Effect of hydrogen sulfide (H2 S) on the growth and development of tobacco seedlings in absence of stress
作者：
Dai, Jingcheng;Wen, Dingxin;Li, Hao;Yang, Jingpeng;Rao, Xiongfei;...
期刊：
BMC Plant Biology ,2024年24(1):1-14 ISSN：1471-2229
通讯作者：
Yang, Chunlei;Yu, J
作者机构：
[Dai, Jingcheng; Yang, Jiangke] Wuhan Polytech Univ, Sch Life Sci & Technol, Wuhan 430023, Peoples R China.;[Yu, Jun; Yang, Chunlei; Li, Hao; Yang, Jingpeng; Rao, Xiongfei] Tobacco Res Inst Hubei Prov, Wuhan 430030, Hubei, Peoples R China.;[Wen, Dingxin; Yang, Yong] Hubei Univ, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn, Wuhan 430062, Peoples R China.;[Yang, Jiangke] Wuhan Polytech Univ, Coll Life Sci & Technol, Pilot Base Food Microbial Resources Utilizat Hubei, Wuhan 430024, Peoples R China.
通讯机构：
[Yu, J ; Yang, CL] T;Tobacco Res Inst Hubei Prov, Wuhan 430030, Hubei, Peoples R China.
关键词：
Enzymes activities;Hydrogen sulfide;NaHS;Photosynthetic pigments;Tobacco
摘要：
<jats:title>Abstract</jats:title><jats:sec>
<jats:title>Background</jats:title>
<jats:p>Hydrogen sulfide (H<jats:sub>2</jats:sub>S) is a novel signaling molecule involved in the growth and development of plants and their response to stress. However, the involvement of H<jats:sub>2</jats:sub>S in promoting the growth and development of tobacco plants is still unclear.</jats:p>
</jats:sec><jats:sec>
<jats:title>Results</jats:title>
<jats:p>In this study, we explored the effect of pre-soaking or irrigating the roots of tobacco plants with 0.0, 2.0, 4.0, 6.0, and 8.0mM of sodium hydrosulfide (NaHS) on endogenous H<jats:sub>2</jats:sub>S production, antioxidant enzymatic and cysteine desulfhydrase activities, seed germination, agronomic traits, photosynthetic pigments contents, and root vigor. The results revealed that exogenous NaHS treatment could significantly promote endogenous H<jats:sub>2</jats:sub>S production by inducing gene expression of <jats:italic>D/L-CD</jats:italic> and the activities of D/L-CD enzymes. Additionally, a significant increase in the agronomic traits and the contents of photosynthetic pigments, and no significant difference in carotenoid content among tobacco plants treated with 0.0 to 8.0mM of NaHS was observed. Additionally, a significant increase in the germination speed, dry weight, and vigor of tobacco seeds, whereas no significant effect on the percentage of seed germination was observed on NaHS treatment. Furthermore, NaHS treatment could significantly increase the activity of superoxide dismutase (SOD) and peroxidase (POD) enzymes, which reduces damage due to oxidative stress by maintaining reactive oxygen species homeostasis.</jats:p>
</jats:sec><jats:sec>
<jats:title>Conclusions</jats:title>
<jats:p>These results would aid in enhancing our understanding of the involvement of H<jats:sub>2</jats:sub>S, a novel signaling molecule to promote the growth and development of tobacco plants.</jats:p>
</jats:sec>
语种：
英文
展开
A convenient broad-host counterselectable system endowing rapid genetic manipulations of Kluyveromyces lactis and other yeast species
作者：
Zheng, Yanli;Deng, Yuhui;Hu, Ping;Wang, Shiqing;Wu, Jiawen;...
期刊：
Microbial Cell Factories ,2024年23(1):1-9 ISSN：1475-2859
通讯作者：
Peng, WF
作者机构：
[Wu, Jiawen; Wang, Shiqing; Luo, Siqi; Deng, Yuhui; Zheng, Yanli; Hu, Ping; Lei, Lei; Yang, Jiangke] Wuhan Polytech Univ, Coll Life Sci & Technol, Wuhan 430023, Peoples R China.;[Luo, Siqi; Peng, Wenfang; Peng, WF] Hubei Univ, Environm Microbial Technol Ctr Hubei Prov, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn,Hubei Eng, Wuhan 430062, Peoples R China.
通讯机构：
[Peng, WF ] H;Hubei Univ, Environm Microbial Technol Ctr Hubei Prov, Sch Life Sci, State Key Lab Biocatalysis & Enzyme Engn,Hubei Eng, Wuhan 430062, Peoples R China.
