingsong Shi , Zhenghong Xu

Calcidiol (25(OH)VD₃) and calcitriol (1α,25(OH)₂VD₃) are active vitamin D₃ with high medicinal value, which can maintain calcium and phosphorus balance and treat vitamin D deficiency. Microbial synthesis is an important method to produce high-value-added compounds. It can produce active vitamin D₃ through the hydroxylation reaction of P450, which can reduce the traditional chemical synthesis steps, and greatly improve the production efficiency and economic benefits. In this work, Bacillus megaterium H-1 was screened for its ability to produce 25(OH)VD₃ and 1α,25(OH)₂VD₃ from vitamin D₃. A new highly inducible vitamin D₃ hydroxylase CYP109E1-H was identified from B. megaterium H-1 through searching for transcripts with cytochrome P450 structural domains, combining the transcriptome sequencing with functional expression in Bacillus subtilis WB600. Biotransformation in recombinant B. subtilis confirmed that CYP109E1-H has C-25 hydroxylase activity towards vitamin D₃. CYP109E1-H is a natural mutant of CYP109E1 with greater stereoselectivity and it is a new vitamin D₃ mono-hydroxylase. The cloning and characterization of the CYP109E1-H gene provide useful information on the structural basis for improving the regional and stereoselectivity of the CYP109E gene.

钙二醇（25(OH)VD3）和骨化三醇（1α,25(OH)2VD3）是具有较高药用价值的活性维生素D3，可维持钙磷平衡，治疗维生素D缺乏症。微生物合成是生产高附加值化合物的重要方法。通过P450的羟基化反应可以生产活性维生素D3，减少了传统的化学合成步骤，大大提高了生产效率和经济效益。本研究筛选了巨芽孢杆菌H-1从维生素D3中产生25(OH)VD3和1α，25(OH)2VD3的能力。通过寻找含有细胞色素P450结构域的转录本，结合转录组测序和枯草芽孢杆菌WB600的功能表达，从巨芽孢杆菌H-1中鉴定出一个新的高诱导性维生素D3羟化酶CYP109E1-H。在重组枯草芽孢杆菌中的生物转化证实了CYP109E1-H对维生素D3具有C-25羟化酶活性。CYP109E1- h是CYP109E1的天然突变体，具有较高的立体选择性，是一种新的维生素D3单羟化酶。CYP109E1-H基因的克隆和鉴定为提高CYP109E基因的区域选择性和立体选择性提供了有益的结构依据。

{"title":"Identification of a bacteria P450 enzyme from B. megaterium H-1 with vitamin D3 C-25 hydroxylation capabilities","authors":"Yulin He , Yina Hou , Hui Li , Fan He , Jingyi Zhou , Xiaomei Zhang , Jingsong Shi , Zhenghong Xu","doi":"10.1016/j.enzmictec.2024.110578","DOIUrl":"10.1016/j.enzmictec.2024.110578","url":null,"abstract":"<div><div>Calcidiol (25(OH)VD<sub>3</sub>) and calcitriol (1<em>α</em>,25(OH)<sub>2</sub>VD<sub>3</sub>) are active vitamin D<sub>3</sub> with high medicinal value, which can maintain calcium and phosphorus balance and treat vitamin D deficiency. Microbial synthesis is an important method to produce high-value-added compounds. It can produce active vitamin D<sub>3</sub> through the hydroxylation reaction of P450, which can reduce the traditional chemical synthesis steps, and greatly improve the production efficiency and economic benefits. In this work, <em>Bacillus megaterium</em> H-1 was screened for its ability to produce 25(OH)VD<sub>3</sub> and 1<em>α</em>,25(OH)<sub>2</sub>VD<sub>3</sub> from vitamin D<sub>3</sub>. A new highly inducible vitamin D<sub>3</sub> hydroxylase CYP109E1-H was identified from <em>B. megaterium</em> H-1 through searching for transcripts with cytochrome P450 structural domains, combining the transcriptome sequencing with functional expression in <em>Bacillus subtilis</em> WB600. Biotransformation in recombinant <em>B. subtilis</em> confirmed that CYP109E1-H has C-25 hydroxylase activity towards vitamin D<sub>3</sub>. CYP109E1-H is a natural mutant of CYP109E1 with greater stereoselectivity and it is a new vitamin D<sub>3</sub> mono-hydroxylase. The cloning and characterization of the CYP109E1-H gene provide useful information on the structural basis for improving the regional and stereoselectivity of the CYP109E gene.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110578"},"PeriodicalIF":3.4,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892980","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

