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Published in last 1 year |  In last 2 years |  In last 3 years |  All Please wait a minute... For Selected: Download Citations EndNote Ris BibTeX Toggle Thumbnails Select Study on humic acid-like components, molecular structure and physiological activity Bingkai SHANG Haitao MO Zhuowang FU Xiaoyong ZHANG The Chinese Journal of Process Engineering    2021, 21 (8): 969-975.   DOI: 10.12034/j.issn.1009-606X.220291 Abstract （311）   HTML （2）    PDF （905KB）（99）       Knowledge map    Save By means of methods of elemental analysis, FT-IR and solid 13C NMR, a comparative study on the component, elemental composition and molecular structure of humic acid-like substances prepared from four different raw materials and humic acids from mineral sources were carried out. Also the physiological activity of four humic acid-like substances was determined by seed germination experiment. The study results showed that the content of fulvic acid in the humic acid-like materials prepared from herbaceous plants (wheat straw and bamboo) was significantly higher than that from woody plants (pine and poplar). The molecular structure of humic acid-like materials was similar to that of mineral humic acid, because both of them had the characteristic functional groups of humic acid. Compared with mineral humic acids, the structures of humic acid-like materials were more complex, and the types and contents of functional groups were different. They have more aromatic structures and lower oxygen-containing functional groups. Also, humic acid-like materials from herbaceous plants had higher aromatization degree and lower molecular weight than those from woody plants. The results of wheat seed germination experiment showed that, when the concentration of the humic acid-like materials was 20~40 mg/L, it can obviously promote the germination of seeds. However the seed growth will be inhibited if the concentration was too high. Related Articles | Metrics Select Preparation of a Pickering emulsion for treatment of bacterial biofilm infection Xieru BAO Jie WU Guanghui MA The Chinese Journal of Process Engineering    2021, 21 (5): 594-600.   DOI: 10.12034/j.issn.1009-606X.220140 Abstract （331）      PDF （1514KB）（111）       Knowledge map    Save Bacterial biofilms are closely associated with persistent infections. E?ective penetration of antibacterial drugs is necessary for the treatment of bacterial biofilms. In this study, a nanoparticle-stabilized Pickering emulsion with strong permeability and high antibacterial activity was fabricated for the treatment of bacterial biofilms. This Pickering emulsion was composed of clove oil core and chitosan nanoparticles. The clove oil was used as an antibacterial agent, and the chitosan nanoparticles provided a positively charged shell, which could interact with the negatively charged extracellular matrix to achieve strong penetration through bacterial biofilms. Both of the chitosan nanoparticles and the Pickering emulsion with narrow size distribution and good dispersity were prepared successfully. The average size of chitosan nanoparticles was 590.30±3.90 nm with 0.125±0.003 of polydispersity index (PDI), and the average Zeta potential was 15.60±0.40 mV. The average size of the Pickering emulsion was 2312±53 nm with 0.137±0.013 of PDI, and the average Zeta potential was 26.45±0.55 mV. The experiments of bacterial biofilms penetration and antibacterial test revealed that the Pickering emulsion could effectively penetrate the bacterial biofilms and kill bacteria. Besides, the cytotoxicity test in vitro showed that the Pickering emulsion had good biosafety. All these results indicated that the Pickering emulsion had promising prospects for the treatment of persistent infections associated with bacterial biofilms. Related Articles | Metrics Select Preparation of submicron Pickering emulsion stabilized by cellulose nanocrystals Yanling QU Jie WU Guanghui MA Chin. J. Process Eng.    2021, 21 (4): 454-462.   DOI: 10.12034/j.issn.1009-606X.220141 Abstract （387）      PDF （1872KB）（124）       Knowledge map    Save Compared with the traditional surfactant-stabilized emulsions, Pickering emulsions stabilized by solid particles have the advantages of strong interfacial stability, versatility, and low toxicity. A number of cases have demonstrated the potential of Pickering emulsions in biomedical applications. Submicron Pickering emulsion has a larger specific surface area and more efficient delivery efficiency than large-sized Pickering emulsions, which is expected to further expand the advantages of Pickering emulsions in the biomedicine fields. However, the size of Pickering emulsions is determined by many factors such as particle properties, characteristics of oil and water phase. And it is difficult to arrange the rigid particles in a limited and small oil–water interface. The above reasons increase the difficulty of preparing the submicron Pickering emulsions. The purpose of this study is to prepare stable submicron-sized Pickering emulsion by utilizing the natural polysaccharide with good biocompatibility-cellulose nanocrystals (CNCs) as particle emulsifier, squalene as oil phase. The effects of preparation conditions such as particle concentration, oil–water ratio, aqueous phase, ultrasonic time and frequency on size distribution and stability of Pickering emulsions were investigated. Finally, the submicron-sized Pickering emulsion (CNCs-PE) with good storage stability and centrifugal resistance was prepared, and the average size of CNCs-PE was about 638.7?8.40 nm. The confocal laser scanning microscopic (CLSM) images revealed that Pickering emulsion was formed successfully by the adsorption of CNCs on the oil–water interface. The cytotoxicity of CNCs and CNCs stabilized Pickering emulsion (CNCs-PE) was evaluated using CCK-8 method, and it showed that there was no significant loss of cell viability. In addition, the vaccine formulation was prepared by absorbing antigen protein-OVA. The adsorption efficiency of OVA was about 80%, and the histological micrographs of the intramuscular injection site section also showed the injection safety. It is expected to expand the applications of submicron-sized Pickering emulsion based on CNCs in biomedicine fields. Related Articles | Metrics Select Preparation and characterization of amphiphilic lipopeptide nanosuspension lyophilized powder Mengqiu ZHANG Huijuan JIN Fangling GONG Youhong ZHANG Yi WEI Yuxian HE Guanghui MA Chin. J. Process Eng.    2021, 21 (4): 463-470.   DOI: 10.12034/j.issn.1009-606X.220144 Abstract （275）      PDF （536KB）（115）       Knowledge map    Save AIDS has spread widely and become a serious public health problem around the world. Membrane fusion inhibitor LP-98 shows strong antiviral activity among the anti-AIDS drugs under study in China, and has broad clinical application prospects. However, the clinical application of LP-98 is limited by its low solubility and poor suspension ability in aqueous phase, which could lead to blocked needle during injection and patient's pain. To improve solubility of LP-98, high-pressure homogenization technology was used to prepare LP-98 nanosuspension lyophilized powder (LP-98 NSLP). The optimal preparation process was as follow: the optimum stabilizer was sodium dodecyl sulfate (SDS), the concentration of SDS was 0.80wt%, the high-pressure homogenization pressure was 150 MPa, and it was repeated 5 times. The physical and chemical characterization and pharmacokinetics study of LP-98 NSLP were investigated. The LP-98 nanosuspension lyophilized powder had an average particle size of 261.5±1.1 nm and a Zeta potential of ?31.5±0.2 mV. Circular dichroism spectrometer and single-cycle virus infection experiments showed that the structure and biological activity of active pharmaceutical ingredients (API) were unchanged. The pharmacokinetic results showed that the bioavailability of the LP-98 NSLP was 98.1% of API. The solubility of LP-98 in water has been increased from 184 μg/mL to 1733 μg/mL, which was 8 times higher than that of the API. The drug activity in the LP-98 nanosuspension lyophilized powder was well preserved, and the solubility of the drug was improved. As a result it solved the problem of blocked needle during injection due to poor suspension of LP-98, hence reduced patient's pain. This research promoted the development of LP-98 application in clinical. Related Articles | Metrics Select Enzymatic hydrolysis of penicillin mycelium protein Zhan'ao ZHANG Qingfen LIU Chin. J. Process Eng.    2021, 21 (4): 471-478.   