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The rapid development of bioeconomy urgently needs the support of biotechnology talents. Establishing an innovative training mode of biotechnology talents can provide support for regional economic development and industrial upgrading. Closely revolved around the concepts of new engineering disciplines development, such as serving the national strategy, docking industry, leading the future development and student-centered, a new economy-oriented training system was developed in School of Bioengineering of Dalian University of Technology. These systems include interdisciplinary curriculum system reconstruction, project-based teaching mode reform, evaluation system implementation and other aspects. The reform and exploration of the first-class biotechnology major under the new economic situation, puts forward the theory of value guidance, deep foundation, strong sense of innovation, technical and non-technical core ability literacy. This reform meets the industry demand for talent diversification, personalization, and dynamic change, helps the merge of industry and education, which provides a way for fostering first-class biotechnology-majored undergraduates.
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Humans , Biotechnology , Bioengineering , Biomedical Engineering , Students , CurriculumABSTRACT
Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.
Subject(s)
Sphingobacterium/metabolism , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Amino Acids , Adenosine Triphosphate , Regeneration , Polyphosphates/metabolismABSTRACT
Objective:To evaluate the application value of metagenomic next-generation sequencing(mNGS) for patients with severe pneumonia of unknown pathogen.Methods:The clinical data of 8 patients with unknown pneumonia pathogens who were assisted by the alveolar lavage fluid mNGS technology treated in Affiliated Hospital of Yangzhou University from June 2019 to February 2020 were retrospectively analyzed, including alveolar lavage fluid smear, culture, and the sequencing result.The clinical characteristics and mNGS detection results were comprehensively analyzed.According to the results of mNGS and the judgment of the clinician, the patients were divided into confirmed pathogens group and undetermined pathogens group, and the Simpson's diversity index of two groups was compared.Results:Among 8 cases, there were 5 males and 3 females, and the median age was 62.5 years (ranged from 41 to 71 years). All of them had chronic diseases except 1 case, including diabetes, chronic obstructive pulmonary disease and kidney transplantation.mNGS was positive in 8 patients, and conventional test was positive in 3 patients.The comparison of the Simpson's diversity index between the identified pathogenic group and the undetermined pathogenic group showed statistically significant difference [(0.398 ± 0.222) vs.(0.763 ± 0.061), t=2.709, P=0.035]. The positive results of mNGS were different from that of conventional test in 2 patients.The nucleic acid number in 3 cases of Pneumocystis carinii with mNGS positive results ranged from 120 to 15 580, with genomic coverage rates from 24.6% to 99.8%. Conclusion:mNGS can help physicians acquire information of the pathogenic bacteria quickly and comprehensively, especially for patients with unknown pneumonia pathogenic bacteria.
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Objective Through regulatory study on the common defects of air purification system, this paper provides valuable reference for practitioners in medical device industry. Methods More than 100 verification results of different companies had been collected during 2015 to 2018, followed by systematically analysis of the defects related to air purification system. Result 70 types of common defects in 13 areas had been summarized, and 20 key points in verification had been briefly concluded. Conclusion Recognizing and understanding these summarized defects and key points will not only promote the unification of criterion scale, but also benefit enterprises for themselves, inspection, quality management improvement, and the plant transformation as well.
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Air Pollution , Equipment and Supplies , IndustryABSTRACT
With the discovery of the significant medicinal value of alginate oligosaccharides and bioethanol produced by microalgae, alginate lyase has been the focus of research in all fields. Five alginate lyase genes in cluster from Vibrio alginolyticus were cloned and expressed in Escherichia coli. SDS-PAGE and enzyme activity showed that four of the five genes have the activity to degrade alginate. Optimization of the induction conditions, protein purification and enzyme properties of rAlgV3 with the highest enzyme activity were studied. The results showed that the enzyme activity of recombinant enzyme rAlgV3 increased from 2.34×10⁴ U/L to 1.68×10⁵ U/L, which was 7.3 times higher than before. The optimal reaction temperature was 40 °C, and the enzyme was relatively stable between 4 °C and 20 °C. The enzyme had a higher activity between pH 6.5 and 9.0, with the optimum pH 8.0. It showed a wide range of pH that the alginate lyase can exist stably between pH 4.5 and 9.5. Appropriate concentrations of NaCl and Fe²⁺, Fe³⁺ ions promoted enzyme activity. SDS and Cu²⁺ ions inhibited the enzyme activity. The enzyme degraded Poly-M fragments and Poly-G fragments, with a wide range of substrate properties. The degraded product of sodium alginate of rAlgV3 analyzed by ESI-MS mainly was oligosaccharides with a polymerization degree of 2 to 3, which means that rAlgV3 was an endo-type alginate lyase. This enzyme has the potential in the development of third-generation bioethanol and the production of alginate oligosaccharides.
