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

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

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

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

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

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

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

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^T were characterized. Structure modeling and structure-based sequence alignment indicated that laXynA belongs to the glycoside hydrolase family 10, and it is 20&ndash;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&nbsp;&deg;C toward beechwood xylan and retained important percentage of optimal activity at low temperatures (64, 55, and 29% at 10, 5, and 0&nbsp;&deg;C, respectively). A remarkable characteristic of laXynA was extreme halophilicity as demonstrated by fourfold enhancement on xylanase activity at 0.5&nbsp;M NaCl and by maintaining nearly 100% activity at 4&nbsp;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/> &copy;2018, Springer Nature B.V.

语种： 英文

作者： Ding, Wentao;Cheng, Jian;Guo, Dan;Mao, Ling;Li, Jingwei;...

期刊： ACS Synthetic Biology,2018年7(12):2709-2714 ISSN：2161-5063

通讯作者： Jiang, Huifeng

作者机构： [Lu, Lina; Cheng, Jian; Jiang, Huifeng; Ding, Wentao; Guo, Dan] Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Key Lab Syst Microbial Biotechnol, Tianjin 300308, Peoples R China.;[Ding, Wentao] Beijing Univ Chem Technol, Beijing Adv Innovat Ctr Soft Matter Sci & Engn, Beijing 100029, Peoples R China.;[Mao, Ling; Yang, Jiangke] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Hubei, Peoples R China.;[Zhang, Yunxin; Li, Jingwei] Fudan Univ, Shanghai Key Lab Contemporary Appl Math, Sch Math Sci, Lab Math Nonlinear Sci,Ctr Computat Syst Biol, Shanghai 200433, Peoples R China.

通讯机构： [Jiang, Huifeng] C;Chinese Acad Sci, Tianjin Inst Ind Biotechnol, Key Lab Syst Microbial Biotechnol, Tianjin 300308, Peoples R China.

关键词： 5' untranslated region;Saccharomyces cerevisiae;directed evolution;translational regulation

摘要： The 5′ untranslated region (5′UTR) plays a key role in post-transcriptional regulation, but interaction between nucleotides and directed evolution of 5′UTRs as synthetic regulatory elements remain unclear. By constructing a library of synthesized random 5′UTRs of 24 nucleotides in Saccharomyces cerevisiae, we observed strong epistatic interactions among bases from different positions in the 5′UTR. Taking into account these base interactions, we constructed a mathematical model to predict protein abundance with a precision of R 2 = 0.60. On the basis of this model, we developed an approach to engineer 5′UTRs according to nucleotide sequence activity relationships (NuSAR), in which 5′UTRs were engineered stepwise through repeated cycles of backbone design, directed screening, and model reconstruction. After three rounds of NuSAR, the predictive accuracy of our model was improved to R 2 = 0.71, and a strong 5′UTR was obtained with 5-fold higher protein abundance than the starting 5′UTR. Our findings provide new insights into the mechanism of 5′UTR regulation and contribute to a new translational elements engineering approach in synthetic biology. © 2018 American Chemical Society.

语种： 英文

作者： Yang, Jiang-Ke*;Liang, Jian-Fang;Xiao, Lu-Mei;Yang, Yang;Chao, Qun-Fang

期刊： PLOS ONE,2018年13(9):e0203919 ISSN：1932-6203

通讯作者： Yang, Jiang-Ke

作者机构： [Yang, Jiang-Ke] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Hubei, Peoples R China.;[Chao, Qun-Fang; Liang, Jian-Fang; Yang, Yang; Xiao, Lu-Mei] Xinjiang Univ, Coll Life Sci & Technol, Urumqi, Xinjiang, Peoples R China.

通讯机构： [Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Hubei, Peoples R China.

关键词： Crude oil;Petroleum;Bacteria;Community ecology;Community structure;Ribosomal RNA;Gas chromatography-mass spectrometry;Soil ecology

摘要： The largely semi-deserted and deserted Dzungharian Basin sites in the northwest of China geologically represent an extension of the Paleozoic Kazakhstan Block and were once part of an independent continent. For reasons of overdevelopment and unreasonable operations during the process of exploitation and transportation, oil pollutants that were discharged into the soil environment caused serious pollution in this weak ecosystem. To explore the bacterial community composition in detail and their possible origination and potential during the natural attenuation of petroleum contaminants in this type of ecologic niche, GC-MS and high-throughput sequencing techniques were used to resolve the organic compounds and bacterial communities in vertical soil layers. The degradation of petroleum contaminants in semi-deserted and deserted soils mainly occurred in the layer at a depth of 45–55 cm. During this process, aromatic and heterocyclic compounds were significantly enriched in soils. The bacterial communities in this basin exhibited a distinct vertical stratification from the surface layer down to the bottom soil layer. Considering the interaction between the community composition and the geochemical properties, we speculate that the degradation of petroleum contaminants in this semi-deserted and deserted soil might represent a microorganism-mediated process and mainly occur in the deeper soil layer.

