Development of a whole-cell biosensor for detecting organophosphorus pesticide methyl parathion in the farmland soil / 生物工程学报

The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21-6.66 µg/L and a linear range of 10-10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.

Genotyping Characteristics of Human Fecal Escherichia coli and Their Association with Multidrug Resistance in Miyun District, Beijing / 生物医学与环境科学（英文）

OBJECTIVE@#To explore the genotyping characteristics of human fecal Escherichia coli( E. coli) and the relationships between antibiotic resistance genes (ARGs) and multidrug resistance (MDR) of E. coli in Miyun District, Beijing, an area with high incidence of infectious diarrheal cases but no related data.@*METHODS@#Over a period of 3 years, 94 E. coli strains were isolated from fecal samples collected from Miyun District Hospital, a surveillance hospital of the National Pathogen Identification Network. The antibiotic susceptibility of the isolates was determined by the broth microdilution method. ARGs, multilocus sequence typing (MLST), and polymorphism trees were analyzed using whole-genome sequencing data (WGS).@*RESULTS@#This study revealed that 68.09% of the isolates had MDR, prevalent and distributed in different clades, with a relatively high rate and low pathogenicity. There was no difference in MDR between the diarrheal (49/70) and healthy groups (15/24).@*CONCLUSION@#We developed a random forest (RF) prediction model of TEM.1 + baeR + mphA + mphB + QnrS1 + AAC.3-IId to identify MDR status, highlighting its potential for early resistance identification. The causes of MDR are likely mobile units transmitting the ARGs. In the future, we will continue to strengthen the monitoring of ARGs and MDR, and increase the number of strains to further verify the accuracy of the MDR markers.

Immobilization of mutated xylanase from Neocallimastix patriciarum in E. coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E. coli e aplicação para polpa kraft

Abstract As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Resumo Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Analysis of endogenous plasmids in Lacticaseibacillus paracasei ZY-1 and development of expression vectors / 生物工程学报

The construction of efficient and stable Lactobacillus expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from Lacticaseibacillus paracasei ZY-1 and subjected to functional analysis. The Escherichia coli-Lactobacillus shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon rep from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene cat from pNZ5319 and the replicon ori from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter Pldh3 of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into Lacticaseibacillus, and the transformation efficiency of pLPZ4N (5.23×102-8.93×102 CFU/μg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into L. paracasei S-NB. The β-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E-lacG constructed with Pldh3 as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of Lacticaseibacillus strains.

Construction of "lock-key" biological living material based on double engineered bacteria and its application on intestinal retention in vivo / 生物工程学报

At present, the research of biological living materials mainly focuses on applications in vitro, such as using a single bacterial strain to produce biofilm and water plastics. However, due to the small volume of a single strain, it is easy to escape when used in vivo, resulting in poor retention. In order to solve this problem, this study used the surface display system (Neae) of Escherichia coli to display SpyTag and SpyCatcher on the surface of two strains, respectively, and constructed a double bacteria "lock-key" type biological living material production system. Through this force, the two strains are cross-linked in situ to form a grid-like aggregate, which can stay in the intestinal tract for a longer time. The in vitro experiment results showed that the two strains would deposit after mixing for several minutes. In addition, confocal imaging and microfluidic platform results further proved the adhesion effect of the dual bacteria system in the flow state. Finally, in order to verify the feasibility of the dual bacteria system in vivo, mice were orally administrated by bacteria A (p15A-Neae-SpyTag/sfGFP) and bacteria B (p15A-Neae-SpyCatcher/mCherry) for three consecutive days, and then intestinal tissues were collected for frozen section staining. The in vivo results showed that the two bacteria system could be more detained in the intestinal tract of mice compared with the non-combined strains, which laid a foundation for further application of biological living materials in vivo.

Immobilizing engineered Escherichia coli cells into zeolitic imidazolate framework 8 for efficient biosynthesis of Ala-Gln / 生物工程学报

