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Synergistic effects of cellobiose dehydrogenase and manganese-dependent peroxidases during lignin degradation

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Chinese Science Bulletin 2001年第23期46卷 1956-1962页

作者： HUANG Feng FANG Jing LU Xuemei GAO Peiji CHEN Jiaxiang State Key Laboratory of Microbial Technology Shandong UniversityJinan 250100China State Key Lab of Pulp and Paper Engineering South China University of TechnologyGuangzhou 510641China

The synergistic effects of cellobiose dehydro-genase (CDH) and manganese-dependent peroxidases (MnP) on the degradation of kraft pulp cellulolytic enzyme lignin (CEL) were investigated. Addition of CDH significantly increased the amount of water-soluble products reduced from CEL by MnP. CDH facilitated the reduction of the contents of methoxyl, carboxyl, phenolic hydroxyl and total hydroxyl groups of CEL by MnP. 1H-NMR analysis showed that addition of CDH also decreased further the amount of protons of CEL degraded by MnP. The results proved for the first time that CDH could promote degradation of lignin by MnP and suggest that CDH could not only promote degradation of cellulose but also is an important part of the lignin biodeg-radation system.

关键词： cellobiose dehydrogenase manganese-dependent per- oxidase lignin degradation.

Lactate dehydrogenase:An important molecule involved in acetamizuril action against Eimeria tenella

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Journal of Integrative Agriculture 2020年第5期19卷 1332-1339页

作者： LIU Li-li FEI Chen-zhong DONG Hui ZHANG Ke-yu Fu Jian-jun LI Tao XUE Fei-qun Key Laboratory of Animal Parasitology Ministry of Agriculture/Shanghai Veterinary Research InstituteChinese Academy of Agricultural SciencesShanghai 200241P.R.China East China University of Science and Technology Shanghai 200237P.R.China

Lactate dehydrogenase(LDH),a vital enzyme in anaerobic glycolysis,is closely associated with the survival of *** studies of some parasites have shown that LDH exhibits unique physicochemical properties and molecular structures and may be an ideal potential target for diagnosis and drug *** this study,we aimed to investigate the effects of acetamizuril,a novel anticoccidial compound,on LDH in the second-generation merozoites of Eimeria tenella(mz-LDH).Quantitative real-time PCR,Western blot,immunofluorescence and enzyme activity assays were each applied to detect the changes of *** results indicated that the mRNA and protein levels of mz-LDH were reduced upon acetamizuril *** of mz-LDH demonstrated that considerable amount of mz-LDH was distributed around or in the nuclei of second-generation merozoites within the untreated group;in contrast,the acetamizuril-treated group had very low level of ***,LDH enzyme activity assay suggested that a decreased LDH enzyme activity in both cytoplasm and nucleus of merozoites in the acetamizuril-treated ***,the induced apoptosis in second-generation merozoites by the acetamizuril was evaluated by detecting caspase 3 activity,and acetamizuril was found to significantly increase caspase 3 *** above findings show that LDH may play an important role in the mediating the activity of acetamizuril against coccidiosis,and further investigation into this aspect might contribute to new light on the pathogenesis of *** during its interaction with acetamizuril.

关键词： acetamizuril lactate dehydrogenase E.tenella second-generation merozoites

Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6×His tag in Pichia pastoris

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Science China(Life Sciences) 2009年第10期52卷 935-941页

作者： ZHAO YuFeng1,2, LEI MingKe2, WU YuanXin2, ZHANG ZiSheng3 & WANG CunWen2 1 State key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agriculture University, Wuhan 430070, China 2 Key Laboratory for Green Chemical Processes of the Ministry of Education, Hubei Key Lab of Novel Reactor & Green Chemical Technology, School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430074, China 3 Department of Chemical Engineering, University of Ottawa, Ottawa, Canada K1N 6N5 1. State key Laboratory of Agricultural Microbiology College of Life Science and Technology Huazhong Agriculture University Wuhan 430070 China 2. Key Laboratory for Green Chemical Processes of the Ministry of Education Hubei Key Lab of Novel Reactor & Green Chemical Technology School of Chemical Engineering and Pharmacy Wuhan Institute of Technology Wuhan 430074 China 3. Department of Chemical Engineering University of Ottawa Ottawa Canada K1N 6N5