语种： 英文

作者： Yang, Jiang-Ke*;Xiong, Wei;Chen, Fang-Yuan;Xu, Li;Han, Zheng-Gang

期刊： PLOS ONE,2017年12(5):e0176444 ISSN：1932-6203

通讯作者： Yang, Jiang-Ke

作者机构： [Xu, Li; Han, Zheng-Gang; Chen, Fang-Yuan; Yang, Jiang-Ke; Xiong, Wei] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Peoples R China.

通讯机构： [Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan, Peoples R China.

关键词： Cellulose;Aromatic amino acids;Cell binding;Cell binding assay;Cellulases;Medical facies;Filter paper;Binding analysis

摘要： The cellulose binding domain (CBD) of cellulase binding to cellulosic materials is the initiation of a synergistic action on the enzymatic hydrolysis of the most abundant renewable biomass resources in nature. The binding of the CBD domain to cellulosic substrates generally relies on the interaction between the aromatic amino acids structurally located on the flat face of the CBD domain and the glucose rings of cellulose. In this study, we found the CBD domain of a newly cloned Penicillium crustosum endoglucanase EGL1, which was phylogenetically related to Aspergillus, Fusarium and Rhizopus, and divergent from the well-characterized Trichoderma reeseis cellulase CBD domain, contain two conserved aromatic amino acid-rich regions, Y451-Y452 and Y477-Y478-Y479, among which three amino acids Y451, Y477, and Y478 structurally sited on a flat face of this domain. Cellulose binding assays with green fluorescence protein as the marker, adsorption isotherm assays and an isothermal titration calorimetry assays revealed that although these three amino acids participated in this process, the Y451-Y452 appears to contribute more to the cellulose binding than Y477-Y478-Y479. Further glycine scanning mutagenesis and structural modelling revealed that the binding between CBD domain and cellulosic materials might be multi-amino-acids that participated in this process. The flexible poly-glucose molecule could contact Y451, Y477, and Y478 which form the contacting flat face of CBD domain as the typical model, some other amino acids in or outside the flat face might also participate in the interaction. Thus, it is possible that the conserved Y451-Y452 of CBD might have a higher chance of contacting the cellulosic substrates, contributing more to the affinity of CBD than the other amino acids.

语种： 英文

作者： Xiong, Wei;Yang, Jiang-Ke*;Chen, Fang-yuan;Han, Zheng-gang

期刊： Enzyme and Microbial Technology,2017年97:71-81 ISSN：0141-0229

通讯作者： Yang, Jiang-Ke

作者机构： [Han, Zheng-gang; Chen, Fang-yuan; Yang, Jiang-Ke; Xiong, Wei] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.

通讯机构： [Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.

关键词： Catalytic domain;Cellulase binding assay;Endoglucanase;Glycine scanning mutagenesis;Hydrophobic core

摘要： The cellulase-mediated degradation of cellulosic materials, which is initiated by endoglucanases by the random cleavage of the glycosidic bonds between glucose units to break long cellulose molecules into shorter ones, represents a major carbon flow in the global carbon cycle. The structure of a typical endoglucanase contains a classical (&alpha;/&beta;)<inf>8</inf> barrel fold catalytic domain, a linker region and a cellulose-binding domain. In this study, we found that both the full-length enzyme and the catalytic domain of endoglucanase EGL1 cloned from Penicillium crustosum strain 601 have CMCase and FPase activity. A cellulose-binding assay using green fluorescent protein as a marker further showed that the catalytic domain could also bind the cellulose substrate. The three-dimensional structure of the catalytic domain of EGL1 revealed that this cellulose substrate-binding capacity of the catalytic domain may come from the hydrophobic core formed by aromatic amino acids distributed in or outside the (&alpha;/&beta;)<inf>8</inf> barrel fold. A glycine scanning mutagenesis assay further found that the aromatic amino acids at the bottom of the barrel fold and those adjacent to the catalytic site significantly affect the cellulolytic activity and the cellulose binding affinity of the catalytic domain. Thus, it could be speculated that the aromatic amino acids in the bottom of the barrel fold might be the main contributors in the binding capacity of the catalytic domain with the cellulose substrate, and those distributed around the active sites on the top of the enzyme might participate in moving the cellulose substrate to the active site in the barrel fold or releasing the hydrolysis products.<br/> &copy;2016 Elsevier Inc.