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Objective:To regulate operation requirement for electric instrument using in surgery and eliminate hidden danger so as to ensure their safety in using.Methods: Through discussed the regulation of operative process for electric instrument using in surgery to develop a insulation detector which can adapt the particular case of hospital, and then to carry out relevant management for this kind of electric instrument.Results: The simplest insulation monitoring method got best reliable data, and it provide accuracy basis for clinical electric instrument. Therefore, it ensured the safety of clinical application of electric instrument.Conclusion: The special insulation monitor detects electric instrument in surgery has series of merits including low investment, fast detection and reliable performance, it not only can ensure the safety of surgery but also can provide traceable basis for preventing relevant medical dispute.
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Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.
ABSTRACT
To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.
Subject(s)
Glycoside Hydrolases , Chemistry , Genetics , Industrial Microbiology , Inulin , Kluyveromyces , Genetics , Pichia , Sucrose , TemperatureABSTRACT
Consolidated bioprocessing technology can be used for Kluyveromyces marxianus YX01 to produce ethanol from Jerusalem artichoke, which is one of the potential processes to produce biofuel from non-cereal crops. In this study, we combined the aeration rate with the substrate concentration to conduct cross-over experiments for K. marxinaus YX01, and studied ethanol fermentation and the influence of inulin enzyme activity. The substrate concentration had a little repressive effect on ethanol productivity. When substrate concentration reached 250 g/L under anaerobic conditions, ethanol concentration was 84.8 g/L, and ethanol yield was reduced from 86.4% (50 g/L substrate concentration) to 84.7% of the theoretical value. Aeration rate could accelerate K. marxinaus YX01 ethanol fermentation, but reduced ethanol yield. When substrate concentration reached 250 g/L under aeration at 1.0 vvm, ethanol yield was reduced from 84.7% under anaerobic conditions to 73.3% of the theoretical value. With increased concentration of the carbon source and reduced aeration rate, the inulinase of K. marxinaus YX01 reduced and the concentration of glycerol increased, however, the acetic acid increased with the increased concentration of the carbon source and aeration rate. When substrate concentration reached 250 g/L under anaerobic conditions, inulinase activity was only 6.59 U/mL; when substrate concentration reached 50 g/L under aeration at 1.0 vvm, inulinase activity was 21.54 U/mL.
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Ethanol , Metabolism , Fermentation , Glycoside Hydrolases , Metabolism , Helianthus , Metabolism , Inulin , Metabolism , Kluyveromyces , Classification , Metabolism , Substrate SpecificityABSTRACT
Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.
Subject(s)
Amino Acid Sequence , Catalysis , Coloring Agents , Metabolism , Escherichia coli , Genetics , Metabolism , Molecular Sequence Data , Peroxidases , Genetics , Recombinant Proteins , Genetics , ZymomonasABSTRACT
We used ribosome engineering technology, with which antibiotic-resistant strains are resulted from mutations on microbial ribosome, to screen a high butanol-producing Clostridium strain. A novel mutant strain S3 with high butanol production and tolerance was obtained from the original Clostridium acetobutylicum L7 with the presence of mutagen of streptomycin. Butanol of 12.48 g/L and ethanol of 1.70 g/L were achieved in S3, 11.2% and 50%, respectively higher than the parent strain. The conversion rate of glucose to butanol increased from 0.19 to 0.22, and fermentation time was 9 h shorter. This caused an increase in butanol productivity by 30.5%, reaching 0.24 g/(Lh). The mutant butanol tolerance was increased from 12 g/L to 14 g/L, the viscosity of fermentation broth was dramatically decreased to 4 mPa/s, 60% lower than the parent strain. In addition, the genetic stability of mutant strain S3 was also favorable. These results demonstrate that ribosome engineering technology may be a promising process for developing high butanol-producing strains.
Subject(s)
Butanols , Metabolism , Clostridium acetobutylicum , Genetics , Metabolism , Fermentation , Genetic Engineering , Mutation , Recombinant Proteins , Genetics , Ribosomes , Genetics , Streptomycin , PharmacologyABSTRACT
Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.