DOI: 10.12034/j.issn.1009-606X.220122 Abstract （265）      PDF （406KB）（97）       Knowledge map    Save More than 2000000 tons of antibiotics fermentation residues (AFRs) were produced in China every year, which were hazardous solid waste, and how to safely dispose of them is attracting attention. AFRs are rich in organic substances, such as protein and carbohydrates, which accounts for about 50% of dry penicillin AFRs. So it is significant to develop new methods for recycling and utilization of mycelium protein in AFRs. In this work, penicillin mycelium was dissolved by alkali-thermal method, and penicillin mycelium protein solution was prepared, and then enzymatic hydrolysis performance of penicillin mycelium protein was explored in this solution using enzyme as catalyst, such as alcalase (A), bromelain (B), neutrase (C), and acid protease (D). The effects of types of enzymes, pH of the solution, the ratio of enzyme to protein, reaction temperature and time on protein hydrolysis process were systematically studied, and an optimization scheme was proposed. In a single enzyme-catalyzed hydrolysis process, alcalase showed the optimal performance, the hydrolysis degree of protein reached 31.43% at following conditions, the mass ratio of alcalase to protein as 6%, pH=11, the reaction time of 3 h and temperature of 50℃. In the complex enzyme-catalyzed hydrolysis process, the mixture of alcalase and bromelain as catalysts was demonstrated got the best efficiency. The optimum parameters were as follows: the mass ratio of complex enzyme to protein was 9% (A:B=2:1), pH=10, the reaction time of 3 h and temperature of 50℃. Under these optimal conditions, the protein hydrolysis degree reached 42.73%, which was 11.30 percentage points higher than the best single-enzyme method. The high performance liquid chromatography (HPLC) analysis found the enzymatic hydrolysate contained 16 amino acids, such as glutamic acid, aspartic acid, glycine, leucine and alanine, the ratio was 15.47wt%, 11.94wt%, 9.41wt%, 9.23wt%, 9.22wt%, respectively. This work provides a high efficient enzyme-catalyzed hydrolysis method of penicillin mycelium protein for preparing amino acids, and to carry out utilization of penicillin mycelium protein in AFRs. Related Articles | Metrics Select Optimization of preparation of sulforaphane proliposome in broccoli seeds by response surface method Huanpu XU Zhaoyang PEI Shijie SUN Yingxue WU Lulu XU Jing HAN Hui XU Chin. J. Process Eng.    2021, 21 (3): 305-313.   DOI: 10.12034/j.issn.1009-606X.220055 Abstract （324）      PDF （1513KB）（113）       Knowledge map    Save Sulforaphane is made into proliposomes to improve the stability of liposomes, while improving the water solubility and bioavailability of sulforaphane. Taking the encapsulation efficiency and particle size as indicators, the carrier materials, the type and amount of surfactants and the effects of lipid-drug ratios were investigated respectively. The optimal formulation of proliposome was optimized by response surface method. The stability of liposomes and proliposomes was investigated through room temperature stability experiments. The results showed that the optimal prescription was that the mass ratio of lipid phase to sulforaphane was 6.5:1, the mass ratio of NaCl to sulforaphane was 105:1, and the mass ratio of poloxamer-188 to sulforaphane was 1.5:1. The maximum average encapsulation rate was 77.43%, and the average particle size was 160.5 nm. Stability experiment results showed that when the drug retention rate was used as an indicator, liposomes and proliposomes had good stability within 60 days and a high retention rate of sulforaphane. When the encapsulation rate was used as an indicator, the liposome suspension produced precipitated, the encapsulation rate decreased, and the encapsulation rate of the proliposome did not decrease significantly within 60 days. This indicated that the proliposome can solve the oxidative deterioration of sulforaphane and the decrease of the encapsulation rate of the liposome due to precipitation, flocculation and other reasons. The proliposome has high encapsulation efficiency and simple preparation. It not only improves the stability of sulforaphane, but also improves the water solubility of sulforaphane and has broad application prospects. Related Articles | Metrics Select Effects of two-compartment gas interflow on the performance of photosynthetic microbe fuel cell Xintong ZHU Huan HE Runyun ZHU Zhiang XU Fengxia HAN Hongping PU Chin. J. Process Eng.    2021, 21 (3): 314-322.   DOI: 10.12034/j.issn.1009-606X.220088 Abstract （380）      PDF （1356KB）（107）       Knowledge map    Save Microbial carbon capture cell (MCC) was assembled with a photo biocathode where growing Scenedesmus obliquus to produce oxygen as an electron acceptor after the operation of photo-microbe fuel cell (PMFC) and an added CO2 photo-MFC (AC-PMFC). The voltage generation, dissolved oxygen and pH were measured over each day in the different systems. It was demonstrated that cell voltage produced by MFC was in line with the oxygen concentration in all systems with algae cathode. The pH of the electrolyte can also affect voltage generation. The highest voltage and power density of MCC were obtained in the three types of MFC with 492 mV and 102.3 mW/m2, respectively. Its maximum power density was higher than that of PMFC and AC-PMFC. The three systems received different concentrations of carbon dioxide for photosynthesis. The AC-PMFC achieved the lowest voltage and power density due to the excessive concentration of CO2, which could inhibit the biological activity and photosynthesis of microalgae. The scanning electron microscope (SEM) was measured to observe the morphology characteristics of algae on the cathode surface of MCC after long-term operation. A layer of in situ oxygen film with high concentration could be generated on the surface of algae biofilm and electrode plate. The electrochemical analysed demonstrated that the biofilm could not directly receive the electrons from the plate and had no biocatalytic activity. This biofilm could increase the rate of oxygen reduction, which can effectively reduce the resistance of the battery surface. Polymerase chain reaction (PCR) and 16S rRNA gene detection technology indicated that the Chao1 index in MFC was 170, while the PMFC was 152 and the MCC was 145. The oversaturated oxygen in the cathode could be transported to the anode by pipeline and affect the microbial community in the anode. This study could provide a basis for further understanding of algae-based microbial carbon-trapping cells to improve MCC performance. Related Articles | Metrics Select Molecular dynamics simulation and calculation of binding free energy of a HBc-VLP Yanyan MA Zhengjun LI Songping ZHANG Wei CHEN Ying REN Chin. J. Process Eng.    2021, 21 (2): 219-229.   DOI: 10.12034/j.issn.1009-606X.220085 Abstract （332）      PDF （1019KB）（222）       Knowledge map    Save Hepatitis B core antigen virus-like particles (HBc-VLPs) are widely used as vaccine vectors due to their good stability and easy modification, and the investigations of VLPs is one of the hot spots in the field of bio-pharmaceutical engineering. However, VLPs may disassemble or aggregate due to their sensitivity to temperature, pH and other factors, which becomes the bottleneck hindering the widely application, and the underlying mechanisms which governs the structure and thus stability of VLPs is still ambiguous. In this work, molecular dynamics simulation was utilized to investigate the stability of the dimer, pentamer and hexamer formed by protein subunits in HBc-VLP. Instead of using empirical values in previous studies, the parameters of protein dielectric constant in aqueous solution were obtained by molecular dynamics simulations, and the results suggested that both the aqueous solvent and the arrangement of protein subunits in the complex could significantly change the dielectric constant, which further affected the binding free energy. Furthermore, with the dielectric constant of protein subunits, the binding free energy between protein subunits were calculated by the molecular mechanics-Poisson Boltzmann solvent accessible surface area (MM-PBSA) method. Finally, according to the calculation results, it was speculated that the stability of the hexamer was better than the pentamer, and the dimers formed between two adjacent hexamers or between a pentamer and a hexamer can further lead to a more stable structure. These understandings could provide theoretical guidance for the modification of candidate vaccine with HBc-VLPs as the carrier. Related Articles | Metrics Select Role of Rcs phosphorelay system on capsule synthesis of Klebsiella pneumoniae Shaoqi SUN Yike WANG Yang YANG Chenguang ZHU Jiping SHI Jian HAO Chin. J. Process Eng.    2021, 21 (2): 230-239.   DOI: 10.12034/j.issn.1009-606X.220104 Abstract （447）      PDF （654KB）（82）       Knowledge map    Save Klebsiella pneumoniae, capsulated bacterium, is an important industrial microorganism. Roles of the Rcs phosphorelay system on K. pneumoniae capsular polysaccharide synthesis were investigated in this work. In this research, rcsA and rcsB in K. pneumoniae were knocked out individually with the Red recombinase assisted gene replacement method. Capsule synthesized by K. pneumoniae ΔrcsA and K. pneumoniae ΔrcsB were both weak, therefore the transformation efficiency of cells was significantly increased. When these two strains were cultured with glucose or glycerol as a main carbon source, the yields of 2,3-butanediol or 1,3-propanediol produced by the cells were both higher than that of the wild-type strain. Cells of the two strains were more likely to agglutinate in the broth, which is favored for the downstream process. rcsA and rcsB over-expression strains were constructed, and the growth of the two strains were both slower than the wild-type strain. The transformation efficiency of rcsA and rcsB over-expressing strains both decreased compared with the wild-type strain. The titer of extracellular polysaccharide and the viscosity of fermentation broth of rcsA or rcsB over-expression strains increased. Especially, 10.33 g/L of extracellular polysaccharide was produced by the rcsA over-expression strain, which was five times of that of the wild type strain. On the whole, the regulation of rcsA and rcsB expression provides a novel way to influence the performance of K. pneumoniae. Related Articles | Metrics Select Analysis of preparation factors of ROP-PLGA microspheres with high encapsulation efficiency based on response surface method Kang WEN Yi WEI Guanghui MA Chin. J. Process Eng.    2021, 21 (1): 83-91.   