摘要：
Being generally regarded as safe, Kluyveromyces lactis has been widely taken for food, feed, and pharmaceutical applications, owing to its ability to achieve high levels of protein secretion and hence being suitable for industrial production of heterologous proteins. Production platform strains can be created through genetic engineering; while prototrophic cells without chromosomally accumulated antibiotics resistance genes have been generally preferred, arising the need for dominant counterselection. We report here the establishment of a convenient counterselection system based on a Frs2 variant, Frs2v, which is a mutant of the alpha-subunit of phenylalanyl-tRNA synthase capable of preferentially incorporating a toxic analog of phenylalanine, r-chloro-phenylalanine (4-CP), into proteins to bring about cell growth inhibition. We demonstrated that expression of Frs2v from an episomal plasmid in K. lactis could make the host cells sensitive to 2 mM 4-CP, and a Frs2v-expressing plasmid could be efficiently removed from the cells immediately after a single round of cell culturing in a 4-CP-contianing YPD medium. This Frs2v-based counterselection helped us attain scarless gene replacement in K. lactis without any prior engineering of the host cells. More importantly, counterselection with this system was proven to be functionally efficient also in Saccharomyces cerevisiae and Komagataella phaffii, suggestive of a broader application scope of the system in various yeast hosts. Collectively, this work has developed a strategy to enable rapid, convenient, and high-efficiency construction of prototrophic strains of K. lactis and possibly many other yeast species, and provided an important reference for establishing similar methods in other industrially important eukaryotic microbes.
语种：
英文
展开
枯草芽孢杆菌漆酶的理性设计及在霉菌毒素降解的应用
作者：
乐琛;王晓;谢珂;郑艳丽;杨江科;...
期刊：
食品与发酵工业 ,2024年CSCD ISSN：0253-990X
作者机构：
武汉轻工大学生命科学与技术学院
摘要：
为解决目前制约食品等领域发展的霉菌毒素污染问题，选取了枯草芽孢杆菌来源的BsCotA漆酶并且通过理性设计提高酶活、稳定性，从而实现对霉菌毒素的降解效率的提高。利用理性设计改造BsCotA漆酶，获得了4个突变体漆酶，包括3个点突变体：CotAA344K、CotAA317T、CotAT36Y和突变整合体CotAgold，使用毕赤酵母进行异源表达，测定其酶学性质，并检测其对霉菌毒素的降解效率。基于蛋白质结构进行设计的3个单点突变，均从不同程度上提高漆酶的稳定性。而将3个突变位点同时整合到1个漆酶突变体上时，3个突变位点间未发生显著拮抗作用，使得突变整合体CotAgold展示出最好的稳定性以及催化能力；CotAgold比酶活为99.4 U/mg，相比BsCotA的比酶活提高了10倍，对于ABTS的kcat/KM 达到了7.12×105 M-1/s，相比BsCotA提高了1.9倍；突变体CotAgold的最适反应pH值为4，最适反应温度均为70 ℃，与野生型BsCotA催化条件保持一致；CotAgold在pH 2～12，孵育30 min，剩余酶活均在70%以上，相比野生型BsCotA在酸性条件下的稳定性有明显提高；CotAgold在40～90 ℃，孵育30 min，残留酶活均在90%以上，相比野生型BsCotA在高温条件下的稳定性有明显提高；并在ABTS＋自由基作为介体的条件下，漆酶CotAgold能完全降解黄曲霉毒素B1和玉米赤霉烯酮；相比之下，野生型漆酶BsCotA的降解率仅为5%。通过蛋白质的理性设计得到了漆酶突变体CotAgold，表现出显著提高的pH稳定性以及热稳定性，蛋白表达量也有一定的提高，在霉菌毒素降解等领域具有良好的应用前景。
语种：
中文
展开
弧菌DS32褐藻胶裂解酶基因vralg1的异源表达和酶学性质研究
作者：
李慧敏;邵宗泽;路瑶;杨江科;周梅先
期刊：
应用海洋学学报 ,2024年43(2):255-265 ISSN：2095-4972
作者机构：
[李慧敏] 武汉轻工大学生命科学与技术学院,湖北武汉 430023;[李慧敏] 自然资源部第三海洋研究所、自然资源部海洋生物遗传资源重点实验室,福建厦门 361005;[路瑶; 邵宗泽; 周梅先] 自然资源部第三海洋研究所;[杨江科] 武汉轻工大学
关键词：
海洋生物学;褐藻胶裂解酶;褐藻寡糖;克隆表达;酶学性质;聚古罗糖醛酸偏好性
摘要：
对从福建省东山湾沉积物样品中筛选到的菌株Vibrio sp. DS32的褐藻胶裂解酶基因vralg1进行克隆和异源表达,并对其酶学性质进行评估。以DS32基因组为模板,克隆褐藻胶裂解酶基因vralg1,构建了pET-vr...展开更多 对从福建省东山湾沉积物样品中筛选到的菌株Vibrio sp. DS32的褐藻胶裂解酶基因vralg1进行克隆和异源表达,并对其酶学性质进行评估。以DS32基因组为模板,克隆褐藻胶裂解酶基因vralg1,构建了pET-vralg1重组表达载体,并在大肠杆菌中实现了异源表达,对重组酶VRALG1的酶学性质、底物特异性和完全降解产物等进行了测定。结果表明:重组酶VRALG1最适温度为35℃,在5~50℃范围内相对酶活力达到80%以上,最适pH为6.5~7.5,在pH为6.0~9.0范围内保温1 h后相对酶活力在90%以上;重组酶VRALG1最大反应速率为5.919 mmol/(L·min),米氏常数为3.712 mmol/L,最适条件下比活力为5.874 U/mg;K+、Cs+、Na+、咪唑和乙醇对酶活性影响较小,5 mmol/L或50 mg/mL浓度下相对酶活力保持90%以上,EDTA对酶的抑制作用明显,1 mmol/L浓度下可使酶完全失活;重组酶VRALG1对海藻酸钠和聚古罗糖醛酸具有较高的降解活性,TLC分析显示产物主要为单糖、二糖和三糖混合物,结合底物特异性分析,推测重组酶VRALG1是具有明显聚古罗糖醛酸偏好性的内切型双功能褐藻胶裂解酶。本研究成功克隆了弧菌DS32中褐藻胶裂解酶基因并实现了其在大肠杆菌中的异源表达,所得重组酶VRALG1具有优良的海藻酸钠降解活性和明显的聚古罗糖醛酸偏好性,可以用于制备低聚合度的褐藻寡糖。收起