Acidogenic fermentation of Ulva in a fed-batch reactor system: tubular versus foliose biomass 进料批式反应器系统中Ulva的产酸发酵：管状与卵泡生物量。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-24 DOI: 10.1016/j.enzmictec.2024.110580

James Lawrence , Armando Oliva , Jerry D. Murphy , Piet N.L. Lens

The present study proposes a biorefinery of the macroalgae Ulva, focusing on evaluating two different morphologies of the species (foliose and tubular) during acidogenic fermentation in fed-batch reactors. Stage 1 of the study evaluates lyophilised foliose and tubular Ulva, whilst Stage 2 analyses the impact of ulvan extraction on volatile fatty acids yield and changes in carbohydrate availability. Acetic, propionic, and butyric acids were produced from each substrate, with peak concentrations of total VFAs recorded at 2179.5 mg HAc/L (foliose Ulva) and 2029.3 mg HAc/L (tubular Ulva) when ulvan was present. After ulvan extraction, the acidogenic fermentation of the foliose morphotype was negatively affected, reaching at most 315.3 mg HAc/L. In contrast, the extraction showed no influence on the tubular morphotype, peaking at 2165.0 mg HAc/L. Additional variations were noted in the availability of carbohydrates in each substrate during the acidogenic fermentation process. The ulvan-extracted tubular morphotype exhibited the highest peak in carbohydrate concentration (9.8 g glucose/L), whilst the ulvan-extracted foliose morphotype yielded up to 8.5 g glucose/L. This study highlights the biorefinery potential of Ulva biomass, proposing a multiple cascading approach linking multiple energy and biomolecule applications to maximise the valorisation of the biomass.

本研究提出了一种大型藻类Ulva的生物精制方法，重点研究了在进料间歇反应器中产酸发酵过程中该物种的两种不同形态（叶状和管状）。研究的第一阶段评估了冻干的folose和管状Ulva，而第二阶段分析了ulvan提取对挥发性脂肪酸产量和碳水化合物可用性变化的影响。每种底物都产生乙酸、丙酸和丁酸，当存在ulvan时，总VFAs的峰值浓度为2179.5 mg HAc/L （foliose Ulva）和2029.3 mg HAc/L（管状Ulva）。经ulvan提取后，对卵泡叶形态的产酸发酵产生不利影响，最高可达315.3 mg HAc/L。相比之下，提取物对管状形态没有影响，峰值为2165.0 mg HAc/L。在产酸发酵过程中，每种底物中碳水化合物的可用性也发生了额外的变化。紫菀提取的管状形态碳水化合物浓度最高（9.8 g葡萄糖/L），而紫菀提取的叶状形态碳水化合物浓度最高可达8.5 g葡萄糖/L。这项研究强调了Ulva生物质的生物精炼潜力，提出了一种连接多种能源和生物分子应用的多重级联方法，以最大限度地提高生物质的价值。

