The temporal and spatial growth behaviour of protein crystals, subject to different cooling strategies in protein crystallisation was investigated. Although the impact of temperature and cooling rate on crystal growth...The temporal and spatial growth behaviour of protein crystals, subject to different cooling strategies in protein crystallisation was investigated. Although the impact of temperature and cooling rate on crystal growth of small molecules was well documented, much less has been reported on their impact on the crystallisation of proteins. In this paper, an experimental set-up is configured to carry out such a study which involves an automatic temperature controlled hot-stage crystalliser fitted with a real-time imaging system. Linbro parallel crystallisation experiments(24-well plate) were also conducted to find the suitable initial conditions to be used in the hot-stage crystallisation experiments, including the initial concentration of HEW lysozyme solutions, precipitate concentration and pH value. It was observed that fast cooling rates at the early stage led to precipitates while slow cooling rates produced crystal nuclei, and very slow cooling rates, much smaller than for small molecules are critical to the growth of the nuclei and the crystals to a desired shape. The interesting results provide valuable insight as well as experimental proof of the feasibility and effectiveness of cooling as a means for achieving controlled protein crystallisation, compared with the evaporation approach which was widely used to grow single large crystals for X-ray diffraction study. Since cooling rate control can be easily achieved and has good repeatability, it suggests that large-scale production of protein crystals can be effectively achieved by manipulating cooling rates.展开更多
The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high final refolded HEWL concentration (1 mg/mL). Hexadecyltrimethylammonium bromide (CTAB) and sucrose fatty acid monoester (D...The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high final refolded HEWL concentration (1 mg/mL). Hexadecyltrimethylammonium bromide (CTAB) and sucrose fatty acid monoester (DK-SS) were used to dissolve denatured HEWL without denaturants such as guanidine hydrochloride (GuHCl) and urea. When denatured HEWL was perfectly dissolved in buffer solutions containing surfactants and dithiothreitol (DTT), the concentration of CTAB was about one-twentieth times less than that of DK-SS. The concentration of CTAB strongly affected the refolding yield, and the maximum refolding yield was obtained at 0.88 mM CTAB, which is around the critical micelle concentration of CTAB. The refolding yield was influenced by the molar ratio of oxidized glutathione (GSSG) to DTT, and the maximum refolding yield was obtained when [GSSG]/[DTT] was 1.5. The refolding yield was markedly dependent upon the solution pH of HEWL, and exhibited 80% at pH 5.2.展开更多
The influence of minor environmental factors,such as the geomagnetic field,on the biomineralization of nacres,is often ignored but a great deal of research has confirmed its important role in the normal mineralization...The influence of minor environmental factors,such as the geomagnetic field,on the biomineralization of nacres,is often ignored but a great deal of research has confirmed its important role in the normal mineralization of calcium carbonate.Although the geomagnetic field is weak,its cumulative effects need to be considered given that the biomineralization process can take years.Accordingly,the authors of this paper have investigated the effects of weak magnetic fields(25 Gs or 50 Gs)on calcium carbonate mineralization and analyzed the mechanism involved.The results show that even a weak magnetic field conduces to the formation of vaterite or aragonite,in the induction order of precursor→vaterite→aragonite.The stronger the magnetic field and the longer the time,the more obvious the induction effect.The effect of a magnetic field is strongest in the aging stage and weakest in the solution stage.Inductions by egg-white protein and by a magnetic field inhibit each other,but they both restrict particle growth.These findings highlight the importance of minor environmental factors for biomineralization and can serve as a reference for biomimetic preparation of a CaCO_(3)nacre-like structure and for anti-scale technology for circulating cooling water.展开更多
探讨了单酶及双酶分步酶解鸭蛋清蛋白制备多肽的工艺和水解效果。在7种不同来源的蛋白酶中,蛋清水解专用蛋白酶、碱性蛋白酶Alcalase AF 2.4L和胰蛋白酶均显示出了较好的酶解效果,其中以碱性蛋白酶Alcalase AF 2.4L酶解效果最佳,酶解的...探讨了单酶及双酶分步酶解鸭蛋清蛋白制备多肽的工艺和水解效果。在7种不同来源的蛋白酶中,蛋清水解专用蛋白酶、碱性蛋白酶Alcalase AF 2.4L和胰蛋白酶均显示出了较好的酶解效果,其中以碱性蛋白酶Alcalase AF 2.4L酶解效果最佳,酶解的最佳工艺条件为:底物浓度为50g/L,反应时间4 h,加酶量8.0×104 U/g,pH值8.3、60℃,最大水解度为10.0%;而以蛋清水解专用蛋白酶和胰蛋白酶为催化剂时最大水解度分别为8.8%和7.2%。根据单酶酶解结果探讨了双酶分步酶解工艺,确定了以"蛋清水解专用蛋白酶-碱性蛋白酶Al-calase AF 2.4L"和"碱性蛋白酶Alcalase AF 2.4L-胰蛋白酶"的双酶组合较好,鸭蛋清蛋白在酶解8h后水解度可达到26.2%和26.6%,明显优于单酶水解效果。展开更多
Animal lysozymes can selectively cleave a kind of glycosidic bond ofN-acetylglucosamine in the bacterial cell wall peptidoglycan. The hen egg-white lysozymeconsists of a single polypeptide chain containing 129 amino a...Animal lysozymes can selectively cleave a kind of glycosidic bond ofN-acetylglucosamine in the bacterial cell wall peptidoglycan. The hen egg-white lysozymeconsists of a single polypeptide chain containing 129 amino acid residues and fourdisulfide bonds. The three-dimensional structure of this enzyme was determined展开更多
基金Supported by the China One Thousand Talent Scheme,the National Natural Science Foundation of China under its Major Research Scheme of Meso-scale Mechanism and Control in Multi-phase Reaction Processes(91434126)the Natural Science Foundation of Guangdong Province(2014A030313228)+1 种基金benefited from early work funded by UK Engineering and Physical Science Research Council(EP/H008012/1EP/H008853/1)
文摘The temporal and spatial growth behaviour of protein crystals, subject to different cooling strategies in protein crystallisation was investigated. Although the impact of temperature and cooling rate on crystal growth of small molecules was well documented, much less has been reported on their impact on the crystallisation of proteins. In this paper, an experimental set-up is configured to carry out such a study which involves an automatic temperature controlled hot-stage crystalliser fitted with a real-time imaging system. Linbro parallel crystallisation experiments(24-well plate) were also conducted to find the suitable initial conditions to be used in the hot-stage crystallisation experiments, including the initial concentration of HEW lysozyme solutions, precipitate concentration and pH value. It was observed that fast cooling rates at the early stage led to precipitates while slow cooling rates produced crystal nuclei, and very slow cooling rates, much smaller than for small molecules are critical to the growth of the nuclei and the crystals to a desired shape. The interesting results provide valuable insight as well as experimental proof of the feasibility and effectiveness of cooling as a means for achieving controlled protein crystallisation, compared with the evaporation approach which was widely used to grow single large crystals for X-ray diffraction study. Since cooling rate control can be easily achieved and has good repeatability, it suggests that large-scale production of protein crystals can be effectively achieved by manipulating cooling rates.