语种：
中文
展开
The Motion Paradigm of Pre-Dock Zearalenone Hydrolase Predictions with Molecular Dynamics and the Docking Phase with Umbrella Sampling
作者：
Hong, Xi-Zhi;Han, Zheng-Gang;Yang, Jiang-Ke;Liu, Yi-Han
期刊：
Molecules ,2023年28(11):4545- ISSN：1420-3049
通讯作者：
Yang, JK;Liu, YH
作者机构：
[Yang, Jiang-Ke; Han, Zheng-Gang; Hong, Xi-Zhi] Wuhan Polytech Univ, Coll Life Sci & Technol, Pilot Base Food Microbial Resources Utilizat Hubei, Wuhan 430024, Peoples R China.;[Liu, Yi-Han] Tianjin Univ Sci & Technol, Coll Biotechnol, Key Lab Ind Fermentat Microbiol, Minist Educ, Tianjin 300453, Peoples R China.
通讯机构：
[Yang, JK ] W;[Liu, YH ] T;Wuhan Polytech Univ, Coll Life Sci & Technol, Pilot Base Food Microbial Resources Utilizat Hubei, Wuhan 430024, Peoples R China.;Tianjin Univ Sci & Technol, Coll Biotechnol, Key Lab Ind Fermentat Microbiol, Minist Educ, Tianjin 300453, Peoples R China.
关键词：
hydrolase;mycotoxin;zearalenone;chemistry;metabolism;molecular dynamics;motion;Hydrolases;Molecular Dynamics Simulation;Motion;Mycotoxins;Zearalenone
摘要：
Zearalenone (ZEN) is one of the most prevalent estrogenic mycotoxins, is produced mainly by the Fusarium family of fungi, and poses a risk to the health of animals. Zearalenone hydrolase (ZHD) is an important enzyme capable of degrading ZEN into a non-toxic compound. Although previous research has investigated the catalytic mechanism of ZHD, information on its dynamic interaction with ZEN remains unknown. This study aimed to develop a pipeline for identifying the allosteric pathway of ZHD. Using an identity analysis, we identified hub genes whose sequences can generalize a set of sequences in a protein family. We then utilized a neural relational inference (NRI) model to identify the allosteric pathway of the protein throughout the entire molecular dynamics simulation. The production run lasted 1 microsecond, and we analyzed residues 139–222 for the allosteric pathway using the NRI model. We found that the cap domain of the protein opened up during catalysis, resembling a hemostatic tape. We used umbrella sampling to simulate the dynamic docking phase of the ligand–protein complex and found that the protein took on a square sandwich shape. Our energy analysis, using both molecular mechanics/Poisson–Boltzmann (Generalized-Born) surface area (MMPBSA) and Potential Mean Force (PMF) analysis, showed discrepancies, with scores of −8.45 kcal/mol and −1.95 kcal/mol, respectively. MMPBSA, however, obtained a similar score to that of a previous report. © 2023 by the authors.
语种：
英文
展开
通过理性设计提高南极假丝酵母脂肪酶B的温度稳定性
作者：
刘桂子;丁睿;胡国帅;杨江科
期刊：
生物技术 ,2022年32(03):284-290+296 ISSN：1004-311X
作者机构：
武汉轻工大学生命科学与技术学院,湖北武汉430023;[胡国帅; 刘桂子; 丁睿; 杨江科] 武汉轻工大学
关键词：
理性设计;温度稳定性;脂肪酶;毕赤酵母;酯化
摘要：
[目的]提高南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的温度稳定性及活性.[方法]基于CALB的结构信息进行了理性设计,最终获得脂肪酶基因calbTm,并构建了 pPICZαA-CALBTm重组表达质粒并实现了在毕赤酵母中的高效表达.[结果]突变体酶CALBTm在摇瓶发酵72 h后酶活达到600 U/mL,且在50℃下保温3 h后仍保留74.8％的酶活;此外,中间突变体酶Tm1和Tm2经摇瓶发酵后酶活分别为220 U/mL、170 U/mL,在50℃下保温3 h后酶活分别保留了 73.9％、10％.通过对蛋白结构的分析发现,引入疏水性强的氨基酸和增加蛋白质三维空间中的氢键数量,能维持蛋白质构象的稳定,进而提高了脂肪酶的温度稳定性.[结论]成功设计并获得一种新型高活性、耐高温的脂肪酶CALB,其酶活达到了 600 U/mL,与野生型CALB相比提高了 10倍,同时温度稳定性也显著提升,在50℃保温3 h后酶活保留了 74.8％,而野生型CALB在保温3 h后酶活仅保留5％.