语种： 英文

作者： 许力;熊炜;杨江科

期刊： 中国生物工程杂志,2016年36(2):43-50 ISSN：1671-8135

作者机构： [许力; 熊炜; 杨江科] 武汉轻工大学生物与制药工程学院, 武汉, 430023

关键词： 空肠弯曲杆菌;血红蛋白;生长能力;启动子;CO差式光谱

摘要： 细菌血红蛋白可增加细胞在低氧条件下的氧气利用率,提高细胞的增长速率。为提高E. coli在现有发酵水平下的生物量,获得新型的基因工程底盘细胞,基于空肠弯曲杆菌(Campylobacter jejuni)血红蛋白基因,比较分析了组成型和诱导型启动子对血红蛋白在E. coli基因工程菌中的表达状况及对E. coli生长的影响。首先合成了空肠弯曲杆菌血红蛋白基因chb,并让chb基因在诱导型启动子(PT7和Pvgh)和组成型启动子(P2和PspoI-II)控制下表达;随后,测定了由四种类型启动子控制的血红蛋白工程菌在摇瓶和蓝盖瓶中的生长状况。结果表明四种重组菌可以表达出有活性的血红蛋白,并可显著提高大肠杆菌的生长;在发酵罐中的试验也表明四种工程菌同样对细胞生长具有出促进作用;进一步比较分析了菌体密度、细胞鲜重和CO差式光谱值间的相互关系,讨论了四种类型启动子控制的基因工程菌在诱导表达和生长调控等方面的特点,并为在微氧和好氧等不同发酵条件下选用基因工程底盘细胞提供了参考。

语种： 中文

作者： 杨江科;程占冰

期刊： 微生物学报,2016年56(6):943-955 ISSN：0001-6209

作者机构： [杨江科; 程占冰] School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Hubei, Wuhan, 430023

关键词： eutrophic lake;community composition;spatial variation;principal coordinates analysis (PCoA);canonical correspondence analysis (CCA)

摘要： [Objective] Sediment bacteria are the important biological factors for remediating of eutrophic environments. To enrich our understanding of the bacteria communities in eutrophic urban lake sediments for better environment protection and pollution control in urban lake eco-systems, we resolved the composition of bacteria communities and their spatial variation in the sediments of a middle-size eutrophic urban lake, East Lake. [Methods] We used 16S rRNA gene RFLP and sequencing methods to generate the phylogeny information of the bacteria community, used principal coordinates analysis (PCoA) and canonical correspondence analysis (CCA) methods to resolve the relationship between East Lake and other lakes, and the relationship between environmental factors and the bacteria communities. [Results] Sediments inhabited 13 phyla and 2 unclassified clusters. PCoA further revealed that the bacteria communities in three sub-lakes of East Lake sediments were closely related to the communities in similar eutropic lake environments, and divergent from the hypereutrophic sub-lake Miao Lake, which was also found to inhabit a relative abundant amount of Thermogymnomonas-type archaea. CCA further revealed that the distribution of bacteria was closely correlated with the carbon, nitrogen and phosphate contents in the sediments. [Conclusion] The environment factors regulated the bacteria community composition and distribution. The results of this study providereference to the research, protection and pollution control on urban lake eco-systems.

语种： 英文

作者： 杨杨;杨江科;熊炜;梁建芳;段魏魏;...

期刊： 中国生物工程杂志,2016年36(5):59-67 ISSN：1671-8135

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;[杨杨; 梁建芳; 段魏魏; 晁群芳] 新疆大学生命科学与技术学院, 乌鲁木齐, 830046;[杨江科; 熊炜] 武汉轻工大学生物工程与制药学院, 武汉, 430023