The α-amino acid ester acyltransferase (SAET) from Sphingobacterium siyangensis is one of the enzymes with the highest catalytic ability for the biosynthesis of l-alanyl-l-glutamine (Ala-Gln) with unprotected l-alanine methylester and l-glutamine. To improve the catalytic performance of SAET, a one-step method was used to rapidly prepare the immobilized cells (SAET@ZIF-8) in the aqueous system. The engineered Escherichia coli (E. coli) expressing SAET was encapsulated into the imidazole framework structure of metal organic zeolite (ZIF-8). Subsequently, the obtained SAET@ZIF-8 was characterized, and the catalytic activity, reusability and storage stability were also investigated. Results showed that the morphology of the prepared SAET@ZIF-8 nanoparticles was basically the same as that of the standard ZIF-8 materials reported in literature, and the introduction of cells did not significantly change the morphology of ZIF-8. After repeated use for 7 times, SAET@ZIF-8 could still retain 67% of the initial catalytic activity. Maintained at room temperature for 4 days, 50% of the original catalytic activity of SAET@ZIF-8 could be retained, indicating that SAET@ZIF-8 has good stability for reuse and storage. When used in the biosynthesis of Ala-Gln, the final concentration of Ala-Gln reached 62.83 mmol/L (13.65 g/L) after 30 min, the yield reached 0.455 g/(L·min), and the conversion rate relative to glutamine was 62.83%. All these results suggested that the preparation of SAET@ZIF-8 is an efficient strategy for the biosynthesis of Ala-Gln.

Protein expression, purification and mouse antiserum preparation of monkeypox virus A23R / 细胞与分子免疫学杂志

Objective To express the monkeypox virus (MPXV) A23R protein in Escherichia coli and purify by Ni-NTA affinity column, and to prepare mouse antiserum against MPXV A23R. Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein. After optimizing the conditions of expression, A23R protein was highly expressed. Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis. The purified protein was used to immunize mice for preparing the A23R polyclonal antibody, and the antibody titer was detected by ELISA. Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-β-D-thiogalactoside (IPTG), 37 DegreesCelsius and 20 hours. The purity of the protein was about 96.07% and was identified by Western blot analysis. The mice were immunized with recombinant protein, and the titer of antibody reached 1:102 400 at the 6th week after immunization. Conclusion MPXV A23R is expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.

Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein / 细胞与分子免疫学杂志

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

Preparation and identification of rabbit anti-mouse coiled-coil domain containing 189(Ccdc189)polyclonal antibody / 细胞与分子免疫学杂志

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.

Antibiotic susceptibility and fosfomycin resistance characterization in a cohort of children older than 6 years of age with urinary tract infection / Sensibilidad antibiótica y caracterización de la resistencia a fosfomicina en una cohorte de niños mayores de 6 años con infección urinaria

Fosfomycin tromethamol (FT) was reintroduced as an option for the treatment of low urinary tract infection (UTI) in children. In this study, we described the antibiotic sensitivity and mechanisms of resistance to fosfomycin in isolates from children older than 6 years with UTI. Urine culture and antibiotic susceptibility study were performed. In fosfomycin resistant strains, PCR for fos, blaCTX-M was performed followed by classification by phylogenetic group and sequencetyping. Escherichia coli was the most frequent etiological agent (89.2%). The susceptibility percentages were: fosfomycin 97.9%; amoxicillin-clavulanate 92.7%; cefuroxime and ceftriaxone 99%; nitrofurantoin 94.4%. An E. coli strain (ST69, phylogenetic group D) was resistant to fosfomycin (MIC 256mg/l) and carried the blaCTX-M-14 and fosA3 genes in a 45kb IncN-type plasmid.

La fosfomicina-trometamol (FT) se reintrodujo como una opción para el tratamiento de la infección del tracto urinario (ITU) baja en niños. En este estudio describimos la sensibilidad antibiótica y los mecanismos de resistencia a FT en aislamientos de niños mayores de 6 anos con ITU. Se realizaron urocultivos y estudios de sensibilidad antibiótica. En las cepas resistentes a fosfomicina se realizó la técnica de PCR para fos, blaCTX-M, y su identificación según su grupo filogenéticoy secuenciotipo. Escherichiacoli fue el agente etiológico más frecuente (89,2%). Los porcentajes de sensibilidad fueron: fosfomicina 97,9%; amoxicilina-clavulánico 92,7%; cefurox-ima y ceftriaxona 99%; nitrofurantoína 94,9%. Una cepa de E. coli (ST69, grupo filogenético D) fue resistente a fosfomicina (CIM 256mg/l) y portaba los genes blaCTX-M-14 y fosA3 en un plás-mido de 45 kb del tipo IncN. Este es el primer reporte de E. coli ST69 con blaCTX-M-14/fosA3 de origen humano.