语种：
中文
展开
海洋细菌粪产碱菌J481中DMS趋化受体的鉴定
作者：
赵丽君;谢珂;杨江科;雷磊
期刊：
微生物学报 ,2022年62(12):5029-5042 ISSN：0001-6209
作者机构：
[谢珂; 雷磊; 杨江科; 赵丽君] 武汉轻工大学生命科学与技术学院
关键词：
粪产碱菌J481;基因组挖掘;趋化作用;信号转导
摘要：
【目的】β-二甲基巯基丙酸（β-dimethylsufoniopropionate,DMSP）是海洋环境中重要的含硫有机化合物，会被海洋中的微生物裂解并释放出挥发性气体二甲基硫醚（dimethylsulfide,DMS）。该反应的生物学意义尚不明确，前人曾有少量研究表明DMSP可能是趋化效应物。本研究旨在鉴定DMSP趋化作用中的信号分子以及该过程中的趋化受体基因。【方法】挖掘出基因组中含有甲基趋化受体蛋白结构域（methyl-accepting chemotaxis protein,MCP）的全部基因，并预测趋化受体蛋白的结构特征及功能。通过同源重组构建所有趋化受体的缺失突变株，并通过软琼脂平板实验观察趋化表型确定DMS的趋化受体。【结果】对不同化合物DMSP、DMS、丙烯酸进行鉴别后，明确趋化信号分子为DMS。从粪产碱菌（Alcaligenes faecalis） J481基因组中一共挖掘到8个潜在编码趋化受体蛋白的基因，分别构建对应的缺失突变株，并通过实验筛选出D6I95＿17420基因为编码识别DMS的趋化受体蛋白基因。【结论】D6I95＿17420基因为编码DMS的趋化受体蛋白的基因，为之后进一步阐明DMS作为信号分子在细胞内的调控作用奠定了基础。
语种：
中文
展开
枯草芽孢杆菌漆酶BsCotA的定向进化及其在靛蓝脱色中的应用
作者：
张俊雄;乐琛;赵丽君;雷磊;杨江科
期刊：
生物技术 ,2022年32(4):409-414+426 ISSN：1004-311X
作者机构：
武汉轻工大学生命科学与技术学院,湖北武汉430023;[雷磊; 乐琛; 张俊雄; 赵丽君; 杨江科] 武汉轻工大学
关键词：
枯草芽孢杆菌;漆酶;定向进化;酶学性质;染料脱色
摘要：
[目的]获得枯草芽孢杆菌(Bacillus subtilis)CotA漆酶活性及热稳定性提高的突变体并用于靛蓝脱色.[方法]利用易错PCR技术筛选获得2株酶活提高的漆酶突变体A1、A2,测定酶学性质,并进行靛蓝脱色研究.[结果]突变体A1、A2的最适pH值均为5.5,最适温度均为70℃;A1、A2在pH值2～7范围内,孵育30 min,剩余酶活均在40％以上,相比野生型(35％以下)有明显提高;A1、A2的比酶活为74.31 U/mg和80.96 U/mg,相比野生型(27.04 U/mg)分别提高了 1.75倍、1.99倍;酶的三维结构分析显示,形成的4个突变位点中,1个突变位点通过氨基酸的替换,降低了主肽链的构象自由度,其余3个突变位点在漆酶的整体结构上引入了新的氢键;靛蓝染料脱色结果表明,3种漆酶对靛蓝的12 h脱色率均超过60％.[结论]筛选出来的漆酶突变体A1、A2,在pH值2～7范围内和65～80℃范围内的活性和稳定性比野生型显著提高,在靛蓝脱色等领域具有良好的应用基础.
语种：
中文
展开
米根霉脂肪酶的改造及其在废弃油脂转化中的应用
作者：
罗思曼;胡博洋;胡国帅;赵丽君;柳庆月;...
期刊：
生物化工 ,2022年8(3):9-12,35 ISSN：2096-0387
作者机构：
武汉轻工大学 生命科学与技术学院,湖北武汉 430023;[胡国帅; 柳庆月; 胡博洋; 赵丽君; 杨江科; 罗思曼] 武汉轻工大学
关键词：
米根霉;脂肪酶;分子改造;生物柴油
摘要：
利用液态脂肪酶将废弃油脂转换为清洁能源生物柴油，对环境保护以及工业生产具有实际意义与价值。通过分子改造米根霉脂肪酶（ROL），将61位上的丝氨酸突变成苏氨酸（S61T），将150位点上的谷氨酸胺突变为亮氨酸（G150L），提高其在毕赤酵母X-33中的表达，并利用高效的液态脂肪酶将废弃油脂转化为生物柴油。改造后的米根霉表达菌株在摇瓶条件下发酵酶活最高达到1 430 U/mL，酶活提高了5.1倍。
语种：
中文
展开
Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31
作者：
Ding, Rui;Xie, Huifang;Han, Zhenggang;Yang, Jiangke
期刊：
PROTEIN AND PEPTIDE LETTERS ,2022年29(8):692-701 ISSN：0929-8665
作者机构：
[Ding, Rui; Xie, Huifang; Yang, Jiangke; Han, Zhenggang] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.