{"title":"Acidogenic fermentation of Ulva in a fed-batch reactor system: tubular versus foliose biomass","authors":"James Lawrence , Armando Oliva , Jerry D. Murphy , Piet N.L. Lens","doi":"10.1016/j.enzmictec.2024.110580","DOIUrl":"10.1016/j.enzmictec.2024.110580","url":null,"abstract":"<div><div>The present study proposes a biorefinery of the macroalgae <em>Ulva,</em> focusing on evaluating two different morphologies of the species (foliose and tubular) during acidogenic fermentation in fed-batch reactors. Stage 1 of the study evaluates lyophilised foliose and tubular <em>Ulva</em>, whilst Stage 2 analyses the impact of ulvan extraction on volatile fatty acids yield and changes in carbohydrate availability. Acetic, propionic, and butyric acids were produced from each substrate, with peak concentrations of total VFAs recorded at 2179.5 mg HAc/L (foliose <em>Ulva</em>) and 2029.3 mg HAc/L (tubular <em>Ulva</em>) when ulvan was present. After ulvan extraction, the acidogenic fermentation of the foliose morphotype was negatively affected, reaching at most 315.3 mg HAc/L. In contrast, the extraction showed no influence on the tubular morphotype, peaking at 2165.0 mg HAc/L. Additional variations were noted in the availability of carbohydrates in each substrate during the acidogenic fermentation process. The ulvan-extracted tubular morphotype exhibited the highest peak in carbohydrate concentration (9.8 g glucose/L), whilst the ulvan-extracted foliose morphotype yielded up to 8.5 g glucose/L. This study highlights the biorefinery potential of <em>Ulva</em> biomass<em>,</em> proposing a multiple cascading approach linking multiple energy and biomolecule applications to maximise the valorisation of the biomass.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110580"},"PeriodicalIF":3.4,"publicationDate":"2024-12-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142946515","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

Green and sustainable production of isofraxidin from Acanthopanax senticosus with cellulose-based immobilized probiotics 纤维素基固定化益生菌绿色可持续生产刺五加异黄酮。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-23 DOI: 10.1016/j.enzmictec.2024.110574

Shuang Jin , Hongyao Cai , Cailiang Peng , Yupeng Cheng , Yubin Ren , Weili Liu , Yujie Fu , Chen Lv

This study utilizes deep eutectic solvent (DES)-assisted enhancement of cellulose-based immobilized probiotics for efficient biotransformation of isofraxidin from Acanthopanax senticosus. Among seven probiotic strains tested, Lactiplantibacillus plantarum CICC 20767 exhibited the best catalytic activity. We explored the effects of 12 different DESs with varying hydrogen bond donors on the conversion capacity of the immobilized probiotics, with the highest efficiency observed using ChCl/EG (4.0 wt %). The optimized process, with a solid-to-liquid ratio of 1:5 (g/mL), a temperature of 35.6 °C, a reaction time of 4 d, and a pH of 6.9, resulted in a 5.53-folds increase in isofraxidin yield, reaching 0.4034 mg/g, compared to the untreated sample (0.0729 mg/g). The immobilized probiotics retained excellent catalytic activity after 12 cycles of use, demonstrating their stability and potential for large-scale, green production of isofraxidin. This study presents a valuable method for industrial isofraxidin production and highlights the broad potential of this environmentally friendly bioconversion process in the pharmaceutical industry.

本研究利用深度共熔溶剂（DES）辅助强化纤维素基固定化益生菌，对刺五加中异黄皮苷进行高效生物转化。7株益生菌中，植物乳杆菌CICC 20767的催化活性最好。我们探索了12种不同氢键供体的DESs对固定化益生菌转化能力的影响，其中使用ChCl/EG（4.0 wt %）的效率最高。在料液比为1:5 （g/mL）、温度为35.6 ℃、反应时间为4 d、pH为6.9的条件下，异黄皮苷的得率达到0.4034 mg/g，是未处理样品（0.0729 mg/g）的5.53倍。在12个循环使用后，固定化益生菌仍保持了良好的催化活性，证明了其稳定性和大规模绿色生产异黄菌素的潜力。本研究提出了一种有价值的工业异黄酮生产方法，并强调了这种环境友好型生物转化工艺在制药工业中的广泛潜力。