DOI: 10.12034/j.issn.1009-606X.220050 Abstract （453）      PDF （2092KB）（141）       Knowledge map    Save As a novel amide local anesthetic, ropivacaine (ROP) is widely used in postoperative pain management. However, ROP has a short half-life (t1/2=1.8 h), and multiple doses in clinical is needed to meet the demand for analgesia, resulting in poor patient compliance. In this study, ROP-PLGA microspheres were prepared by using the premix membrane emulsification technique combined with the double emulsion-solvent evaporation method. Finally, the uniform microspheres with particle size of 7.831 μm and Span value of 0.874 were prepared under 1.5% (w/v) of PVA in external aqueous phase, 1:7.5 of volume ratio of oil phase-external aqueous phase (O/W2), 300 r/min of stirring rate of pre-double emulsion as well as 10 kPa of trans-membrane pressure. Additionally, factors such as the pH of external water phase, PLGA concentration in oil phase and internal water phaseoil phase volume ratios (W1/O) on the effect of encapsulation efficiency (EE) and the drug loading (DL) were investigated based on response surface method (RSM). The optimal formulation and process parameters designed by RSM were as follow: the pH of the external water phase was 11, PLGA concentration in oil phase was 15%(w/v), internal water phaseoil phase volume ratios (W1/O) were 1:10. Moreover, the model predicted the DL of 17.6 μg/mg and the EE of 53.89% were predicted by RSM model. In the meanwhile, repeatability test showed that the DL was (18.0±0.5) μg/mg and the EE was (55.7±2.69)%. Eventually, the relative error was insignificant (less than 7%), which meant the model was reliable. In vitro release profile showed that the cumulative release of ropivacaine loaded microspheres at three and five days was about 50% and 70% respectively, which indicated that the prepared ROP-PLGA microspheres had a stable sustained release effect and microsphere bioformulation had great potential in the field of sustained release of local anesthetics. Related Articles | Metrics Select Direct separation of human serum albumin from Cohn fraction V supernatant by one-step ion exchange chromatography Jie XIANG Songping ZHANG Guifeng ZHANG Jian LUO Rong YU Chin. J. Process Eng.    2021, 21 (1): 92-99.   DOI: 10.12034/j.issn.1009-606X.220073 Abstract （596）      PDF （489KB）（184）       Knowledge map    Save Fraction V supernatant is an effluent of Cohn fractionation in plasma protein industry. Due to its high ethanol concentration, further recovery of the residual protein has been regarded as non-economical. In this work, a recovery of human serum albumin (HSA) from fraction V supernatant by ion exchange chromatography was reported, which had not been reported in the literature to our knowledge. Firstly, bovine serum albumin (BSA) was used as model protein to compare the adsorption capacity of three different types of chromatographic media in different ethanol-aqueous solutions. The adsorption capacity of the hydrophobic medium to BSA in ethanol-aqueous solution was very weak, and the increase of ethanol concentration led to the adsorption capacity approaching to 0. The cation exchange medium had a high adsorption capacity at low ethanol concentration, but decreased quickly with the increase of the ethanol concentration. In contrast, the anion exchange medium showed the best adsorption performance, and the adsorption capacity in 40% ethanol-aqueous solution still reached 34.66 mg/mL. Further experiments showed that the adsorption of BSA on the anion exchange medium in the presence of ethanol could be described by Langmuir isothermal adsorption equation. The anion exchange medium DEAE Sepharose Fast Flow was packed into a chromatographic column. Real purification of Cohn fraction V supernatant was performed. The Cohn fraction V supernatant, containing about 40% ethanol, was directly loaded to the anion exchange column. A two-step elution strategy was used. The first elution was pH change from 7.0 to 4.5 to obtain the target product human serum albumin, and the second elution was to increase the concentration of sodium chloride from 0 to 1 mol/L to elute glycoproteins. The purity of HSA was 96.35% by electrophoresis, and the activity of binding to the ligand warfarin was comparable to that of commercial HSA product by Cohn fractionation. The total recovery was 43 mg/L Cohn fraction V supernatant. Related Articles | Metrics Select Cross-flow ultrafiltration refolding of ribonuclease A Xiangjuan WANG Xiunan LI Chao CHEN Zhiguo SU Guanghui MA Dawei ZHAO Rong YU Chin. J. Process Eng.    2020, 20 (12): 1455-1462.   DOI: 10.12034/j.issn.1009-606X.220038 Abstract （299）      PDF （557KB）（130）       Knowledge map    Save Performance of cross-flow ultrafiltration refolding strategy based on hollow fiber membrane for ribonuclease A was investigated. To improve the refolding efficiency, effects of operating conditions of ultrafiltration refolding on the activity yield and mass recovery were studied. Parameters of RNase A concentration (A), transmembrane pressure (B), circulation velocity (C) were chosen as the three test factors, with three level for each factor an L9(34) orthogonal array was implemented. Results of the orthogonal test showed that these three factors had a significant influence on the activity yield of the ultrafiltration refolding of ribonuclease A, whether based on variance analysis or range analysis. However, results of ANOVA (Analysis of Variance) showed that the three factors had no significant effect on mass recovery rate of ultrafiltration of ribonuclease A. Therefore, the optimal conditions for ultrafiltration of ribonuclease A were A1B1C2, that was, the concentration of refolding was 0.3 mg/mL, the transmembrane pressure was 34.0 kPa and the circulation velocity was 935 cm/min. Verification test was performed under the optimal conditions with the active yield of 92.31% and the mass recovery rate of 77.56%, respectively. Furthermore, the reliability of ultrafiltration refolding was confirmed by characterization of refolding products with circular dichroism, reversed-phase high performance liquid chromatography and high-performance gel filtration chromatography. Related Articles | Metrics Select Vaccine particle integrity-based purification of recombinant hepatitis B surface antigen using silica gel adsorption/desorption Shengjie HU Yongdong HUANG Lan ZHAO Kai ZHU Zhuang MIAO Fei WANG Hongchao JIN Jian LI Jun YANG Hemu WANG Guanghui MA Hongshui YUAN Chin. J. Process Eng.    2020, 20 (10): 1198-1209.   DOI: 10.12034/j.issn.1009-606X.219329 Abstract （575）      PDF （1198KB）（113）       Knowledge map    Save Low stability was one of the biggest problems during traditional purification process of recombinant hepatitis B surface antigen (rHBsAg). A combination process of silica gel adsorption/desorption and hydrophobic interaction chromatography was proposed in this work. The effect of pH on HBsAg stability was studied from the particle integrity of view using static light scattering, fluorescence spectrum and dynamic light scattering, respectively. HBsAg was purified by silica gel adsorption/desorption using response surface methodology for experimental design, followed by hydrophobic interaction chromatography, and both its morphology and particle integrity were studied. In acidic solution, the electrostatic repulsion of HBsAg particles decreased leading to aggregation when pH of solution was close to the isoelectric point of HBsAg. In alkaline solution, hydrophobic patches inside HBsAg particles were likely to be exposed leading to disaggregation. The silica gel adsorption/desorption process was optimized. When HBsAg activity recovery was taken as the response value, the optimum operating condition was as follows: adsorption pH was 7.43, desorption pH was 10.48 and desorption temperature was 55.4℃, and HBsAg activity recovery was 39.1%. When purification fold was taken as the response value, the optimum operating condition was as follows: adsorption pH was 7.16, desorption pH was 10.52 and desorption temperature was 55.1℃, and purification fold was 1.90. After hydrophobic interaction chromatography, HBsAg activity recovery was 49.73% and the particle integrity was 85.79% respectively. Compared with conventional hydrophobic interaction chromatography, the efficiency of HBsAg purification was improved greatly using this combination method, etc., HBsAg activity recovery increased by 31.99%, the particle integrity by 20.90%, and the particle stability by 22.93%, respectively. It provided a new idea for both high efficient recombinant HBsAg purification and improvement of antigen particle integrity. Related Articles | Metrics Select Secretory of a multicopper oxidase in Escherichia coli Tao YANG Jian CHEN Fang FANG Chin. J. Process Eng.    2020, 20 (10): 1210-1217.   