关键词： 双加氧酶;纯化;苯环

摘要： 目的:从Aeromonas sp. XJ-6中克隆双加氧酶基因,初步探索该酶的功能,为芳香烃化合物的生物降解提供基因资源。方法:PCR扩增双加氧酶基因dio6,并实现该基因在大肠杆菌(Escherichia coli)中的诱导表达。产物经Ni-NTA柱纯化后,通过薄层层析(TLC)和HPLC检测双加氧酶dio6对Tyr的降解效果,再结合LC-MS检测降解产物,并分析其可能的降解途径。结果:Aeromonas sp. XJ-6双加氧酶基因dio6大小为1 194bp;通过金属鳌合亲和层析(MCAC)纯化后dio6表达产物的大小为44.9kDa。双加氧酶dio6对Tyr具有较强的降解作用。TLC和HPLC检测表明,在60μl酶量和30℃反应温度等条件下,Tyr降解较快;Mg2+、Ca2+略微抑制酶促反应,Mn2+、Zn2+、Cu2+、Fe2+、Ca2+促进底物降解,其中Mn2+对双加氧酶影响最大。LC-MS分析表明,在双加氧酶dio6作用下,Tyr被降解为延胡索酸。结论:Aeromonas sp. XJ-6双加氧酶dio6是一种苯环开环酶,为芳香烃化合物的生物降解提供了良好的基因资源。

语种： 中文

作者： 梁建芳;杨江科;杨杨;晁群芳;殷亚兰;...

期刊： 微生物学报,2016年56(8):1301-1313 ISSN：0001-6209

作者机构： 新疆大学生命科学与技术学院, 新疆, 乌鲁木齐, 830046;武汉轻工大学生物工程与制药学院, 湖北, 武汉, 430023;[梁建芳; 杨杨; 晁群芳; 殷亚兰; 赵亚光] 新疆大学生命科学与技术学院, 新疆, 乌鲁木齐, 830046;[杨江科] 武汉轻工大学生物工程与制药学院, 湖北, 武汉, 430023

关键词： 石油污染土壤;RFLP分析;细菌多样性;系统发育分析;CCA分析

摘要： 【目的】以16S rRNA为分子标记,探讨克拉玛依油田石油污染土壤中细菌群落多样性和系统发育,并分析环境因子对群落分布的影响,为生物降解石油污染物提供理论基础。【方法】在克拉玛依油田分别采集深度为5、20、50 cm的石油污染土壤样品,测定环境参数;提取石油污染土壤细菌群落基因组DNA,分别构建3个土层细菌16S rRNA基因文库,利用限制性片段长度多态性分析(Restriction fragment length polymorphisms RFLP)技术初步分群,确定各文库中的代表菌株并测定16S rRNA基因序列;利用软件Biodap计算各群落多样性和丰富度指数,以Neighbor-Joining法构建3个土层细菌的系统发育树;运用软件CANOCO 4.5结合不同样品环境因子的差异进行典型对应分析(CCA),并探讨了环境因子对细菌多样性的影响。【结果】环境参数结果表明20 cm土层总磷(TP)、总氮(TN)含量最低,50 cm含量最高;5 cm土层中有机碳(TOC)含量最高,50 cm含量最低。基于16S rRNA序列的生物多样性和物种丰富度指数表明20 cm土层生物多样性和丰富度指数较高,而50 cm土层各项指数均较低。各土层供试序列RFLP聚类分析表明,克拉玛依油田石油污染土壤细菌种群具有丰富的多样性。Neighbor-Joining构建的系统发育分析表明,石油污染土壤被分为5个类群(I–V),分别为变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicute)、拟杆菌门(Bacteroidetes)、浮霉状菌门(Planctomycetes),其中群Ⅰ占78.57%,广泛分布于不同的生态环境;其中来自5 cm土层代表菌的69.23%分布于群Ⅰ。CCA分析结果显示TN、TP和TOC对大部分细菌影响较大;TOC含量对Pseudomonas影响明显。【结论】克拉玛依油田石油污染土壤细菌群落具有丰富的多样性;环境因子是影响石油污染土壤细菌群落空间分布的重要因素。

语种： 中文

作者： Yang, Jiang-Ke*;Zhang, Ji-Wen;Mao, Lin;You, Xun;Chen, Guang-Jun

期刊： Journal of Molecular Catalysis B: Enzymatic,2016年134:225-232 ISSN：1381-1177

通讯作者： Yang, Jiang-Ke

作者机构： [You, Xun; Chen, Guang-Jun; Zhang, Ji-Wen; Yang, Jiang-Ke; Mao, Lin] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.

通讯机构： [Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.