Subject(s)
Ethanol , Metabolism , Fermentation , Glycoside Hydrolases , Genetics , Bodily Secretions , Helianthus , Metabolism , Kluyveromyces , Genetics , Metabolic Engineering , Methods , Plant Tubers , Metabolism , Recombination, Genetic , Saccharomyces cerevisiae , GeneticsABSTRACT
This research aimed to study the effect of distillage recycling on ethanol fermentation, the key glycolytic enzymes and cell composition of the self-flocculating yeast. With the self-flocculating yeast SPSC01 and medium composed of 220 g/L glucose, 8 g/L yeast extract and 6 g/L peptone, continuous ethanol fermentation was carried out at the dilution rate of 0.04 h(-1) with a 1.5 L tank bioreactor. Fermentation broth was collected every 3 days, and ethanol and other volatile byproducts were removed by distillation, but the stillage with high boiling byproducts was recycled to prepare the medium instead of fresh water. The system was run for 20 days, during which ethanol and biomass concentrations in the effluent decreased continuously, indicating the significant inhibition of the high boiling byproducts accumulated within the system. Thus, the activities of the key enzymes of the glycolytic pathway: hexokinase, 6-phosphofructose kinase, and pyruvate kinase were analyzed, and it was observed that all of them were inhibited. Furthermore, the biosynthesis of the stress response metabolites glycerol and trehalose was investigated, and it was found that glycerol production that can protect yeast cells against osmotic pressure stress was enhanced, but trehalose biosynthesis that can protect yeast cells against ethanol inhibition was not improved, correspondingly. And in the meantime, the biosynthesis of the major intracellular components proteins and hydrocarbons was adjusted, correspondingly.
Subject(s)
Bioreactors , Microbiology , Ethanol , Metabolism , Fermentation , Flocculation , Glycerol , Metabolism , Glycolysis , Hexokinase , Metabolism , Industrial Microbiology , Methods , Phosphofructokinase-1 , Metabolism , Saccharomyces cerevisiae , Genetics , Metabolism , Schizosaccharomyces , Genetics , Metabolism , Trehalose , Metabolism , Triticum , Metabolism , Zea mays , MetabolismABSTRACT
Purpose:To evaluate the role of multi - detector row CT(MDCT) using reconstruction techniques in the assessment of patients with obstructed diseases of biliary tract.Materials and Methods: 47 Patients with obstructed diseases of biliary tract confirmed clinically underwent MDCT and their reconstructed images of biliary tract including multi-planar reconstruction (MPR) images and curved planar reconstruction(CPR) images were compared with those of 50 patients without obstruction and dilatation of biliary tract.The display effect of biliary duct structure and biliary duct wall and the display ability of biliary tract by MPR and CPR images between the 2 groups were compared and analysed.The reconstruction images of biliary tract were analysed retrospectively to evaluate the location and possible causes of biliary obstruction.Results: The display effect of biliary duct structure and biliary duct wall in MPR and CPR images of the group with biliary obstruction is better than that of control group,and the display ability of biliary tract in CPR images of the group with biliary obstruction is also better than that of control group.The accuracy of localization and cause evaluation of obstruction by MPR and CPR images is 100% and 89.4% respectively.Conclusions: The MPR and CPR images of MDCT provide a good display of biliary duct structure,biliary duct wall and an accurate evaluation of obstruction localization.The reconstruction technique of MDCT such as MPR and CPR should be widely applied in the evaluation of biliary obstruction.
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A unique one-step ethanol fermentation process was developed with the inulinase-producing strain Kluyveromyces marxianus YX01. Firstly, the impact of temperature on ethanol fermentation was investigated through flask fermentation, and the temperature of 35 degrees C was observed to be the optimum to coordinate inulinase production, inulin saccharification and ethanol fermentation. And then, the impact of aeration and substrate concentration was studied through batch fermentation in the 2.5 L fermentor, and the experimental data indicated that the average ethanol fermentation time was decreased at the aeration rates of 50 mL/min and 100 mL/min, but higher ethanol yield was obtained under non-aeration conditions with more substrate directed to ethanol production. The ethanol concentration of 92.2 g/L was achieved with the substrate containing 235 g/L inulin, and the ethanol yield was calculated to be 0.436, equivalent to 85.5% of its theoretical value. Finally, Jerusalem artichoke grown in salina and irrigated with seawater was fermented without sterilization treatment, 84.0 g/L ethanol was obtained with the substrate containing 280 g/L dry Jerusalem artichoke meal, and the ethanol yield was calculated to be 0.405, indicating the Jerusalem artichoke could be an alternative feedstock for grain-based fuel ethanol production.