DOI: 10.12034/j.issn.1009-606X.219370 Abstract （687）      PDF （1070KB）（272）       Knowledge map    Save Biogenic amines (BAs) are organic compounds that present in fermented foods. The excessive intake of BAs is harmful to human health. Some enzymes belonging to the multicopper oxidase (MCO) family exhibit the activity of degrading a variety of BAs. Thus, they may have good application prospects in reducing ammonia (amine) hazards levels in fermented foods. It is of great significance to accomplish the secretion of multicopper oxidase for the purpose of modification of enzyme catalytic properties for its industrial production and applications. In this work, the secretion of multicopper oxidase in Escherichia coli was achieved by fusing the signal peptide PhoA to the N-terminal of MCOB from Bacillus amyloliquefaciens with an extracellular activity of 69.8 U/L. Secretory of MCOB was improved by optimizing the induction and secretion conditions. The optimal fermentation conditions for MCOB were determined to be: the induction temperature was 25℃, the IPTG concentration was 0.05 mmol/L, induced when the cell density (OD600) reached 1.0, and 150 mmol/L glycine was added after 6 h of induction. After 40 h of fermentation, the extracellular activity of MCOB reached 238.1 U/L, which was 3.4 times of that before optimization. Related Articles | Metrics Select Isolation and identification of XXG a strain of Paenibacillus with algae-lysing ability and study on algae-lysing characteristics Jingjing XUE Meijuan WANG Linqiang MAO Mingfei ZHAN Jun NING Wenyi ZHANG Chin. J. Process Eng.    2020, 20 (9): 1097-1105.   DOI: 10.12034/j.issn.1009-606X.219303 Abstract （351）      PDF （1108KB）（164）       Knowledge map    Save A strain of bacteria XXG was isolated from rice fields irrigated from Taihu Lake. The bacterial genus was determined by analyzing its morphological characteristics, physiology biochemistry experiment and 16SrDNA sequence. Single factor test was conducted on the addition ratio of medium, the volume ratio of bacteria to algae, the density of bacteria and density of algae to study the effect of each factor on the effect of lysing algae. Box-Behnken Design (BBD) was used to design three major factors, namely, temperature (X1), pH (X2) and shaker speed (X3), to optimize the environmental factors that affect the lysing rate of algae. The method of lysing algae was initially examined by comparing the effect of bacterial weight suspension and sterile fermentation liquid on algal liquid. The experimental results showed that the similarity degree between XXG and Paenibacillus sp. KU573975 was 99.27%, and was initially identified as Paenibacillus. When the optimum volume ratio of bacterial to algal was 5.6%, the lysing rate of XXG bacteria to Microcystis aeruginosa solution at the early logarithmic growth stage reached at 77.1% after 6 d. Three important environmental factors that affected the efficiency of XXG algal lysing had a cross effect. The quadratic regression model of lysing rate of algae with the temperature, pH and shaker speed was established with P<0.0001 and R2 was 0.9727. Under the optimal algae-lysing characteristics, the lysing rate of algae was the highest when the temperature was 30℃, the pH was 7.5, and the shaking speed was 150 r/min, and the lysing rate of algae was up to 92.02%. Aseptic fermentation liquid without algal bacterial still had algae-lysing effect. It was speculated that the main algae-lysing processing of XXG bacteria was indirect effect. The reduction in algae-lysing effect produced by aseptic fermentation liquid compared with bacterial fermentation liquid may be achieved by the bacteria in bacterial fermentation liquid continuing to secrete some algae-lysing active substances. Related Articles | Metrics Select Preparation and evaluation of polyvinyl alcohol/bovine type I collagen scaffolds Di MENG Xiongxin LEI Yang LI Dawei HUANG Guifeng ZHANG Chin. J. Process Eng.    2020, 20 (9): 1106-1113.   DOI: 10.12034/j.issn.1009-606X.219312 Abstract （382）      PDF （819KB）（87）       Knowledge map    Save Collagen is an excellent biomedical material with biocompatibility and biodegradability. However, the poor stability and low mechanical strength of collagen limit its application. Polyvinyl alcohol has good physical and chemical properties which can form a good bio-scaffold material by chemically coupling with collagen. Bioartificial liver (BAL) support has been researched and widely used in providing a bridge to patients waiting for a liver transplant or promoting liver regeneration, which can prevent complications caused by liver failure and improve survival rate. The scaffolds of BAL are important for the reproduction and biological function of liver cells. Polyvinyl alcohol (PVA) and collagen (COL) composite scaffolds was prepared in this work. PVA was modified by the amino silane and collagen crosslinked by glutaraldehyde. Then the heterogeneous mixture was treated by lysine and freeze-dried to obtain the PVA/COL composite scaffolds. The physical and chemical properties of the scaffolds were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). The biological properties of the scaffolds were evaluated by laser confocal, scanning electron microscopy (SEM) and CCK-8 experiments. FT-IR and XPS experiments demonstrated that collagen was successfully coupled to the PVA sponge and maintained a good triple helix structure. The results showed that the pore diameter of PVA modified by COL had uniform pore distribution with porosity of 21.33% and pore diameter of 168.68 μm. The contact angle of the scaffold material was 20.03°. The biological evaluation of scaffold materials showed that C3A cells adhered well on the PVA/COL scaffold and the proliferation test showed that the cells grew well on the composites. The proliferation of cells on the composite scaffolds was significantly different from the control group (P?0.01). The scaffolds prepared by the combination of PVA and COL had a promising application with the good physical and chemical properties. Related Articles | Metrics Select The effects of salinity on microbial activity and N 2O release in anoxic–aerobic sequencing batch biofilm reactor Youkui GONG Yinglong YUE Yongzhen PENG Chin. J. Process Eng.    2020, 20 (8): 970-978.   DOI: 10.12034/j.issn.1009-606X.219325 Abstract （525）      PDF （604KB）（126）       Knowledge map    Save Accumulation of nitrites is frequently observed in saline wastewater nitrification processes, which often results in the release of the strong greenhouse gas, nitrous oxide (N2O). This study investigated the effect of salinity on microbial activity and N2O release characteristics during the simultaneous nitrification and denitrification processes in a sequencing batch biofilm reactor (SBBR). The result showed that salinity inhibited the microbial activities of each bacterial group in increasing, sequential order as follows: nitrite oxidizing bacteria (NOB)>ammonia oxidizing bacteria (AOB)>carbon oxidizing bacteria. The effluent COD was stable at about 50.0 mg/L in the range of salinity from 0 to 20 g NaCl/L. The average NH4+ removal efficiency reduced from more than 98% to 70.5%, and TN removal efficiency reduced from 42.4% to 16.9% respectively, while the N2O yield increased from 3.9% to 13.3%. Similar to SND efficiency, the internal concentration of carbon sources (PHA and Gly) first increased and then decreased with increase in salinity. The N2O release was mainly produced in AOB aerobic and endogenous denitrification processes. The low N2O release could be ascribed to the anoxic zone deep in the SBBR at low salinity (≤10 g NaCl/L). As the salinity was increased to more than 10 g NaCl/L, there was a decrease in internal carbon source synthesis, which aggravated the electron competition between each bacterial reductases in the denitrification process. Further increase in salinity led to an increase in extracellular polymer substances (EPS) synthesis and the proportion of polysaccharides (PS) in the EPS. A decrease in the anoxic area deep in the membrane led to the inhibition of the N2O reduction. Related Articles | Metrics Select Optimization of reaction conditions for preparation of penicillin G sulfoxide by direct oxidation of penicillin fermentation broth Feng YAN Weige ZHANG Chin. J. Process Eng.    2020, 20 (6): 711-717.   DOI: 10.12034/j.issn.1009-606X.219233 Abstract （531）      PDF （677KB）（189）       Knowledge map    Save The process of direct oxidation of penicillin G sulfoxide with penicillin fermentation broth was studied with peroxyacetic acid as oxidant, and a series of experiments were conducted to investigate the effects of different influencing factors on the conversion rate of penicillin G sulfoxide. The residual penicillin in the mycelium after oxidation was analyzed. The direct oxidation process of penicillin fermentation broth was established and optimized. The results showed that the stirring speed, reaction temperature, peroxyacetic acid feeding dosage and peroxyacetic acid concentration were the key factors affecting the conversion rate of penicillin G sulfoxide, other factors had little effect on the process. Peroxyacetic acid directly oxidized the penicillin fermentation broth and released penicillin remaining in the mycelium. So its conversion rate was higher than the conversion of penicillin G potassium salt. The optimum oxidation process conditions were reaction temperature of 5~10℃, stirring speed of 100 r/min, adding a high concentration of peroxyacetic acid in a molar ratio of 1.3 times of penicillin at 30 minutes, stirring reaction for 10 minutes after adding peroxyacetic acid. The conversion rate of penicillin G sulfoxide reached 98.6%, and the conversion rate increased by 1.2% than that of penicillin G potassium salt. Related Articles | Metrics Select Study of immobilized glycerol dehydrogenase on modified agarose microspheres Min ZHANG Lin HAN Yueyue HU Jian LI Bing YAN Chin. J. Process Eng.    2020, 20 (5): 591-598.   