关键词： Gene expression;Genes;Genetic engineering;Polysaccharides;Genetic modifications;High level expression;Inulinase;Oligo-fructose;Parameter optimization;Cloning;edetic acid;endo inulinase;fructose oligosaccharide;glycosidase;inulin;inulinase;methanol;proteinase;unclassified drug;amino acid sequence;Article;Aspergillus;bioreactor;controlled study;DNA modification;enzyme activity;enzyme stability;enzyme substrate complex;enzyme synthesis;Fusarium oxysporum;heterologous expression;high performance liquid chromatography;Jerusalem artichoke;Kluyveromyces;Komagataella pastoris;nonhuman;Penicillium;phylogeny;Pichia;polymerization;protein content;reverse transcription polymerase chain reaction;Saccharomyces;substrate concentration;thermostability

摘要： The enzymatic hydrolyzation of inulin by endo-inulinase to produce oligofructoses, a new type of food additive and health product, is a promising, “green”, and environmentally friendly technique. To identify novel genetic sources of endo-inulinase genes and facilitate their industrial application for oligofructose production, we cloned an endo-inulinase gene from a Fusarium oxysporum strain and achieved high-level expression in the genetically modified Pichia pastoris strain in a pilot-scale bioreactor by using strategies such as C-terminal truncation and mutagenesis of protease-sensitive sites. We then optimized the parameters of the inulinase reaction and the amount of enzyme used to inulin hydrolysis and oligofructose production. The results of this study should facilitate the bulk production of inulinase and provide a reference for the industrial production of oligofructose from inulin. © 2016 Elsevier B.V.

语种： 英文

作者： Guang-Jun Chen;Jiang-Ke Yang;Xiao-Bo Peng;Jing-Ren He

期刊： Journal of Molecular Catalysis B: Enzymatic,2016年133(Supplement 1):S543-S551 ISSN：1381-1177

通讯作者： Yang, Jiang-Ke(jiangke.yang@gmail.com)

作者机构： [Chen G.-J.; Yang J.-K.; Peng X.-B.] College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, 430023, China;[He J.-R.] College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan, 430023, China

通讯机构： [Jiang-Ke Yang] C;College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China

关键词： Aspergillus;Cell membranes;Fructose;Gene expression;Polysaccharides;Codon optimization;Endoplasmic reticulum;Enzymatic preparation;Incubation temperatures;Inducible expression;Inulin;Inulinase;State-of-the-art techniques;Enzymatic hydrolysis;calcium ion;carbohydrate;copper ion;cyclodextrin;dissolved oxygen;fructose;glycerol;inulin;inulinase;iron;magnesium ion;manganese;mannitol;messenger RNA;microcrystalline cellulose;monosaccharide;polygalacturonic acid;raffinose;sucrose;zinc ion;Article;Aspergillus;biomass;codon usage;comparative study;controlled study;endoplasmic reticulum;enzyme activity;enzyme chemistry;fresh weight;gene dosage;high performance liquid chromatography;hydrolysis;incubation temperature;nonhuman;nucleotide sequence;pH;Pichia;protein content;protein expression;protein secondary structure;reaction time;syrup;temperature sensitivity

摘要： Inulin is a type of fructose polymer that is commonly present in plants as a storage carbohydrate. Enzymatic hydrolysis of inulin via exo-inulinase to produce fructose is an efficient, green and state-of-the-art technique. To achieve the high-level secretory expression of inulinase and to realize enzymatic preparation of fructose syrup from inulin, an Aspergillus exo-inulinase gene inu was codon-optimized and co-expressed with the endoplasmic reticulum secretion protein in Pichia cells. After inducible expression in a 500-L pilot scale bioreactor, the inulinase activity of the recombinant strains reached 10,480 U/mL of cultivation broth. Next, according to the determined enzymatic characteristics of inulinase INU, we optimized the parameters for inulinase to hydrolyse inulin. Under the optimal condition of the enzyme/inulin ratio of 5000 U/g, 15% substrate and an incubation temperature of 50 °C for 4 h, the hydrolysis ratio of inulin reached 100%. The hydrolysis products of inulin contain two components, 95% fructose, and 5% glucose. This study has fulfilled the scaled-up production of inulinase and facilitated its industrial application for enzymatic preparation of fructose from inulin. © 2017 Elsevier B.V.