关键词：
Auto-induction;Escherichia coli expression;glycoside hydrolase family 5;mannanase;mannooligosaccharides;Pichia pastoris expression.
摘要：
<jats:sec>
<jats:title>Background:</jats:title>
<jats:p>Mannans are the main components of hemicellulose in nature and serve as
the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their
functional characteristics are an important basis for mannan biotechnological applications.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Objective:</jats:title>
<jats:p>In this study, a putative mannanase gene (ManBs31) from the genome of the marine
bacterium Alteromonadaceae Bs31 was characterized.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Methods:</jats:title>
<jats:p>Amino acid sequence analysis and protein structural modeling were used to reveal the
molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced
using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the
enzymes were determined by reducing sugar assay and thin-layer chromatography.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Results:</jats:title>
<jats:p>Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a
catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding
domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and
pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity
up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain
modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and
ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides,
and no monosaccharide was generated. Structural analysis suggested that
ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Conclusion:</jats:title>
<jats:p>ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative
for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.</jats:p>
</jats:sec>
语种：
英文
展开
海洋微生物Cellulophaga algicola DSM 14237木聚糖酶的分子鉴定及酶学性质
作者：
丁睿;谢会芳;韩正刚;杨江科
期刊：
武汉轻工大学学报 ,2022年41(01):16-24 ISSN：2095-7386
作者机构：
[丁睿; 谢会芳; 韩正刚; 杨江科] 武汉轻工大学生命科学与技术学院
关键词：
海洋微生物;木聚糖酶;10家族糖苷水解酶;酶学性质;原核表达;亲和纯化;蛋白质三维结构模拟
摘要：
从海洋微生物中挖掘酶基因资源是获取新型工业酶的重要途径。从海洋微生物基因组资源库中发掘到一个来源于海洋微生物Cellulophaga algicola DSM 14237的木聚糖酶基因（Xyn14237）。通过氨基酸序列比对和三维结构模拟分析得知Xyn14237属于糖苷水解酶第10家族。将部分截短并密码子优化后的Xyn14237在大肠杆菌BL21内进行重组表达，通过镍柱亲和纯化获得重组Xyn14237,采用还原糖法测定其酶学性质。结果显示Xyn14237的最适催化温度和pH分别40℃和7.0。酶分子在pH 5～10之间稳定性较好，相对酶活均保持在90%以上。酶分子不耐热，当温度超过30℃时其活性急剧下降。以榉木木聚糖为底物，Xyn14237的最大反应速度为1627.31 U/mg,Km为3.15 mg/mL、kcat为975.98 s-1。薄层层析分析显示Xyn14237水解木聚糖释放出的产物为木三糖、木二糖及木糖。从分子和酶学性质层面上分析了来自海洋微生物C.algicola的第一个木聚糖酶基因。
语种：
中文
展开
疏棉状嗜热丝孢菌(Thermomyces lanuginosus)脂肪酶的理性设计提高其活性和温度稳定性
作者：
魏子翔;张柳群;雷磊;韩正刚;杨江科
期刊：
中国生物工程杂志 ,2021年41(Z1):63-69 ISSN：1671-8135
作者机构：
[雷磊; 魏子翔; 杨江科; 韩正刚; 张柳群] 武汉轻工大学生物与制药工程学院 武汉430023
关键词：
脂肪酶;理性设计;高活性;高温稳定性
摘要：
目的:通过对疏棉状嗜热丝孢菌（Thermomyces lanuginosus）脂肪酶的理性设计,获得高酶活与耐高温的脂肪酶品种,为脂肪酶在饲料、油脂加工和生物柴油等领域的应用奠定基础。方法:对脂肪酶典型结构域lid和loop区域的系统发育分析,找到候选的位点,理性设计并通过实验验证,获得脂肪酶活性和耐高温特性显著提高的脂肪酶重组子,并构建多拷贝载体,完成50L发酵罐中进行产酶能力评价。结果:经过设计的脂肪酶高温稳定性显著提升。其在80℃下放置12h后仍保留78.94%的酶活。脂肪酶重组子在50L发酵罐中发酵诱导168h,其上清液酶活达到29 000U/m L。结论:成功设计并获得了一种新型的高活性、耐高温的脂肪酶品种TLL,并实现了高效表达,为其产业化和工业应用奠定了基础。
语种：
中文
展开
High-level secretive expression of a novel achieved Talaromyces cellulolyticus endo-polygalacturonase in Pichia pastoris by improving gene dosage for hydrolysis of natural pectin
作者：
Peng, Xiao-Bo;Chen, Guang-Jun;Han, Zhen-Gang;Yang, Jiang-Ke*
期刊：
World Journal of Microbiology and Biotechnology ,2019年35(6):1-12 ISSN：0959-3993
通讯作者：
Yang, Jiang-Ke
作者机构：
[Chen, Guang-Jun; Peng, Xiao-Bo; Han, Zhen-Gang; Yang, Jiang-Ke] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
通讯机构：
[Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
关键词：
Pectinase;Gene dosage;Real time quantitative PCR;Thermal stable;Parameters optimization
摘要：
Abstract: Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides. Graphical abstract: [Figure not available: see fulltext.]. © 2019, Springer Nature B.V.