文摘The refolding of denatured hen egg white lysozyme (HEWL) was examined by surfactants at a high final refolded HEWL concentration (1 mg/mL). Hexadecyltrimethylammonium bromide (CTAB) and sucrose fatty acid monoester (DK-SS) were used to dissolve denatured HEWL without denaturants such as guanidine hydrochloride (GuHCl) and urea. When denatured HEWL was perfectly dissolved in buffer solutions containing surfactants and dithiothreitol (DTT), the concentration of CTAB was about one-twentieth times less than that of DK-SS. The concentration of CTAB strongly affected the refolding yield, and the maximum refolding yield was obtained at 0.88 mM CTAB, which is around the critical micelle concentration of CTAB. The refolding yield was influenced by the molar ratio of oxidized glutathione (GSSG) to DTT, and the maximum refolding yield was obtained when [GSSG]/[DTT] was 1.5. The refolding yield was markedly dependent upon the solution pH of HEWL, and exhibited 80% at pH 5.2.
基金supported by the National Natural Science Foundation of China(12272329)the Sichuan University Student Innovation and Entrepreneurship Training Program(S202110619066)+2 种基金the Project of State Key Laboratory of Environment-friendly Energy Materials,Southwest University of Science and Technology(No.20fksy18)the Undergraduate Innovation Fund Project by Southwest University of Science and Technology(CX21-098)the NHC Key Laboratory of Nuclear Technology Medical Transformation(Mianyang Central Hospital)(21HYX019)。
文摘The influence of minor environmental factors,such as the geomagnetic field,on the biomineralization of nacres,is often ignored but a great deal of research has confirmed its important role in the normal mineralization of calcium carbonate.Although the geomagnetic field is weak,its cumulative effects need to be considered given that the biomineralization process can take years.Accordingly,the authors of this paper have investigated the effects of weak magnetic fields(25 Gs or 50 Gs)on calcium carbonate mineralization and analyzed the mechanism involved.The results show that even a weak magnetic field conduces to the formation of vaterite or aragonite,in the induction order of precursor→vaterite→aragonite.The stronger the magnetic field and the longer the time,the more obvious the induction effect.The effect of a magnetic field is strongest in the aging stage and weakest in the solution stage.Inductions by egg-white protein and by a magnetic field inhibit each other,but they both restrict particle growth.These findings highlight the importance of minor environmental factors for biomineralization and can serve as a reference for biomimetic preparation of a CaCO_(3)nacre-like structure and for anti-scale technology for circulating cooling water.
文摘探讨了单酶及双酶分步酶解鸭蛋清蛋白制备多肽的工艺和水解效果。在7种不同来源的蛋白酶中,蛋清水解专用蛋白酶、碱性蛋白酶Alcalase AF 2.4L和胰蛋白酶均显示出了较好的酶解效果,其中以碱性蛋白酶Alcalase AF 2.4L酶解效果最佳,酶解的最佳工艺条件为:底物浓度为50g/L,反应时间4 h,加酶量8.0×104 U/g,pH值8.3、60℃,最大水解度为10.0%;而以蛋清水解专用蛋白酶和胰蛋白酶为催化剂时最大水解度分别为8.8%和7.2%。根据单酶酶解结果探讨了双酶分步酶解工艺,确定了以"蛋清水解专用蛋白酶-碱性蛋白酶Al-calase AF 2.4L"和"碱性蛋白酶Alcalase AF 2.4L-胰蛋白酶"的双酶组合较好,鸭蛋清蛋白在酶解8h后水解度可达到26.2%和26.6%,明显优于单酶水解效果。
基金Project supported by the National High-Tech Program of China.
文摘Animal lysozymes can selectively cleave a kind of glycosidic bond ofN-acetylglucosamine in the bacterial cell wall peptidoglycan. The hen egg-white lysozymeconsists of a single polypeptide chain containing 129 amino acid residues and fourdisulfide bonds. The three-dimensional structure of this enzyme was determined