{"title":"Green and sustainable production of isofraxidin from Acanthopanax senticosus with cellulose-based immobilized probiotics","authors":"Shuang Jin , Hongyao Cai , Cailiang Peng , Yupeng Cheng , Yubin Ren , Weili Liu , Yujie Fu , Chen Lv","doi":"10.1016/j.enzmictec.2024.110574","DOIUrl":"10.1016/j.enzmictec.2024.110574","url":null,"abstract":"<div><div>This study utilizes deep eutectic solvent (DES)-assisted enhancement of cellulose-based immobilized probiotics for efficient biotransformation of isofraxidin from <em>Acanthopanax senticosus</em>. Among seven probiotic strains tested, <em>Lactiplantibacillus plantarum</em> CICC 20767 exhibited the best catalytic activity. We explored the effects of 12 different DESs with varying hydrogen bond donors on the conversion capacity of the immobilized probiotics, with the highest efficiency observed using ChCl/EG (4.0 wt %). The optimized process, with a solid-to-liquid ratio of 1:5 (g/mL), a temperature of 35.6 °C, a reaction time of 4 d, and a pH of 6.9, resulted in a 5.53-folds increase in isofraxidin yield, reaching 0.4034 mg/g, compared to the untreated sample (0.0729 mg/g). The immobilized probiotics retained excellent catalytic activity after 12 cycles of use, demonstrating their stability and potential for large-scale, green production of isofraxidin. This study presents a valuable method for industrial isofraxidin production and highlights the broad potential of this environmentally friendly bioconversion process in the pharmaceutical industry.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 110574"},"PeriodicalIF":3.4,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142892979","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

Tailoring of levansucrase product size by a comparative molecular dynamics approach 用比较分子动力学方法裁剪左旋蔗糖酶产物大小。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-23 DOI: 10.1016/j.enzmictec.2024.110577

Zhiwei Li, Tong Bao, Kaiwen Chen, Chao Hu, Xinyu Zhang, Xueqin Hu, Jingwen Yang, Hongbin Zhang

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.

莱文被广泛用作食品添加剂。其分子量对其利用有显著影响。枯草芽孢杆菌（Bacillus subtilis, Bs-SacB）和巨型葡萄球菌（Priestia megaterium, Pm-SacB）的左万蔗糖酶产量不同。为了深入研究这两种酶产物分子量差异的分子基础，我们对Bs-SacB和Pm-SacB活性位点周围的8个环进行了重点研究，并确定了Loop3和loop4是改变Bs-SacB产物分子量的关键决定因素。随后，利用突变能量分析和非同源替代策略，我们对loop3和loop4进行了定制修饰，得到了具有不同分子量谱的突变酶谱，包括F182Y（3698 Da）、CYTI（3093 Da）、3-Pbl（2776 Da）、4-Bml（1845 Da）和F182K（1571 Da）。本研究为改变产物分子量提供了一种新的比较分子动力学方法，并成功应用于左旋蔗糖酶的改性。

{"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":"10.1016/j.enzmictec.2024.110577","url":null,"abstract":"<div><div>Levan is widely used as food additives. Its utilization is significantly influenced by its molecular weight. <em>Bacillus subtilis</em> levansucrase (Bs-SacB) and <em>Priestia megaterium</em> 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.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 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}

引用次数: 0

Cloning, purification and characterization of a novel thermostable recombinant tannase from Galactobacillus timonensis timmonensis半乳杆菌重组单宁酶的克隆、纯化及特性研究。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-23 DOI: 10.1016/j.enzmictec.2024.110575

Jingya Wu , Huan Zeng , Xinyan Zhong , Xi Chen , Peng Zhang , Zeyuan Deng

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.

与天然鞣酶相关的高昂生产成本对其广泛的工业利用构成了重大挑战。微生物表达系统为酶的生产提供了一种经济有效的方法。本研究从提蒙半乳杆菌中克隆了一个推测为编码B型单宁酶（Gt-Tan）的基因，并在大肠杆菌BL21 （DE3）细胞中异源表达。通过金属亲和层析纯化得到分子量为55 kDa的Gt-Tan单体结构。Gt-Tan在温度50 ℃、pH 6.0时活性最佳。在45 ℃孵育2 h和40 ℃孵育48 h后，其最大活性分别保持在68.3% %和54.7% %左右。低浓度的Mn2+、Zn2+、Al3+、尿素、正丁醇和二甲亚砜对Gt-Tan的活性有微弱的增强作用，而Cu2+、Fe3+、Fe2+、Co2+、SDS、十六烷基三甲基溴化铵、DTT、吐温80和β-巯基乙醇对其活性有显著的抑制作用。没食子酸甲酯的Km和kcat/Km值分别为0.83 mM和19.7 s1 mM1，没食子酸丙酯的Km和kcat/Km值分别为0.67 mM和65.4 s1 mM1，单宁酸的Km和kcat/Km值分别为0.22 mM和240.8 s1 mM1。这些结果增强了我们对单宁酶的理解，并为其在化学、饲料和食品工业中的应用提供了潜在的来源。