语种：
英文
展开
Molecular and Biochemical Characterization of a Bimodular Xylanase From Marinifilaceae Bacterium Strain SPP2
作者：
Han, Zhenggang;Shang-guan, Fang;Yang, Jiangke*
期刊：
Frontiers in Microbiology ,2019年10(JUL):457951 ISSN：1664-302X
通讯作者：
Yang, Jiangke
作者机构：
[Shang-guan, Fang; Yang, Jiangke; Han, Zhenggang] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Hubei, Peoples R China.
通讯机构：
[Yang, Jiangke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Hubei, Peoples R China.
关键词：
FN3 domain;glycoside hydrolase;Marinifilaceae bacterium;xylanase;Xylooligosaccharide
摘要：
In this study, the first xylantic enzyme from the family Marinifilaceae, XynSPP2, was identified from Marinifilaceae bacterium strain SPP2. Amino acid sequence analysis revealed that XynSPP2 is a rare Fn3-fused xylanase, consisting of a signal peptide, a fibronectin type-III domain (Fn3), and a C-terminal catalytic domain belonging to glycoside hydrolase family 10 (GH10). The catalytic domain shared 17-46% identities to those of biochemically characterized GH10 xylanases. Structural analysis revealed that the conserved asparagine and glutamine at the glycone -2/-3 subsite of GH10 xylanases are substituted by a tryptophan and a serine, respectively, in XynSPP2. Full-length XynSPP2 and its Fn3-deleted variant (XynSPP2δFn3) were overexpressed in Escherichia coli and purified by Ni-affinity chromatography. The optimum temperature and pH for both recombinant enzymes were 50°C and 6, respectively. The enzymes were stable under alkaline condition and at temperature lower than 50°C. With beechwood xylan as the substrate, XynSPP2 showed 2.8 times the catalytic efficiency of XynSPP2δFn3, indicating that the Fn3 module promotes xylanase activity. XynSPP2 was active toward xylooligosaccharides (XOSs) longer than xylotriose. Such a substrate preference can be explained by the unique -2/-3 subsite composition in the enzyme which provides new insight into subsite interaction within the GH10 family. XynSPP2 hydrolyzed beechwood xylan into small XOSs (xylotriose and xylotetraose as major products). No monosaccharide was detected by thin-layer chromatography which may be ascribed to putative transxylosylation activity of XynSPP2. Preferring long XOS substrate and lack of monosaccharide production suggest its potential in probiotic XOS manufacture. © 2007 - 2019 Frontiers Media S.A. All Rights Reserved.
语种：
英文
展开
尿黑酸双加氧酶基因工程菌的构建
作者：
肖路梅;杨江科;晁群芳;彭小波;马腾飞
期刊：
基因组学与应用生物学 ,2019年38(6):2627-2634 ISSN：1674-568X
作者机构：
新疆大学生命科学与技术学院,乌鲁木齐,830046;武汉轻工大学生物工程与制药学院,武汉,430023;[马腾飞; 肖路梅; 晁群芳] 新疆大学;[彭小波; 杨江科] 武汉轻工大学
关键词：
基因工程菌;组成型启动子;协同效应
摘要：
为了构建不同表达类型的尿黑酸双加氧酶(Dio6)基因工程菌,本研究利用Over-lap PCR技术使双加氧酶基因在组成型启动子(P2和P_(spoⅠ-Ⅱ))的控制下表达;利用高效液相色谱(HPLC)技术测定XJ-6、XJ-6+P_(T7)、 XJ-6+P2、XJ-6+P_(spoⅠ-Ⅱ)、P_(spoⅠ-Ⅱ)、P2、P_(T7)在7 d对芘的降解。研究结果表明:构建得到3种启动子控制表达的基因工程菌,协同效应研究表明工程菌在XJ-6的协助下可以在含芘的无机盐培养基中生长,并且组成型启动子工程菌的菌数远高于诱导型启动子工程菌的菌数。组成型启动子相较于诱导型启动子工程菌能更稳定表达外源双加氧酶基因,由于诱导型启动子工程菌需要诱导剂IPTG,并且表达双加氧酶基因会受到诱导剂浓度、诱导温度等条件的影响,所以在实际应用中组成型启动子工程菌优于诱导型启动子工程菌。
语种：
中文
展开
Gene dosage and coexpression with endoplasmic reticulum secretion-associated factors improved the secretory expression of α-galactosidase
作者：
Han, Zheng-Gang;Zhang, Ji-Wen;Jiang, Xiao-Fang;Yang, Jiang-Ke*
期刊：
Protein Expression and Purification ,2019年153:83-91 ISSN：1046-5928
通讯作者：
Yang, Jiang-Ke
作者机构：
[Han, Zheng-Gang; Zhang, Ji-Wen; Jiang, Xiao-Fang; Yang, Jiang-Ke] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
通讯机构：
[Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
关键词：
*Copy number;*Endoplasmic reticulum secretion associated factors;*Galactosidase;*Gene dose;*Quantitative PCR
摘要：
The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications.