DOI: 10.12034/j.issn.1009-606X.219263 Abstract （478）      PDF （1360KB）（186）       Knowledge map    Save 1,3-dihydroxyacetone (DHA) is one of the most valuable chemicals with a wide range of applications in cosmetics and pharmaceutical industry. Glycerol dehydrogenase (GlyDH) is able to selectively catalyze the transformation of glycerol to DHA, by which glycerol, the oversupply by-product of the biodiesel industry is highly valued. To make enzymatic production of DHA practically feasible, immobilizing GlyDH onto a suitable insoluble support is critical for facilitating biocatalyst recovery and improving stability of the enzyme. Agarose microspheres as carrier were prepared by membrane emulsification method. Then the agarose microspheres were activated with epichlorohydrin and reacted with polyethyleneimine (PEI) to introduce the amino group into the agarose microspheres. The morphology and structure of the microspheres were characterized by scanning electron microscope (SEM), energy dispersion spectrum (EDS), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). GlyDH was covalently fixed with glutaraldehyde and then catalyzed the formation of 1,3-dihydroxyacetone. The results showed that the spherical degree of microspheres was enhanced due to the membrane emulsification process. The N content in grafted products was obviously effected by the relative molecular weight of PEI. The N content of PEI graft was the highest when the relative molecular mass of PEI was 10000. The effect of pH and temperature dependence, thermal stability and the reusability of GlyDH were systematically investigated. The optimum pH value of free enzyme and immobilized enzyme activity was 10.0. The storage efficiency of immobilized GlyDH was higher than that of free GlyDH after storage at 4℃ for 21 days. After 7 cycles, the activity of GlyDH was still partly remained. Related Articles | Metrics Select Stability and biofouling behavior of plastic films in microalgae cultivation Yixuan WANG Chenghu YAN Wei CONG Chin. J. Process Eng.    2020, 20 (1): 74-83.   DOI: 10.12034/j.issn.1009-606X.219175 Abstract （630）      PDF （2483KB）（190）       Knowledge map    Save The stability of six typical kinds of plastic films including polyethylene (PE), polypropylene (PP), ethylene/vinyl acetate (EVA), polyvinyl chloride (PVC), polyurethane (PU) and polyethylene terephthalate (PET) in NaClO, NaOH and HCl solutions and biofouling behavior in microalgae cultivation (Chlorella vulgaris) system were studied. The results showed that PVC film performed the poorest stability, and the transmittance decreased about 50% after immersing in NaClO, NaOH and HCl solutions. The transmittance of PU film and EVA film declined 10%?15% in NaClO and NaOH solutions, respectively. The transmittance of the other three kinds of plastic films has no significant change when immersed in NaClO, NaOH and HCl solutions for 24 h. Obvious microalgae biofouling behavior happened in six kinds of films, which was a typical biofilm formation process. Adhesion behavior of PVC surface was the most distinct, and the transmittance declined to about 0 on the 7th day, and the amount of adsorption solids increased with time and reached 3069 ?g/cm2 on the 45th day. However, the amount of adsorption solids on PU, EVA, PE, PP and PET film surface increased sharply and then decreased with time, the maximum amounts were 292, 375, 292, 194 and 236 ?g/cm2, respectively. The increase of chlorophyll content on the surface of the plastic films was not obvious in the first 4 d, which indicated that the initial stage of the adhesion was mainly the adhesion of protein and extracellular polymeric substances (EPS), and then microalgae cells began to adhere. The attachment of protein, EPS and microalgae cells on the plastic films was a typical biofilm formation process. The adhesion behavior of microalgae was affected by chemical structure and surface properties of the films, such as hydrophilicity, roughness and surface charge. For Chlorella vulgaris, polyolefin (PE, PP, etc.) and PET film surface with high hydrophilicity, low roughness and negatively charged will perform good anti-biofouling properties. Reference | Related Articles | Metrics Select Preparation of biomimetic peptide attached supermacroporous poly(glycidyl methacrylate) microspheres for monoclonal antibody purification and its performance Jia GE Xiangming NA Xuexing WU Weixing YANG Dongxia HAO Guanghui MA Chin. J. Process Eng.    2020, 20 (1): 84-90.   DOI: 10.12034/j.issn.1009-606X.219166 Abstract （589）      PDF （1346KB）（145）       Knowledge map    Save Recently, immunoglobulin G (IgG) has attracted great attentions in clinical medicine, biotechnology, stimulated the development of downstream purification technology of monoclonal antibodies. Now, the protein based affinity ligands during traditional chromatographic purification of IgG exist drawbacks including the high cost and low stability, which leads to the secondary contamination and biological toxicity to purified sample. Also the chromatographic agarose microspheres present shortcomings such as the gel compressibility and the mass transfer limitations. In this work, biomimetic peptide as the protein based ligands alternative, grafting on the supermacroporous dextran-poly(glycidyl methacrylate) microspheres (Dextran-PGMA) to overcome above drawbacks were prepared and to efficiently improve the IgG chromatographic separation. The hydroxy on dextran-grafted polymer chromatographic microspheres were converted to the epoxy groups in epichlorohydrin solution with 2 mol/L NaOH, and the epoxy groups were opened and coupled with biomimetic peptide, FYEILCH, to achieve the purpose of affinity modification. The SEM observed that the pore structure of macroporous was mostly maintained after coupling with the biomimetic peptide. The change of 10% dynamic binding capacity (DBC10%) of FYEILHC based on Dextran-PGMA microspheres and agarose microspheres under different flow rate (92~923 cm/h) was compared. The DBC10% of dextran-PGMA was declined only 8% under 923 cm/h, whereas the DBC10% of agarose was sharply declined about 25%. This phenomenon might be related to better mass transfer for IgG at higher flow rate on the Dextran-PGMA microspheres. The DBC10% of Dextran-PGMA microspheres maintained (21±1) mg/mL after cleaning-in-place (CIP) 40 cycles using 0.1 mol/L NaOH, indicating that the affinity medium had good chemical stability. The purity of recovered antibodies in plasma was 95.0%, demonstrating that this biomimetic peptide based affinity medium had great potential to purify IgG from complex biological samples. This biomimetic peptide grafted supermacroporous poly(glycidyl methacrylate) microspheres could meet the requirement for rapid and high-throughput separation of monoclonal antibody. Reference | Related Articles | Metrics Select Optimization of enzymatic hydrolysis conditions for antioxidant peptide preparation from velvet antler collagen by response surface methodology Yaru LAN Shuo HUANG Fei ZHAO Hongyu WU Yongxue GUO Chin. J. Process Eng.    2020, 20 (1): 91-98.   DOI: 10.12034/j.issn.1009-606X.219174 Abstract （546）      PDF （562KB）（129）       Knowledge map    Save It is believed that the velvet antler head has the best health benefits in traditional Chinese medicine. But its output is small, and the price is high. The first section of velvet antler is used frequently, and its protein/peptide content is highest among all sections. The polypeptide molecular weight is about 5 kDa. In this work, in order to make full use of the velvet antler resources, the water extract of the middle and low sections collagen of velvet antler was used as an enzymatic substrate to prepare an antioxidant peptide using papain. Enzymatic hydrolysis conditions (time, enzyme addition ratio, pH and temperature) were optimized by response surface methodology (RSM). The 1,1-diphenyl-2-picrylhydrazyl (DPPH?) radical-scavenging activity rate was used as an index. The target antioxidant peptide (molecular weight less than 5 kDa) obtained by separation through an ultrafiltration membrane was further separated by reversed-phase high-performance liquid chromatography. And 10 components, S1?S10, were obtained respectively and the components were lyophilized. Component S3 was further subjected by reversed-phase ultra-high performance liquid chromatography (UPLC) on-line electrospray ionization mass spectrometry (ESI-MS) for molecular weight determination. The polypeptide with the highest antioxidant activity was fractionated by ultrafiltration membranes of molecular weight cut off of 50, 10 and 5 kDa respectively. The results showed that the optimal experimental conditions were time of 56 min, enzyme addition of 1.40wt%, pH of 5.60, temperature of 60℃, DPPH? radical-scavenging activity rate was 83.09%. The molecular weight of polypeptide was 0.2?0.6 kDa. The resulting small molecule peptide has similar health benefits as the head section, is more easily absorbed by the body, and is easier to further process and store. Reference | Related Articles | Metrics Select Molecular weight and molecular weight distribution of alginate determination by gel permeation chromatography Ruixin ZHANG Zhihui WANG Wei CONG Fengji HUI Bin WANG Chin. J. Process Eng.    2020, 20 (1): 99-107.   