Human mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) catalyzes the oxidation of acetaldehyde to acetic acid. Therefore, ALDH2 has therapeutic potential in detoxification of acetaldehyde. Further-more, ALDH2 catalyzes nitroglycerin to nitrate and 1, 2-glyceryldinitrate during therapy for angina pectoris, myocardial infarction, and heart failure. Large quantities of ALDH2 will be needed for potential clinical practice. In this study, Pichia pastoris was used as a platform for expression of human ALDH2. Based on the ALDH2*1 cDNA sequence, we designed ALDH2 cDNA by choosing the P. pastoris preferred codons and by decreasing the G + C content level. The sequence was synthesized using the overlap extension PCR method. The cDNA and 6×His tags were subcloned into the plasmid pPIC9K. The recombinant protein was expressed in P. pastoris GS115 and purified using Ni2+-Sepharose affinity chromatography. The amount of secreted protein in the culture was 80 mg/L in shake-flask cultivation and 260 mg/L in high-density bioreactor fermentation. Secreted ALDH2 was easily purified from the culture supernatant by using Ni2+-Sepharose affinity chromatography. After purification of the fermentation supernatant, the enzyme had a specific activity of 1.2 U/mg protein. The yield was about 16 mg/L in a shake flask culture of P. pastoris GS115 which contained the original human ALDH2*1 cDNA.

关键词： acetaldehyde dehydrogenase codon optimization overlap extension PCR Pichia pastoris.

SPECTRA PROPERTIES OF RAT LIVER MITOCHONDRIAL CHOLINE dehydrogenase

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Science China Chemistry 1990年第8期33卷 955-963页

作者：裘之华林其谁 Shanghai Institute of Biochemistry Academia Sinica Shanghai 200031 PRC

Choline dehydrogenase contains the prosthetic group FAD, non-haem iron and acid labile sulfur. However, the absorption spectra of the purified enzyme do not change after adding substrate. The reduced absorption spectra of choline dehydrogenase can only be determined after the addition of dithionite. Those choline dehydrogenases situated in the mitochondrial inner membrane can be reduced by substrate and exist in the reduced state. When cholate was used to solubilize the substrate-reduced choline dehydrogenase, the reduced spectra will gradually disappear. However, if solubilization is carried out under anaerobic conditions, the reduced spectra can be retained, suggesting that the solubilized choline dehydrogenase can use oxygen as an acceptor.

关键词： mitochondria choline dehydrogenase flavin adenine dinucleotide(FAD)

Cloning, expression and protective immunity evalua-tion of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

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Science China(Life Sciences) 2007年第2期50卷 221-227页

作者： YU JunLong, WANG ShiPing, LI WenKai, DAI Gan, XU ShaoRui, HE Zhuo, PENG XianChu, ZHOU SongHua & LIU XueQin Department of Parasitology, Xiangya School of Medicine, Central South University, Changsha 410078, China 1. Department of Parasitology Xiangya School of Medicine Central South University Changsha 410078 China

1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokary- otic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (posi- tive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also sug- gested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.

关键词： Schistosoma japonicum iron-sulfur protein of succinate dehydrogenase cloning expression protective immunity

Structure of D-glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor in a tetragonal crystal form

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Science China(Life Sciences) 2000年第1期43卷 96-104页

作者：沈月全宋时英林政炯 National Laboratory of Biomacromolecules Institute of Biophysics Chinese Academy of Sciences Beijing 100101China

D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.

关键词： D-glyceraldehyde-3-phosphate dehydrogenase allosteric enzyme and crystal structure.