语种：
英文
展开
Manno‐oligosaccharide preparation by the hydrolysis of konjac flour with a thermostable endo‐mannanase from Talaromyces cellulolyticus
作者：
Yang, J. -K.* ;Chen, Q. -C.;Zhou, B.;Wang, X. -J.;Liu, S. -Q.
期刊：
Journal of Applied Microbiology ,2019年127(2):520-532 ISSN：1364-5072
通讯作者：
Yang, J. -K.
作者机构：
[Yang, J. -K.; Chen, Q. -C.; Zhou, B.] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Hubei, Peoples R China.;[Liu, S. -Q.; Wang, X. -J.] Shandong Longda Bioprod Co Ltd Yishui Cty, Linyi, Shandong, Peoples R China.
通讯机构：
[Yang, J. -K.] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Hubei, Peoples R China.
关键词：
Enzyme activity;Genes;Hydrolysis;Degrees of polymerizations;Enzyme concentrations;Enzymes activity;Gene dosage;Hydrolysis conditions;Konjac flour;Mannanase;Manno-oligosaccharide;Optimal temperature;Protein contents;Oligosaccharides;beta mannosidase;endo mannanase;glucan synthase;mannose oligosaccharide;unclassified drug;(1-6)-alpha-glucomannan;endo-beta-mannosidase;mannan;mannosidase;oligosaccharide;bioreactor;carbohydrate;enzyme activity;fungus;genome;hydrolysis;polymerization;protein;temperature profile;amino acid sequence;Amorphophallus konjac;Article;DNA isolation;enzyme activity;flour;gene expression;high performance liquid chromatography;hydrolysis;konjac flour;nonhuman;phylogeny;polymerization;protein content;real time polymerase chain reaction;Talaromyces;Talaromyces cellulolyticus;thermostability;Amorphophallus;chemistry;enzyme stability;enzymology;flour;genetics;hydrolysis;metabolism;pH;Talaromyces;temperature;Amorphophallus konjac;Bryophyta;Fungi;Talaromyces;Amorphophallus;Enzyme Stability;Flour;Hydrogen-Ion Concentration;Hydrolysis;Mannans;Mannosidases;Oligosaccharides;Polymerization;Talaromyces;Temperature
摘要：
Aims: A thermostable endo-mannanase from the fungus Talaromyces cellulolyticus was identified to facilitate manno-oligosaccharide preparation from Konjac (Amorphophallus konjac) flour. Methods and Results: A putative endo-1,4-β-mannanase from the T. cellulolyticus was obtained and efficiently expressed by improving its gene dosage in the genome of the host. After cultivation in a bench-top bioreactor for about 120h, the protein content and enzyme activity of mannanase increased to 3·4gl−1 and 17500Uml−1 respectively. Enzymatic characterization showed that this enzyme has an optimal temperature of 80°C, optimal pH of 5·0. Under the optimized hydrolysis conditions of pH 5·0, 70°C, and an enzyme concentration of 200Ul−1 solution, this enzyme could efficiently hydrolyse 0·5% konjac flour into manno-oligosaccharides (MOSs) with the degree of polymerization range from 3 to 7. The possible mechanism by which the enzyme produced MOSs was also discussed. Conclusion: Talaromyces cellulolyticus endo-mannanase is thermostable and has a broad pH range adaptability. Method of improving the dosage of mannanase gene in the genome could realized its high-level impression. This enzyme could efficiently hydrolyse konjac flour into manno-oligosaccharide products. Significance and Impact of the Study: This study has enriched endo-mannanase resources, facilitated its bulk production and provided a strong reference for its application in manno-oligosaccharide preparation from the natural glucomannan of konjac flour. © 2019 The Society for Applied Microbiology
语种：
英文
展开
木聚糖酶基因XynB在毕赤酵母中的表达及酶学性质研究
作者：
马腾飞;杨江科;韩正刚;肖璐梅;晁群芳
期刊：
食品科技 ,2018年43(5):1-9 ISSN：1005-9989
作者机构：
新疆大学生命科学与技术学院,乌鲁木齐,830046;武汉轻工大学生物工程与制药学院,武汉,430023;[马腾飞; 肖璐梅; 晁群芳] 新疆大学;[韩正刚; 杨江科] 武汉轻工大学
关键词：
酸性;耐高温;木聚糖酶;酵母表达;酶学性质
摘要：
目的：将来源于Thermotoga maritima的酸性耐高温木聚糖酶基因Xyn B通过密码子优化整合到毕赤酵母GS115基因组上,构建高效表达木聚糖酶Xyn B的毕赤酵母工程菌并研究其酶学性质。方法：通过分子克隆手段构建目的基因重组质粒以及二拷贝重组质粒,并将重组质粒导入毕赤酵母进行发酵表达,用DNS法测定发酵液酶活,并对酵母工程菌分泌的木聚糖酶进行一系列的酶学性质研究。结果：木聚糖酶的最适p H和温度分别为6.0和60℃;在p H46之间,稳定性较好,相对比酶活均在80%以上;在60℃和70℃保温2 h残留酶活分别为80%和70%左右;Co（2＋）对木聚糖酶活性有较明显的促进作用,Fe（2＋）、K＋和Zn（2＋）对酶的活性有微弱的促进作用,而Cu（2＋）、Ca（2＋）、Mg（2＋）、Mn（2＋）、Na＋对木聚糖酶活性有不同程度的抑制作用。TLC检验该木聚糖酶可以将木聚糖水解为二糖和少量单糖。通过10 L发酵罐发酵,最大酶活达到了8000 U/m L。结论：木聚糖酶在毕赤酵母中实现了高效表达,并获得了木聚糖酶的最适反应条件等一系列酶学性质,该酶性质优良适合用于饲料添加剂。