DOI: 10.12034/j.issn.1009-606X.219177 Abstract （583）      PDF （464KB）（177）       Knowledge map    Save A method for the determination of molecular and molecular weight distribution of alginate by gel chromatography and conventional detector was proposed. Alginates in a wide range of viscosities extracted from two dominated raw materials—Lessonia nigrescence and Lessonia trabeculata were taken as the samples. The relative molecular weight of alginate was determined by high-performance gel permeation chromatography (GPC) combined with refractive index detector (RID) with pullulan served as a standard. The corresponding relations of relative weight average molecular weight ( ), relative number average molecular weight ( ) measured by GPC–RID and absolute weight average molecular weight ( ), absolute number average molecular weight ( ) measured by multi-angle laser scattering (GPC–MALLS) were obtained by least square method. The results showed that there was a good linear correlation between the relative molecular weight of alginate determined by GPC–RID and the absolute molecular weight of alginate determined by GPC–MALLS. The correlation index was more than 0.9. The relative error between the absolute weight average molecular weight determined by the established GPC–RID method and the absolute weight average molecular determined by GPC–MALLS was less than ?12%. The relative error between the absolute number average molecular weight determined by the established GPC–RID method and the absolute number average molecular determined by GPC–MALLS was less than ?12%. The polydispersity index calculated by the established GPC–RID method was less than ?12%, compared with that obtained by GPC–MALLS method. The GPC–RID method for the determination of the relative molecular weight of alginate can be used to determinate the absolute molecular weight and the polydispersity index of alginate. It manifested that it was feasible and economical to use the GPC–RID method provided in this work for the determination absolute of the absolute weight molecular weight, absolute number molecular weight and polydispersity index of alginate. The method can save the investment on the instrument and reduce the cost of determination the molecular weight of alginate. Reference | Related Articles | Metrics Select Controllable preparation of novel charged nanodisc and its binding with cytochrome P450 Jiaoli TAO Yongdong HUANG Lan ZHAO Kai ZHU Xuexing WU Danni ZHOU Zhiguo SU Guanghui MA Hongying LIU Chin. J. Process Eng.    2019, 19 (6): 1197-1203.   DOI: 10.12034/j.issn.1009-606X.219121 Abstract （555）      PDF （2840KB）（181）       Knowledge map    Save Membrane proteins are associated with the phospholipid bilayers as integral membrane and have multiple and important functions, such as responsible for carrying out import and export of molecules and communication with the surrounding environment. It has been up to 60% of all currently used drugs target for therapeutic purposes and always been the emphasis of research in the fields of biology, medicine and material science. Membrane proteins are experiencing the bottlenecks due to their low contents, strong hydrophobicity and difficulty in purification. The pretreatment method of membrane proteins is to solubilize the membrane for isolation the membrane proteins using large amounts of detergents firstly, and then purified by reconstitution to restore their functions. Novel charged nanodiscs with both membrane protein binding and protecting were prepared with phospholipid and membrane scaffold protein using both nitrogen blow-drying method and rotary evaporation, respectively in this work. The nanodiscs had clear and transparent appearance, and they were uniform and disc-shaped with an average particle size of 10 nm and a charge of ?19.86 mV under pH 7.4, further size fractionation by gel filtration chromatography. By adjusting the preparation conditions, the properties of nanodiscs could be controlled well. The nanodiscs had a good binding to cytochrome P450 in liver microsome. The CO difference spectrum analysis results showed that the protein?nanodisc system exhibited a strong absorption peak at 450 nm. The content of cytochrome P450 was about 0.10 nmol/mg and the specific activity was improved 13.0 fold, 1.5 times higher than that of the traditional method, the operation time reduced from several days to several hours. It demonstrated that cytochrome P450 can be bound and protected simultaneously. Reference | Related Articles | Metrics Select Facile purification and stabilization of anti-Salmonella pullorum polyclonal immunoglobulin G Xingli YOU Yanli YANG Zhiguo SU Yuan ZHANG Songping ZHANG Chin. J. Process Eng.    2019, 19 (6): 1204-1211.   DOI: 10.12034/j.issn.1009-606X.219153 Abstract （554）      PDF （1222KB）（149）       Knowledge map    Save A single step separation protocol was developed for purification of anti-Salmonella pullorum polyclonal immunoglobulins G (IgG) from rabbit serum. According to the difference of isoelectric point (pI) between IgG and the major impurities, the types of ion exchange media were systematically screened by comparison of their purification results. In order to prevent the IgG from denaturation, different stabilizers were screened by differential scanning fluorimetry (DSF). The results showed that pI of the IgG was 6.04?7.08 determined by capillary iso-electric focusing. After cation exchange chromatography with packing materials of CM Sepharose Fast Flow (CM), the purity analyzed by SDS?PAGE was 63.5% and IgG recovery rate measured by high performance size-exclusion chromatography was 15.5%, respectively. In contrast, the purity of IgG was 99.3% and recovery rate was 67.5% after anion exchange chromatography with packing materials of Q Sepharose XL (Q-XL). 200 g/L sorbitol was found to possess the best protection effect. The two thermal denaturation temperatures of IgG were increased by 5.52 and 8.84℃, respectively, and the stability at 70℃ was significantly improved. The results demonstrated that the one step anion exchange chromatography together with 200 g/L sorbitol protection provided a high purity, high recovery rate, and high stability of the IgG. The whole process is facile and efficient. Reference | Related Articles | Metrics Select Direct production of curdlan oligosaccharides by coupled fermentation system of Agrobacterium sp.- Pichia pastoris Feifei LI Shuxia JIN Li ZHU Xiaobei ZHAN Yue ZHAO Liping LIU Minjie GAO Chin. J. Process Eng.    2019, 19 (4): 801-808.   DOI: 10.12034/j.issn.1009-606X.218319 Abstract （499）      PDF （3423KB）（232）       Knowledge map    Save Curdlan oligosaccharides are widely used in biomedicine and food. Agrobacterium sp. ATCC 31749 can produce curdlan under nitrogen-deficient conditions, and endo-?-1,3-glucanase can hydrolyze curdlan to curdlan oligosaccharides. To improve the production efficiency of curdlan oligosaccharides, a coupled fermentation system of Agrobacterium sp.?Pichia pastoris is constructed, in which the Agrobacterium sp. metabolite curdlan can be directly used to produce curdlan oligosaccharides by endo-β-1,3-glucanase (secreted by Pichia pastoris). This microbial consortium omits the steps of extraction, purification, and drying of curdlan and endo-β-1,3-glucanase, and it has considerable potential for use in the industrial production of curdlan oligosaccharides. The commercially available β-1,3-glucanase is a complex enzyme (it contains a variety of exoglucanases and endoglucanases), and the specific endoglucanase is difficult to obtain. In is work, to avoid the effects of exo-β-1,3-glucanase, the endo-β-1,3-glucanase (BGN13.1) was expressed in Pichia pastoris GS115 by different promoters (AOX1, GAP and FLD), and they were all verified to be effective in hydrolyzing curdlan to curdlan oligosaccharides, the endo-β-1,3-glucanase enzyme activities reached 51.24, 49.64, and 46.99 U/mL, respectively. On this basis, the Pichia pastoris engineered by the GAP promoter was selected for co-culture with Agrobacterium sp. to avoid the methanol-induced process, and soybean sprout extract was the optimal source of nitrogen for both curdlan and endo-?-1,3-glucanase production. In the end, the coupled fermentation system was divided into two stages to produce the best culture effect: Agrobacterium sp. cell growth stage (pH=7.0), and curdlan oligosaccharides production stage (pH=5.6). Potassium phosphate buffer was added to the medium to keep the pH of the fermentation broth within a constant range. When the initial inoculation ratio of Agrobacterium sp. to Pichia pastoris was 1:2, and fermentation time in shaker flasks was 79 h, respectively, the maximum production of curdlan oligosaccharides (degree of polymerization between 17 and 22) reached 4.278 g/L. Reference | Related Articles | Metrics Select Preparation and properties of a new alkali-resistant rProtein A chromatographic medium Wei WEI, Yongdong HUANG Lan ZHAO Xuexing WU Tianxiao ZHU Dongxue LI Haibo JIN Rongyue ZHANG Zhiguo SU Guanghui MA Chin. J. Process Eng.    2019, 19 (3): 609-616.   