Identification and bioinformatics analysis of lactate dehydrogenase genes from Echinococcus granulosus

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Asian Pacific Journal of Tropical Medicine 2010年第10期3卷 757-761页

作者： Gang Lu Yajun Lu Lihua Li Lixian Wu Zhigang Fan Dazhong Shi Hu Wang Xiumin Han Department of Pathogen Biology Hainan Medical University Qinghai Provincial Institute for Endemic Disease Prevention and Control

Objective:To identify full length cDNA sequence of lactate dehydrogenase(LDH) from adult Echinococcus granulosus(***) and to predict the structure and function of its encoding protein using bioinformatics ***:With the help of NCBI,EMBI, Expasy and other online sites,the open reading frame(ORF),conserved domain,physical and chemical parameters,signal peptide,epitope,topological structures of the protein sequences were predicted and a homology tertiary structure model was created:Vector NT1 software was used for sequence alignment,phylogenetic tree construction and tertiary structure prediction. Results:The target sequence was 1 233 bp length with a 996 bp biggest ORF encoding 331 amino acids protein with typical L-LDH conserved *** was confirmed as full length cDNA of LDH from *** and named as EgLDH(GenBank accession number:HM748917).The predicted molecular weight and isoelectric point of the deduced protein were 3 5516.2Da and 6.32 *** with LDHs from Taenia solium,Taenia saginata asiatica,Spirometra *** japonicum,Clonorchis sinensis and human,it showed similarity of 86% ,85% ,55% ,58% ,58% and 53% ,*** contained 3 putative transmembrane regions and 4 major epitopes(54aa-59aa.81aa-87aa,97aa-102aa,307aa-313aa),the latter were significant different from the corresponding regions of human *** addition,some NAD and substrate binding sites located on epitopes 54aa-59aa and 97aa-102aa,*** structure prediction showed that 3 key catalytic residues 105R,165D and 192H forming a catalytic center near the epitope 97aa-102aa,most NAD and substrate binding sites located around the ***:The full length cDNA sequences of EgLDH were *** encoded a putative transmembrane protein which might be an ideal target molecule for vaccine and drugs.

关键词： Echinococcus granulosus Lactate dehydrogenase Bioinformatic method Structure Function

Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

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Science China(Life Sciences) 2000年第3期43卷 254-264页

作者：朱冰俞冠翘朱家璧沈善炯 Shanghai Institute of Plant Physiology Chinese Academy of Sciences Shanghai 200032 China

The gdhA genes of IRC-3 GDH strain and IRC-8 GDH+ strain were cloned, and they both successfully complemented the nutritional lesion of an E. coli glutamate auxotroph, Q100 GDH". However, the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3. The gdhA genes of the wild type and mutant origin were sequenced separately. No nucleotide difference was detected between them. Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant. Additionally, no GDH inhibitor was found in the wild type strain IRC-3. It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression. Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the family I -type hexameric protein, while the GDH of Bacillus subtilis belongs to family II.

关键词： Bacillus licheniformis glutamate dehydrogenase gdhA gene.

Research of 1,3-Dihydroxyacetone Production by Overexpressing Glycerol Transporter and Glycerol dehydrogenase

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Transactions of Tianjin University 2019年第5期25卷 549-558页

作者： Jinxiu Tan Xiaona Yang Wenyu Lu School of Chemical Engineering and Technology Tianjin UniversityTianjin 300072China Key Laboratory of System Bioengineering (Tianjin University) Ministry of EducationTianjin 300072China Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) SynBio Research PlatformTianjin 300072China

1,3-Dihydroxyacetone (DHA), a natural ketose, is widely used in the chemical, cosmetic, and pharmaceutical industries. The current method for DHA production is Gluconobacter oxydans ( G. oxydans ) fermentation, but the high concentration of glycerol in the fermentation broth inhibits cells growth. To overcome this obstacle, in this study, we overexpressed the glycerol transporter (GlpFp) by the use of promoters P tufB , P gmr , P glp1 , and P glp2 in G. oxydans 621H. The results show that the glycerol tolerances of strains overexpressing G lpF were all much better than that of the control strain. The glycerol dehydrogenase gene (G dh) was overexpressed by the promoters P tufB and P gdh , which increased the DHA titer by 12.7% compared with that of the control group. When G lpF and Gdh genes were co-overexpressed in G. oxydans 621H, the OD600 value of the engineered strains all increased, but the DHA titers decreased in di erent degrees, as compared with strains that overexpressed only G dh . This study provides a reference for future research on DHA production.

关键词： G. oxydans 621H 1,3-dihydroxyacetone Glycerol Glycerol transporter Glycerol dehydrogenase