语种：
中文
展开
Characterization of a novel cold-active xylanase from Luteimonas species
作者：
Han, Zhenggang;Shang-guan, Fang;Yang, Jiangke*
期刊：
World Journal of Microbiology and Biotechnology ,2018年34(8):1-13 ISSN：0959-3993
通讯作者：
Yang, Jiangke
作者机构：
[Shang-guan, Fang; Yang, Jiangke; Han, Zhenggang] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
通讯机构：
[Yang, Jiangke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
关键词：
Beechwood xylan;Cold-active;Luteimonas species;Halophilic;Xylanase
摘要：
Abstract: Biotechnological application of xylanolytic enzymes is normally hindered by their temperature-dependent catalytic property. To satisfy the industrial demands, xylanases that can perform catalysis under cold condition are attracting attention. In this study, the biochemical properties of a predicted xylanase (laXynA) encoded in the genome of marine bacterium Luteimonas abyssi XH031<sup>T</sup> were characterized. Structure modeling and structure-based sequence alignment indicated that laXynA belongs to the glycoside hydrolase family 10, and it is 20–26% identical to other characterized cold-active xylanases in the same family. Recombinant laXynA was successfully produced in Escherichia coli system by autoinduction and purified by Ni-affinity chromatography. The isolated enzyme showed an optimum temperature of 30 °C toward beechwood xylan and retained important percentage of optimal activity at low temperatures (64, 55, and 29% at 10, 5, and 0 °C, respectively). A remarkable characteristic of laXynA was extreme halophilicity as demonstrated by fourfold enhancement on xylanase activity at 0.5 M NaCl and by maintaining nearly 100% activity at 4 M NaCl. Thin layer chromatography analysis demonstrated that laXynA is an endo xylanase. This study is the first to report the over-expression and characterization of a cold-active xylanase from Luteimonas species. The enzymatic property revealed the cold-active nature of laXynA. The enzyme is a promising candidate in saline food processing application. Graphical abstract: [Figure not available: see fulltext.].<br/> ©2018, Springer Nature B.V.
语种：
英文
展开
Understanding the Positional Binding and Substrate Interaction of a Highly Thermostable GH10 Xylanase from Thermotoga maritima by Molecular Docking
作者：
Yang, Jiangke;Han, Zhenggang*
期刊：
Biomolecules ,2018年8(3):64 ISSN：2218-273X
通讯作者：
Han, Zhenggang
作者机构：
[Yang, Jiangke; Han, Zhenggang] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
通讯机构：
[Han, Zhenggang] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.
关键词：
AutoDock;Glycoside hydrolase family 10;Molecular docking;Xylanase;Xylooligosaccharide
摘要：
Glycoside hydrolase family 10 (GH10) xylanases are responsible for enzymatic cleavage of the internal glycosidic linkages of the xylan backbone, to generate xylooligosaccharides (XOS) and xyloses. The topologies of active-site cleft determine the substrate preferences and product profiles of xylanases. In this study, positional bindings and substrate interactions of TmxB, one of the most thermostable xylanases characterized from Thermotoga maritima to date, was investigated by docking simulations. XOS with backbone lengths of two to five (X2–X5) were docked into the active-site cleft of TmxB by AutoDock The modeled complex structures provided a series of snapshots of the interactions between XOS and TmxB. Changes in binding energy with the length of the XOS backbone indicated the existence of four effective subsites in TmxB. The interaction patterns at subsites −2 to +1 in TmxB were conserved among GH10 xylanases whereas those at distal aglycone subsite +2, consisting of the hydrogen bond network, was unique for TmxB. This work helps in obtaining an in-depth understanding of the substrate-binding property of TmxB and provides a basis for rational design of mutants with desired product profiles. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.
语种：
英文
展开