DOI: 10.12034/j.issn.1009-606X.218246 Abstract （717）      PDF （714KB）（208）       Knowledge map    Save Monoclonal antibodies (mAb) have been applied for curing a wide range of diseases and is considered to be a major source of new therapies in the next decades. mAb?s production has been received worldwide attention and efficient purification strategies have been always explored. Protein A chromatography was one of the most popular methods for mAb purification. Cleaning-in-place has been widely used in protein A chromatography for meeting the demand of high quality, and therefore an alkali-resistant ligand is necessary. Since protein A from natural sources is not alkali-resistant, it should be genetically modified. In this study, a new alkali-resistant rProtein A was constructed based on C-region gene construction followed by being coupled to epoxy-activated agarose-based microspheres under optimized conditions, and a new rProtein A chromatographic medium was prepared. Both confocal laser scanning microscopy and quartz crystal microbalance were used for analyzing the binding of hIgG to rProtein A chromatographic medium. The results showed that it was transparent and full bright. It had a uniform particle size distribution with an average diameter of 84 μm. This medium had good hydraulics properties with a maximum flow rate of 1400 cm/h. This new rProtein A chromatographic medium had a higher hIgG dynamic binding capacity of 62.0 mg/mL than that of commercial ligand. Also, this medium had a better cleaning-in-place performance and the dynamic binding capacity was kept to 84% of the initial value after 40 cycles, as beneficial for its application on an industrial scale. At the beginning of adsorption to the medium, hIgG was bound quickly to the surface coupled with rProtein A, however, the binding rate decreased gradually due to mass transfer resistance from the inner part of the medium. The desorption rate had a similar tendency to that of the adsorption. The new rProtein A chromatographic medium had great prospects in mAb purification and gave a good basis for its application in the future. Reference | Related Articles | Metrics Select High Cell Density Fermentation of Chlorella Based on Kinetics Model Youcai ZHOU Yongjin HE Linsheng LI Mingzi WANG Bilian CHEN Xing ZHENG Chin. J. Process Eng.    2018, 18 (3): 624-631.   DOI: 10.12034/j.issn.1009-606X.217318 Abstract （845）      PDF （357KB）（315）       Knowledge map    Save The kinetic model of batch fermentation of Chlorella sp. MBFJNU-17 in a 50 L fermenter was built, fed-batch strategy for high cell density cultivation was established. The application of carbon source was investigated. Furthermore, real-time quantitative PCR was used to determine the gene levels of key metabolic enzymes such as diaminopimelate isomerase (dapF), citrate synthase (CS) and glucose?6-phosphate dehydrogenase (G6PDH) in fed-batch fermentation. The results showed that the cell dry weight of Chlorella sp. MBFJNU-17 was 106.65 g/L, the average growth rate was 0.89 g/(L?h) and the yield of cell dry weight on glucose was 0.56 g/g, respectively, after fed-batch culture for 120 h. The gene levels of dapF, G6DPH and CS of Chlorella sp. MBFJNU-17 were strongly related with the concentrations of glucose and urea during the fermentation. Reference | Related Articles | Metrics Select Optimization of Cellulase Assisted Extraction Conditions for Sulfated Polysaccharide (Ascophyllan) from Ascophyllum nodosum by Response Surface Methodology Qingyun BAO Jingliuyi WEI Zedong JIANG Gang YU Gaoling HUANG Yanbing ZHU Anfeng XIAO Hui NI Qingbiao LI Chin. J. Process Eng.    2018, 18 (3): 632-638.   DOI: 10.12034/j.issn.1009-606X.217355 Abstract （834）      PDF （681KB）（336）       Knowledge map    Save In order to obtain the optimum parameters for the cellulase assisted extraction of ascophyllan from brown seaweed Ascophyllum nodosum, the effects of several factors including water to biomass ratio, cellulase concentration, enzymolysis temperature, and enzymolysis time on the extraction yield of ascophyllan were investigated by using a single factor experiment and further optimized by using Box-Behnken experiment and response surface methodology (RSM). The results revealed that the optimal parameters for ascophyllan extraction were ratio of water to biomass 30 mL/g, cellulase concentration 200 IU/mL, enzymolysis temperature 50℃, enzymolysis time 2.0 h. Under these conditions, the actual extraction yield of ascophyllan was 14.65%±0.73%, in keeping approach with the predicted value by RSM (14.75%). Therefore, it is feasible that utilized RSM to optimize the conditions of extracting ascophyllan with the assist of cellulase. Reference | Related Articles | Metrics Select Preparation of Collagen Peptides with Controllable Molecular Weight Range Based on Enzymatic Degradation Coupled with Membrane Separation Hao CUI Yujie MOU Kai WANG Jiyao KANG Guifeng ZHANG Minglin WANG Chin. J. Process Eng.    2018, 18 (2): 427-433.   DOI: 10.12034/j.issn.1009-606X.217123 Abstract （789）      PDF （425KB）（319）       Knowledge map    Save Collagen peptides with controllable molecular weight range were prepared by using enzymatic hydrolysis coupled with membrane separation. The effects of enzyme type, molecular weight and volume of filtration on the enzymolysis?membrane separation process was investigated. The results showed that the molecular weight of the permeated liquid treated with 3 kDa ultrafiltration membrane was mainly distributed in 4.0, 1.6 and 0.6 kDa, share ratio were 13.7%, 34.8% and 51.4%. The molecular weight of the permeated liquid treated with 8 kDa ultrafiltration membrane was mainly distributed in 8.3, 4.0, 1.6 and 0.6 kDa, share ratio were 14.5%, 22.7%, 37.7% and 25.1%. It was found that the protein conversion rate was increased by 15% compared with the traditional enzymatic hydrolysis process, thus, enzymatic degradation coupled membrane separation is possible strategy for preparation of uniform molecular weight collagen peptide. Reference | Related Articles | Metrics Select Preparation of Biochemical Agar Using Gracilaria Agar as Raw Material Jianhua DU Ting LI Hui NI Zedong JIANG Anfeng XIAO Yanbing ZHU Chin. J. Process Eng.    2018, 18 (2): 434-440.   DOI: 10.12034/j.issn.1009-606X.217228 Abstract （789）      PDF （387KB）（192）       Knowledge map    Save The key physicochemical properties of the domestic gracilaria agar and five kinds of commercial biochemical agars were investigated and compared, and the preparation technology of the biochemical agar was further studied by using the carboxymethyl modification method to decrease the melting temperature and promote the transparency of gracilaria agar and further optimized to produce biochemical agar. The results revealed that the gracilaria agar showed related higher melting temperature and lower transparency than these biochemical agars, which were the primary distinctive physicochemical properties between the gracilaria agar and these biochemical agars. The optimized preparation parameters were as follows: the amount of chloroacetic acid was 0.15 g/g and the reaction temperature was 60℃. Under these optimal conditions, 70.8% of Na2SO4 and 57% of NaOH can be reduced respectively through the reutilization of the reaction liquid. The physicochemical properties of synthetic carboxymethyl agar produced by the pilot-scale tests were in accordance with those obtained in the small-scale tests. Meanwhile, the synthetic carboxymethyl agar was consistent with the domestic and international quality standards of the biochemical agars. Moreover, the synthetic carboxymethyl agar possessed a better gel strength and transparency than those commercial biochemical agars. In addition, there is no significant difference between the carboxymethyl agar prepared by the pilot-scale tests and the commercial biochemical agars such as Sigma A6686 and 028990 in the microbial growth tests. The raw materials cost of carboxymethyl agar (1 t/d) is ￥120700. Reference | Related Articles | Metrics Select Influences of Surface Characteristics of Energy Microalgae on Its Harvesting Performance by Air Flotation Zhou SHEN Hao WEN Xiangying REN Jun LIU Liwei YANG Yanpeng LI Chin. J. Process Eng.    2018, 18 (2): 441-446.   DOI: 10.12034/j.issn.1009-606X.217271 Abstract （806）      PDF （382KB）（246）       Knowledge map    Save Two algal species Chlorella vulgaris and Anabaena vasriabilis were chosen as typical energy microalgae to investigate the surface characteristics of microaglae. The surface potential and hydrophobicity was examined based on the extend XDLVO theory. According to the Zeta potential, hexadecyl trimethyl ammonium bromide (C16TAB) was used as cationic surfactant for flotation. The results showed that both two strains carried with negative charge, and free energy of cohesion for C. vulgaris was 1.21 mJ/m2, and ?55.85 mJ/m2 for A. varsriabilis, which meant hydrophilicity and hydrophobicity respectively. A. varsriabilis performed better harvesting rate than C. vulgaris. The maximums concentration factor (12.45 for C. vulgaris and 1.3 for A. varsriabilis) toped at the pH which charged most negatively (pH=7 for C. vulgaris and pH=9 for A. varsriabilis). Nevertheless, harvesting rate toped at pH=10, which meant it could not reach the maximum with the concentration factor in the same condition, may owing to the drainage of the tonoplast. Meanwhile, after adding 80 mg/L C16TAB into the algal solution, the hydrophobicity rate for C. vulgaris raised from 19% to 64%, and the harvesting rate increased by 67.38%. Reference | Related Articles | Metrics Select Effects of Organic Media Treatment Saccharomyces cerevisiae on Its Catalytic Performance to Asymmetric Reduction Reaction Feng LI Shiyin FANG Xianai SHI Chin. J. Process Eng.    2017, 17 (6): 1281-1286.   