语种： 英文

作者： Dai, Jianghong*;Qu, Hong;Yu, Zhisheng;Yang, Jiangke;Zhang, Hongxun

期刊： Journal of Theoretical Biology,2015年379:59-65 ISSN：0022-5193

通讯作者： Dai, Jianghong

作者机构： [Dai, Jianghong; Yang, Jiangke] Wuhan Polytech Univ, Sch Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.;[Dai, Jianghong; Yu, Zhisheng; Zhang, Hongxun] Univ Chinese Acad Sci, Coll Resources & Environm, Beijing 100049, Peoples R China.;[Qu, Hong] Peking Univ, Coll Life Sci, State Key Lab Prot & Plant Gene Res, Ctr Bioinformat, Beijing 100871, Peoples R China.;[Dai, Jianghong] Wuhan Polytech Univ, Sch Biol & Pharmaceut Engn, 68 Xuefu Rd S, Wuhan 430023, Peoples R China.

通讯机构： [Dai, Jianghong] W;Wuhan Polytech Univ, Sch Biol & Pharmaceut Engn, 68 Xuefu Rd S, Wuhan 430023, Peoples R China.

关键词： 1,6 anhydro beta dextro glucosamine;1,6 anhydro n acetylglucosamine;1,6 anhydro n acetylmuramic acid kinase;aspartic acid;glucosamine;levoglucosan kinase;phosphotransferase;unclassified drug;fungal protein;phosphotransferase;chitin;coliform bacterium;fungus;homology;hydrolysis;metabolism;phylogenetics;Article;computational analysis;enzyme analysis;fungal cell wall;hydrolysis;Lipomyces;Lipomyces starkeyi;molecular docking;phylogenetic tree;priority journal;sequence alignment;sequence analysis;cell wall;enzymology;genetics;metabolism;phylogeny;procedures;Escherichia coli;Fungi;Lipomyces starkeyi;Cell Wall;Fungal Proteins;Lipomyces;Phosphotransferases;Phylogeny;Sequence Analysis, Protein

摘要： 1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is the unique enzyme that marks the recycling of the cell wall of Escherichia coli. Here, 81 fungal AnmK-like kinase sequences from 57 fungal species were searched in the NCBI database and a phylogenetic tree was constructed. The three-dimensional structure of an AnmK-like kinase, levoglucosan kinase (LGK) of the yeast Lipomyces starkeyi, was modeled; molecular docking revealed that AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively. Analysis suggests that 1,6-anhydro-N-acetylglucosamine (anhGlcNAc) and/or 1,6-anhydro-β-d-glucosamine (anhGlcN) would be the appropriate substrates of AnmK-like kinases. Also, the counterparts of other characteristic enzymes of cell wall recycling of bacteria were found in fungi. Taken together, it is proposed that a putative recycling of anhGlcNAc/anhGlcN, which is associated with the hydrolysis of cell walls, exists in fungi. This computational analysis will provide new insights into the metabolism of fungal cell walls. © 2015 Elsevier Ltd.

语种： 英文

作者： Zhou, Wen-Jing;Yang, Jiang-Ke*;Mao, Lin;Miao, Li-Hong

期刊： Enzyme and Microbial Technology,2015年71:66-72 ISSN：0141-0229

通讯作者： Yang, Jiang-Ke

作者机构： [Miao, Li-Hong; Zhou, Wen-Jing; Yang, Jiang-Ke; Mao, Lin] Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.

通讯机构： [Yang, Jiang-Ke] W;Wuhan Polytech Univ, Coll Biol & Pharmaceut Engn, Wuhan 430023, Peoples R China.

关键词： De novo design;Fermentation;Lipase;Pichiapink;Synthesis

摘要： Lipase (EC 3.1.1.3) stands amongst the most important and promising biocatalysts for industrial applications. In this study, in order to realize a high-level expression of the Yarrowia lipolytica lipase gene in Pichia pastoris, we optimized the codon of LIP2 by de novo gene design and synthesis, which significantly improved the lipase expression when compared to the native lip2 gene. We also comparatively analyzed the effects of the promoter types (PAOX1 and PFLD1) and the Pichia expression systems, including the newly developed PichiaPink system, on lipase production and obtained the optimal recombinants. Bench-top scale fermentation studies indicated that the recombinant carrying the codon-optimized lipase gene syn-lip under the control of promoter PAOX1 has a significantly higher lipase production capacity in the fermenter than other types of recombinants. After undergoing methanol inducible expression for 96h, the wet cell weight of Pichia, the lipase activity and the protein content in the fermentation broth reached their highest values of 262g/L, 38,500U/mL and 2.82g/L, respectively. This study has not only greatly facilitated the bioapplication of lipase in industrial fields but the strategies utilized, such as de novo gene design and synthesis, the comparative analysis among promoters and different generations of Pichia expression systems will also be useful as references for future work in this field. © 2014 Elsevier Inc.

语种： 英文