The OxyR and SoxR transcriptional regulators are involved in a broad oxidative stress response in Paraburkholderia xenovorans LB400

BACKGROUND: Aerobic metabolism generates reactive oxygen species that may cause critical harm to the cell. The aim of this study is the characterization of the stress responses in the model aromatic degrading bacterium Paraburkholderia xenovorans LB400 to the oxidizing agents paraquat and H 2 O2. METHODS: Antioxidant genes were identified by bioinformatic methods in the genome of P. xenovorans LB400, and the phylogeny of its OxyR and SoxR transcriptional regulators were studied. Functionality of the transcriptional regulators from strain LB400 was assessed by complementation with LB400 SoxR of null mutant P. aeruginosa ΔsoxR, and the construction of P. xenovorans pIZ oxyR that overexpresses OxyR. The effects of oxidizing agents on P. xenovorans were studied measuring bacterial susceptibility, survival and ROS formation after exposure to paraquat and H 2 O2. The effects of these oxidants on gene expression (qRT PCR) and the proteome (LC-MS/MS) were quantified. RESULTS: P. xenovorans LB400 possesses a wide repertoire of genes for the antioxidant defense including the oxyR , ahpC , ahpF , kat , trxB , dpsA and gorA genes, whose orthologous genes are regulated by the transcriptional regulator OxyR in E. coli . The LB400 genome also harbors the soxR, fumC , acnA , sodB , fpr and fldX genes, whose orthologous genes are regulated by the transcriptional regulator SoxR in E. coli . The functionality of the LB400 soxR gene was confirmed by complementation of null mutant P. aeruginosa Δ soxR . Growth, susceptibility, and ROS formation assays revealed that LB400 cells were more susceptible to paraquat than H2O2. Transcriptional analyses indicated the upregulation of the oxyR , ahpC1 , katE and ohrB genes in LB400 cells after exposure to H2O2, whereas the oxyR , fumC , ahpC1 , sodB1 and ohrB genes were induced in presence of paraquat. Proteome analysis revealed that paraquat induced the oxidative stress response proteins AhpCF and DpsA, the universal stress protein UspA and the RNA chaperone CspA. Both oxidizing agents induced the Ohr protein, which is involved in organic peroxide resistance. Notably, the overexpression of the LB400 oxyR gene in P. xenovorans significantly decreased the ROS formation and the susceptibility to paraquat, suggesting a broad OxyR regulated antioxidant response. CONCLUSIONS: This study showed that P. xenovorans LB400 possess a broad range oxidative stress response, which explain the high resistance of this strain to the oxidizing compounds paraquat and H2O2.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi / Estudos sobre a produção recombinante e atividade anticancerígena da L-asparaginase I termoestável de Pyrococcus abyssi

L-Asparaginase catalysing the breakdown of L-Asparagine to L-Aspartate and ammonia is an enzyme of therapeutic importance in the treatment of cancer, especially the lymphomas and leukaemia. The present study describes the recombinant production, properties and anticancer potential of enzyme from a hyperthermophilic archaeon Pyrococcus abyssi. There are two genes coding for asparaginase in the genome of this organism. A 918 bp gene encoding 305 amino acids was PCR amplified and cloned in BL21 (DE3) strain of E. coli using pET28a (+) plasmid. The production of recombinant enzyme was induced under 0.5mM IPTG, purified by selective heat denaturation and ion exchange chromatography. Purified enzyme was analyzed for kinetics, in silico structure and anticancer properties. The recombinant enzyme has shown a molecular weight of 33 kDa, specific activity of 1175 U/mg, KM value 2.05mM, optimum temperature and pH 80°C and 8 respectively. No detectable enzyme activity found when L-Glutamine was used as the substrate. In silico studies have shown that the enzyme exists as a homodimer having Arg11, Ala87, Thr110, His112, Gln142, Leu172, and Lys232 being the putative active site residues. The free energy change calculated by molecular docking studies of enzyme and substrate was found as ∆G ­ 4.5 kJ/mole indicating the affinity of enzyme with the substrate. IC50 values of 5U/mL to 7.5U/mL were determined for FB, caco2 cells and HepG2 cells. A calculated amount of enzyme (5U/mL) exhibited 78% to 55% growth inhibition of caco2 and HepG2 cells. In conclusion, the recombinant enzyme produced and characterized in the present study offers a good candidate for the treatment of cancer. The procedures adopted in the present study can be prolonged for in vivo studies.