{"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":"10.1016/j.enzmictec.2024.110575","url":null,"abstract":"<div><div>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 <em>Galactobacillus timonensis</em> and expressed heterologously in <em>Escherichia coli</em> 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 Mn<sup>2+</sup>, Zn<sup>2+</sup>, Al<sup>3+</sup>, urea, n-butanol, and dimethylsulfoxide at a low concentration slightly enhanced the activity of Gt-Tan, whereas Cu<sup>2+</sup>, Fe<sup>3+</sup>, Fe<sup>2+</sup>, Co<sup>2+</sup>, SDS, cetyltrimethylammonium bromide, DTT, Tween 80, and β-mercaptoethanol significantly inhibited its activity. <em>K</em><sub><em>m</em></sub> and <em>k</em><sub><em>cat</em></sub>/<em>K</em><sub><em>m</em></sub> values were estimated to be 0.83 mM and 19.7 s<sup><img>1</sup> mM<sup><img>1</sup> for methyl gallate, 0.67 mM and 65.4 s<sup><img>1</sup> mM<sup><img>1</sup> for propyl gallate, and 0.22 mM and 240.8 s<sup><img>1</sup> mM<sup><img>1</sup> for tannic acid. These results enhanced our understanding of tannase and provided potential sources for applications in the chemical, feed, and food industries.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 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}

引用次数: 0

Structural and functional insights into recombinant β-glucosidase from Thermothelomyces thermophilus: Cello-oligosaccharide hydrolysis and thermostability 重组β-葡萄糖苷酶的结构和功能研究：纤维寡糖水解和热稳定性。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-17 DOI: 10.1016/j.enzmictec.2024.110572

Ana Luiza da Rocha Fortes Saraiva , Gabriela Leila Berto , Bianca Oliva , Paula Macedo Cunha , Lucas Ramos , Leandro Cristante de Oliveira , Fernando Segato

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.

β-葡萄糖苷酶（β-glucosidases, BGLs）是纤维素生物质解聚的关键酶，可催化纤维素低聚糖转化为葡萄糖。这种转化对于提高生物精炼厂第二代乙醇或其他增值产品的生产至关重要。然而，由于所需的高酶负荷，该过程通常成本过高。因此，新型高效bgl的发现是一个重大的进步。在本研究中，嗜热真菌Thermothelomyces thermophilus （TthBgl3A）的糖苷水解酶家族3 （GH3）中的一个BGL在中性曲霉（Aspergillus nidulans）中异源表达。重组酶在pH 5.0和55°C条件下表现出最佳的活性，在160 h下具有显著的稳定性。在TthBgl3A的催化腔内有一个独特的、广泛的环，促进疏水相互作用，增强长纤维低聚糖的结合和水解。因此，TthBgl3A已被证明是水解木质纤维素生物质的有效酶。这些发现对于扩大嗜热t菌产生的酶的种类具有重要意义，并为TthBgl3A在降解纤维素材料和生产有价值化合物方面的潜在应用提供了新的见解。

{"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":"10.1016/j.enzmictec.2024.110572","url":null,"abstract":"<div><div>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 <em>Thermothelomyces thermophilus</em> (<em>Tth</em>Bgl3A) was heterologously expressed in <em>Aspergillus nidulans</em>. 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 <em>Tth</em>Bgl3A facilitates hydrophobic interactions that enhance the binding and hydrolysis of long cello-oligosaccharides. Consequently, <em>Tth</em>Bgl3A has proven to be an efficient enzyme for the hydrolysis lignocellulosic biomass. These findings are significant for expanding the repertoire of enzymes produced by <em>T. thermophilus</em> and provide new insights into the potential application of <em>Tth</em>Bgl3A in the degradation of cellulosic materials and the production of valuable compounds.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 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}

引用次数: 0

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 一种新分离的噬菌体在控制咖啡植物中的冠突伪单胞杆菌（Pseudomonas coronafaciens pv. garcae）方面的潜力：体外和体内实验的分子特征。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-15 DOI: 10.1016/j.enzmictec.2024.110573

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

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.