DOI: 10.12034/j.issn.1009-606X.217217 Abstract （782）      PDF （452KB）（138）       Knowledge map    Save Different organic media were used to induce changes of cell membrane properties, i.e. the membrane permeability, mobility and integrity. The effect of organic media treatment on the asymmetric reduction of 2-octanone catalyzed by Saccharomyces cerevisiae cells was investigated. The results showed that the reaction yield was significantly decreased when the leakage values of nucleic acid and protein reached 0.057 and 0.046 respectively, or the propidium iodide (PI) uptake factor reached 1.157. When the anisotropy value of cell membrane labeled with diphenylhexatriene (DPH) reached 111.7% of the control group, the cells lost the ability of catalyzing asymmetric reduction. When the ratio of cell with damaged membrane integrity reached 41.8%, the reaction yield was significantly reduced. In the premise of maintaining the integrity of cell membrane, cell membrane permeability and mobility in the appropriate range make Saccharomyces cerevisiae cells show good performance on catalyzing 2-octanone asymmetric reduction reaction. Reference | Related Articles | Metrics Select Expression of CRM197 in E. coli System and Its Application in Universal Influenza Vaccine Lu XU Jing ZHANG Rong YU Zhiguo SU Chin. J. Process Eng.    2017, 17 (5): 1054-1058.   DOI: 10.12034/j.issn.1009-606X.217132 Abstract （1296）      PDF （765KB）（245）       Knowledge map    Save Express the nontoxic mutant of diphtheria toxin (CRM197) in E. coli BL21 (DE3) by gene engineering technique and purify the protein by affinity chromatography under denaturation. As the carrier protein, recombinant CRM197 was conjugated with influenza antigen M2e via BMPH to prepare the conjugate vaccine to immune BALB/c mice. Determine the M2e specific IgG in sera by indirect ELISA. The results showed that rCRM197 was expressed in E. coli system successfully with optimized level (250±30 mg/L) and high purity (>95%). The conjugate vaccine CRM197?M2e prepared by chemical coupling in denaturation could effectively immunize BALB/c mice. And the M2e-specific IgG antibody titer elicited by CRM197?M2e conjugate vaccine was significantly higher than that in healthy, M2e, CRM197 and M2e+CRM197 mixed groups by 430.5, 32, 181 and 215 times. Reference | Related Articles | Metrics Select Pretreatment Method of Lessonia trabeculata Stipe Chuanfeng AN Zhihui WANG Bin WANG Fengji HUI Wei CONG Chin. J. Process Eng.    2017, 17 (5): 1059-1065.   DOI: 10.12034/j.issn.1009-606X.217152 Abstract （880）      PDF （441KB）（145）       Knowledge map    Save The water absorption characteristics of Lessonia trabeculata stipe and the effects of smashing and squeezing pretreatment methods on water absorption were investigated. The results showed that the critical penetration distance of water in the stipe was about 2 mm. When the size of stipe particles was twice times less than the critical penetration distance, the algal particles could be sufficiently penetrated and saturated by water in 6 h. As the crack of stipe was grown and enlarged with the increase of squeezing pressure, the water absorption rate increased to 250% at 149 MPa in 6 h. Since stipe was sufficiently soaked in water after pretreatment, the alkaline extraction speed could be remarkably raised. Especially when some cell walls were destroyed by squeezing, the extraction speed was the highest during the earlier 3 h of extraction. Reference | Related Articles | Metrics Select Optimization of fermentation process for production of glutamic acid oxidase from Rhodococcus opacus Jia LIU Jisi XU Qiuling LUO Xiulai CHEN Liming LIU Chin. J. Process Eng.    2017, 17 (4): 814-820.   DOI: 10.12034/j.issn.1009-606X.216335 Abstract （947）      PDF （358KB）（185）       Knowledge map    Save The optimization of fermentation conditions for the independent screening strain Rhodococcus opacus FMME1-41 was carried out. The optimal nutrition conditions was as follows: 6 g/L yeast powder, 2 g/L soya peptone, 0.8 g/L (NH4)2SO4, 25 g/L glucose, 3 g/L KH2PO4, 0.6 g/L MgSO4, 0.3 g/L MnSO4, 2.5 g/L glutamic acid. In addition, fermentation period was 30 h, the LOGX activity reached 6.12 U/mL. Feeding strategy was optimized in 7.5 L fermentation tank. Under the condition of combination of bat. ch fermentation and DO-stat feeding, the fermentation period was 40 h, and the LOGX activity was 21.5 U/mL, which is 2.8 times of the batch fermentation. LGOX was used to produce α-ketoglutaric acid (α-KG) from L-glutamic acid in 2 L systerm. Finally, the maximum titer of α-KG reached 92 g/L, the molar conversion rate was 92.6%, the production intensity of α-KG was 9.2 g/L/h. Reference | Related Articles | Metrics Select Preparation and Properties of Amphipathy Furcella Microspheres Linlin HU Yi WEI Yusong XU Dianchun JU Guanghui MA Chin. J. Process Eng.    2017, 17 (4): 821-826.   DOI: 10.12034/j.issn.1009-606X.216372 Abstract （1039）      PDF （4348KB）（451）       Knowledge map    Save Furcella methoxy poly(ethylene glycol-L-lactic-co-glycolic acid) (mPEG-PLGA) microspheres were successfully prepared by double emulsion method with solvent evaporate method. The effects of polymer components,LA/GA molar ratio,volume ratio of internal water phase to oil phase,volume ratio of oil phase to external water phase and solidification rate on morphology and encapsulation of microspheres were investigated. The results showed that the microspheres with furcella morphology were prepared by the condition of 1/19 polymer components,50/50 LA/GA molar ratio,0.2/1 volume ratio of internal water phase to oil phase,1/10 volume ratio of oil phase to external water phase and 500 rpm solidification rate. In addition, its encapsulation efficiency is as high as 70%, specifica surface area of furcella microspheres is as fourteen times as the smooth microspheres. Reference | Related Articles | Metrics Select Relationship between Gene Expression of ADH Isozymes and Asymmetric Reduction of 2-Octanone with Saccharomyces cerevisiae xian'ai Shi Haihong LIN Xiaoping WANG Shiyin FANG Chin. J. Process Eng.    2017, 17 (4): 827-833.   DOI: 10.12034/j.issn.1009-606X.216350 Abstract （890）      PDF （370KB）（285）       Knowledge map    Save In the water/n-dodecane biphasic reaction system, the influence of organic solvent treatment on the gene level of isozymes of alcohol dehydrogenase (ADH) 1~3 was investigated to probe their correlation in asymmetric reduction of the substrate 2-octanone by Saccharomyces cerevisiae type II. The results showed that after treatment with organic media, the relative level of isozymes of alcohol dehydrogenase (ADH) varied, along with the different properties shown in asymmetric reduction of 2-octanone treatmented by Saccharomyces cerevisiae, the gene level of ADH 1 was severely inhibited by toluene and 2-octanol, but it was not obviously influenced by n-dodecane, n-octane and n-hexane, while substantially enhanced by 2-octanone. However, the gene level of ADH 2 was highly increased by toluene and 2-octanol, but inhibited by n-dodecane, n-octane, n-hexane, and 2-octanone. Furthermore, the inhibition of ADH 1 gene would reduce the catalytic activity, and inhibition of ADH 2 gene would improve the enantiomeric selectivity, while the gene level of ADH 3 would not influence both the activity and selectivity. The isozyme ADH 1 which has good enantio selectivity is the key oxidoreductase for catalyzing asymmetric reduction of 2-ocatnone by Saccharomyces cerevisiae type II, and the isozyme ADH 2 is of poor enantio selectivity while it conducts the same reaction. Reference | Related Articles | Metrics Select Expression, Purification and Crystallization of Inactivated Serine Protease Domain of Matriptase Tuanyu GUO Baoyu ZHAO Cai YUAN Mingdong HUANG Chin. J. Process Eng.    2017, 17 (4): 834-838.   DOI: 10.12034/j.issn.1009-606X.216364 Abstract （1182）      PDF （525KB）（382）       Knowledge map    Save An inactivated mutant of matriptase serine protease domain (S195A) was constructed, and the recombinant protein in Pichia pastoris was expressed. After captured by anion exchange chromatography, the recombinant protein was further purified by gel-filtration chromatogram column and resource Q anion exchange column with high purity. High quality crystals of this inactivated protein were obtained by sitting-drop vapor diffusion. The results showed that the single point mutant increased the level of matriptase compared with the wild type. This inactivated mutant forms stable complex with its inhibitor HAI-1, although it does not possess catalytic activity. Crystals of this inactivated protein were diffracted to 1.48 ?. The mutant has the same conformation as the wild type. Related Articles | Metrics Select Construction and Analysis on the Kinetic Model of Gibberellin Acid in Batch Fermentation Wei WANG Yaohui WU Jilie LI Yuefei YAO Chin. J. Process Eng.    2017, 17 (3): 605-612.   DOI: 10.12034/j.issn.1009-606X.216304 Abstract （782）      PDF （1593KB）（162）       Knowledge map    Save The concentration changes of mycelium, residual sugar, amino nitrogen and gibberellic acid (GA3) were recorded in a 5 L automatic fermenter. Based on the analysis of fermentation process, a two-stage differential kinetic model, including mycelium growth stage (0~64 h) and GA3 production phase (64~192 h), was constructed. By using the model, the initial carbon, nitrogen concentration and carbon-nitrogen mass ratio (C/N) in the fermentation medium were optimized. Within the scope of the carbon concentration and nitrogen concentration, the calculated results of models were compared satisfactorily with experimental data. 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