A L-asparaginase, que catalisa a degradação da L-asparagina em L-aspartato e amônia, é uma enzima de importância terapêutica no tratamento do câncer, especialmente dos linfomas e da leucemia. O presente estudo descreve a produção recombinante, propriedades e potencial anticancerígeno da enzima de Pyrococcus abyssi, um archaeon hipertermofílico. Existem dois genes que codificam para a asparaginase no genoma desse organismo. Um gene de 918 bp, que codifica 305 aminoácidos, foi amplificado por PCR e clonado na cepa BL21 (DE3) de E. coli usando o plasmídeo pET28a (+). A produção da enzima recombinante foi induzida sob 0,5mM de IPTG, purificada por desnaturação seletiva por calor e cromatografia de troca iônica. A enzima purificada foi analisada quanto à cinética, estrutura in silico e propriedades anticancerígenas. A enzima recombinante apresentou peso molecular de 33 kDa, atividade específica de 1.175 U / mg, valor de KM 2,05 mM, temperatura ótima de 80º C e pH 8. Nenhuma atividade enzimática detectável foi encontrada quando a L-glutamina foi usada como substrato. Estudos in silico mostraram que a enzima existe como um homodímero, com Arg11, Ala87, Thr110, His112, Gln142, Leu172 e Lys232 sendo os resíduos do local ativo putativo. A mudança de energia livre calculada por estudos de docking molecular da enzima e do substrato foi encontrada como ∆G ­ 4,5 kJ / mol, indicando a afinidade da enzima com o substrato. Valores de IC50 de 5U / mL a 7,5U / mL foram determinados para células FB, células caco2 e células HepG2. Uma quantidade de enzima (5U / mL) apresentou inibição de crescimento de 78% a 55% das células caco2 e HepG2, respectivamente. Em conclusão, a enzima recombinante produzida e caracterizada no presente estudo é uma boa possibilidade para o tratamento do câncer. Os procedimentos adotados na presente pesquisa podem ser aplicados para estudos in vivo.

Application of gene editing technology in Escherichia coli / 生物工程学报

Gene editing technology can be used to modify the genome of Escherichia coli for the investigation of gene functions, or to change the metabolic pathways for the efficient production of high-value products in engineered strains with genetic stability. A variety of gene editing technologies have been applied in prokaryotes, such as λ-Red homologous recombination and CRISPR/Cas9. As a traditional gene editing technique, λ-Red recombination is widely used. However, it has a few shortcomings, such as the limited integration efficiency by the integrated fragment size, the cumbersome gene editing process, and the FRT scar in the genome after recombination. CRISPR/Cas9 is widely used for genome editing at specific sites, which requires specific DNA segments according to the editing site. As the understanding of the two technologies deepens, a variety of composite gene editing techniques have been developed, such as the application of λ-Red homologous recombination in combination with homing endonucleaseⅠ-SceⅠ or CRISPR/Cas9. In this review, we summarized the basic principles of common gene editing techniques and composite gene editing techniques, as well as their applications in Escherichia coli, which can provide a basis for the selection of gene editing methods in prokaryotes.

Characterization of a family B DNA polymerase from Thermococcus eurythermalis A501 and its application in PCR / 生物工程学报

DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, 60 mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.

Escherichia coli expression and characterization of alfa-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Prokaryotic expression and polyclonal antibody preparation of human adenovirus type 7 DNA binding protein / 中华预防医学杂志

Objective: To express DNA-binding protein (DBP) of human adenovirus (HAdV) type 7 using the prokaryotic expression system, and product anti-HAdV-7 DBP rabbit polyclonal antibody. Methods: The HAdV-7 DBP gene was synthesized and cloned into prokaryotic expressing vector pET30a, and the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cell. The recombinant protein DBP was expressed by induced Isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with Ni-NTA affinity column. The titer of anti-DBP polyclonal antibody produced in immunized rabbit was measured by indirect ELISA, and the specificity of the antibody was identified by Western blotting and indirect immunofluorescence assay (IFA). In addition, purified rDBP was used as coating antigen for indirect ELISA assay to detect specific IgM and IgG antibodies against DBP in the serum of children infected with HAdV. Results: The HAdV-7 DBP plasmid was constructed successfully. The purified recombinant DBP was more than 95% after purification. The titer of polyclonal antibody was 1∶1 024 000. The polyclonal antibody showed high specificity in vitro using Western blotting and IFA. The positive rate of specific anti-DBP IgM and IgG antibody in acute-phase serum samples collected from children infected with HAdV were 50.0% (19/38) and 63.2% (24/38), respectively, using indirect ELISA. Conclusion: In summary, the HAdV-7 rDBP is expressed using prokaryotic expression system, and the recombinant HAdV-7 DBP protein and the anti-DBP rabbit polyclonal antibody with high titer are prepared.

Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis / 生物工程学报

Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.

Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid / 生物工程学报

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.