传统上，控制由植物病原体冠状假单胞菌引起的咖啡植物细菌性晕疫病（BHB）。garcae （Pcg）涉及经常向咖啡种植园喷洒非环境友好型和可能促进细菌耐药性的铜产品或盐酸卡苏加霉素。在这项研究中，我们报告了在寻求一种新的生态友好方法方面的飞跃，表征（物理化学和生物学）并在体外和体外测试了一种新的Pcg裂解噬菌体。深入的分子（基因组和DNA结构特征）表征噬菌体也进行了。噬菌体PcgS01F属于尾状病毒纲、德雷克勒病毒科、Guelphvirus属，形态类似于siphovirus。噬菌体PcgS01F显示潜伏期为40 min，爆发大小为46 PFU/宿主细胞，可以得出结论，它在Pcg IBSBF-158中复制良好。在感染的多重性（Multiplicity Of Infection，即噬菌体与细菌的比率）为1000时，噬菌体PcgS01F的表现要比MOI为10时好得多，在体外灭活实验结束前，噬菌体PcgS01F的细菌减少量不断增加，稳定在82 %的细菌负荷减少量。噬菌体PcgS01F在人工污染的咖啡树叶片中体外感染并杀死Pcg细胞，孵育36 h后，在MOI 1000下Pcg失活率最高为7.66 log CFU/mL。本研究提供了证据，证明分离的噬菌体是对抗咖啡植物BHB病原体的有希望的候选体。

{"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":"10.1016/j.enzmictec.2024.110573","url":null,"abstract":"<div><div>Traditionally, control of coffee plant bacterial halo blight (BHB) caused by the phytopathogen <em>Pseudomonas coronafaciens</em> pv. <em>garcae</em> (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 <em>in vitro</em> and <em>ex vivo</em> 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, <em>Drexlerviridae</em> family and genus <em>Guelphvirus</em>, 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 <em>in vitro</em> inactivation assays, stabilizing at a significant 82 % bacterial load reduction. Phage PcgS01F infected and killed Pcg cells <em>ex vivo</em> 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.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 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}

引用次数: 0

Improving the anti-autolytic ability of alkaline protease from Bacillus alcalophilus by a rationally combined strategy 合理组合提高嗜钙芽孢杆菌碱性蛋白酶抗自溶能力。

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology

Pub Date : 2024-12-07 DOI: 10.1016/j.enzmictec.2024.110561

Mian Wu, Lin Cao, Wei Tang, Zhemin Liu, Su Feng

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.

洗涤剂酶作为对环境友好的有害化学物质的替代品被广泛开发，其中碱性蛋白酶占洗涤剂酶销售的很大一部分。然而，碱性蛋白酶的自裂功能影响了其活性和整体应用。为此，提出了一种基于自分子对接（Self-ZDOCK）和分子动力学（MD）模拟的合理组合策略。Self-ZDOCK是一种预测蛋白质与自身结合模式的计算方法，对于理解蛋白酶的自裂机制至关重要。另一方面，MD模拟是一种强大的工具，可以深入了解蛋白质随时间的动态行为，从而分析BpAP在各种条件下的结构稳定性和灵活性。实验验证了该策略是提高BpAP抗自溶能力的有效途径。在28个BpAP突变体中，有5个突变体的热稳定性、pH稳定性和洗涤剂储存稳定性均有所提高，表明其抗自溶能力显著增强。结构分析和MD模拟证实了BpAP稳定性的增强是由于抗自溶能力的提高而不是结构稳定性的提高。三点组合突变体（MT5）的自溶能力提高最好，催化效率也较好。失活突变体的低率和阳性突变体的高率表明，新引入的筛选因素（与催化残基的距离、吉布斯自由能项、分子模拟和目测）极大地增强了抗自溶碱性蛋白酶的设计。此外，这些发现增强了碱性蛋白酶在洗涤剂和类似应用中的工业应用。

{"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":"10.1016/j.enzmictec.2024.110561","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":11770,"journal":{"name":"Enzyme and Microbial Technology","volume":"184 ","pages":"Article 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}

引用次数: 0