蚯蚓纤溶酶的纯化及pH值、温度和胰蛋白酶对其纤溶活性的影响

目的纯化并鉴定蚯蚓纤溶酶 ,研究pH、温度和胰蛋白酶对其纤溶活性的影响。方法以赤子爱胜蚓为材料 ,采用硫酸铵盐析、DEAESepharoseCL 6B离子交换色谱、SephadexG 75凝胶过滤和制备电泳等分离技术纯化蚯蚓纤溶酶 ;利用纤维平板和聚丙烯酰胺凝胶电泳研究pH、温度和胰蛋白酶对蚯蚓纤溶酶纤溶活性的影响。结果分离得到纤溶酶 4种单一组分 :EFE 1、EFE 2、EFE 3、EFE 4 ;分子量分别为 2 70 0 0、2 80 0 0、2 30 0 0、330 0 0 ;4种组分对温度的适应范围较广 ;pH值对其纤溶活性的影响不尽相同 ,但都在pH低于 1.5时纤溶活性完全丧失 ;胰蛋白酶对纤溶酶 4种组分均无降解作用 ,对其纤溶活性无影响。结论纤溶酶口服药制剂宜制成肠溶型     

白药子黄酮的抗氧化稳定性

目的 以白药子为原料,研究白药子黄酮各部位的抗氧化稳定性.方法 以大孔吸附树脂分离得到A、B、C部位,以清除DPPH的活性保持率为指标,研究溶液pH值、温度、金属离子、光照对各部位抗氧化稳定性的影响.结果 在pH6及金属离子K+、Na+、Ca2+存在的条件下,各部位均保持较高的清除活性;碱性条件下,清除活性迅速丧失;当加热至70 ℃时,C部位的活性保持较好,大于90％;光照条件下,抗氧化的稳定性依次为:C部位＞A部位＞B部位.结论 C部位具有较好的抗氧化稳定性.     

固定化胰蛋白酶的性质研究

目的　测定固定化胰蛋白酶的催化特性和部分稳定性。方法　在相同的条件下,测定固定化和可溶性胰蛋白酶的活力,得其最适作用pH值和最适作用温度,再测定活力,得到各自的米氏常数(Km)值;测得两种状态酶的热稳定性和酸碱稳定性。同时测定了固定化胰蛋白酶的低温稳定性。结果　固定化和可溶性胰蛋白酶的最适作用pH值分别为8 .0～9. 0和7. 0～9.0 ,最适作用温度分别为6 0℃和5 5℃,Km值分别为2 . 32 6、1 42. 9mg·ml-1 ;固定化胰蛋白酶在5 5℃条件下,保温5h ,仍有78 92 %的活性;在4℃条件下,8d后,活性才略微下降,两周活性保留达90 %以上;在pH 9.0的活性未下降,在pH 7.0～1 0 .0范围内也比较稳定(相对活性>80 %)。结论　胰蛋白酶经固定化后,最适作用pH值范围变窄,最适作用温度和Km值升高;热稳定性和酸碱稳定性均有所增强     

海洋放线菌M326活性代谢产物的初步研究

目的对海洋放线菌M326活性代谢产物进行初步研究。方法以抑菌活性为指标,采用不同的培养基与培养时间研究代谢产物活性与培养条件的关系,用不同温度、pH处理了解活性产物的稳定性,用有机溶剂,大孔树脂对活性物质进行提取分离。结果人工海水、蒸馏水配制的GBP培养基产物活性较强。抗菌物质在pH 2~11时较稳定,强酸性条件下热稳定性好,难以用一般有机溶剂萃取,碱性条件下可被AB-8大孔树脂吸附。结论代谢产物对G 菌有较强的抑菌活性,对G-菌、耐药菌均有不同程度的抑菌活性,且抗菌物质的极性较强。     

溶液pH值对尿囊素稳定性的影响

目的考察溶液pH值对尿囊素稳定性的影响。方法将尿囊素配制成pH值为5．0、5．5、6．0、6．5、7．0的水溶液,分别在60℃条件下放置5d和10d,参照缩泉胶囊质量标准中的山药含量测定项的色谱条件（以丙基酰胺键合硅胶为填充剂;以乙腈-水（90：10）作为流动相;检测波长为191nm;进样量：10μL）测定溶液中的尿囊素含量,并与0d的含量进行比较。结果在60℃条件下放置10d．pH值为5．0、5．5、6．0、6．5、7．0的尿囊素溶液含量分别下降1.3％、5．4％、12．0％、32．0％、68．4％。结论尿囊素在pH值为5．0的溶液中稳定性最好,其pH值越高稳定性越差。     

二氢杨梅素的稳定性研究

目的:研究二氢杨梅素在不同条件下的稳定性。方法:考察二氢杨梅素在不同pH值、温度、光线、金属离子影响下的稳定性,用反相高效液相色谱法测定其含量。结果:在pH值≤4的弱酸性条件下二氢杨梅素能稳定存在,随着pH值的增加,会加速氧化,特别是在pH值≥9的碱性条件下会立即反应而氧化变黄。温度的升高,Fe3 ,Al3 ,Cu2 等金属离子的存在以及光线均可以加速二氢杨梅素的氧化。结论:为使二氢杨梅素能较长时间稳定存在,需保证其生理活性的条件是弱酸性、避免接触Fe3 ,Al3 ,Cu2 等金属离子、避光低温保存。     

溶液pH值对盐酸萘甲唑啉稳定性的影响

目的考察溶液pH值对盐酸萘甲唑啉稳定性的影响。方法将盐酸萘甲唑啉制成pH值为4．0、4．5、5．0、5．5、6．0、6．5、7．0的水溶液,分别在60℃条件下放置5d和10d,参照《中国药典》2010年版二部盐酸萘甲唑啉滴眼液含量测定项的方法测定样品含量。并与0d的含量进行比较。结果在60℃条件下放置10d,pH值为4．0、4．5、5．0、5．5、6．0、6．5、7．0的盐酸萘甲唑啉溶液含量分别下降1.0％、0.8％、0．8％、2．1％、6．3％、22．3％、36．2％。结论盐酸萘甲唑啉在pH值为4．5～5．0的溶液中稳定性最好,其pH值越高稳定性越差。     

可溶性固定化木瓜蛋白酶的制备及性质研究

目的 制备一种可溶性固定化木瓜蛋白酶(PP)并研究其部分酶学性质.方法 将PP偶联在羧甲基纤维素醋酸琥珀酸酯(AS-L)上,制备得到水溶性固定化PP,并测定游离和水溶性固定化PP最适作用pH、最适作用温度、Km值、热稳定性、酸碱稳定性、低温稳定性和回收稳定性.结果 最适作用pH分别为6.0、5.0,最适作用温度分别为60℃、70℃,Km值分别为2.53、3.07 mg·ml-1;可溶性固定化PP在60℃条件下,保温12 h,仍有62%的活性;在4℃条件下,30 d后活性保留达90%以上;在pH6时稳定性最好,pH4～7范围内也较稳定(相对活性＞80%).结论 可溶性固定化PP能够在大于pH5.5的溶液中完全溶解,最适作用pH范围变窄,最适作用温度和Km值升高;热和酸碱的稳定性均明显增强.     

蛋清溶菌酶部分酶学性质及酶活性的影响因素研究

目的探讨将蛋清溶菌酶制成液体制剂的可行性。方法通过改变不同影响因素测定蛋清溶菌酶的活性,观察几种常用辅料对蛋清溶菌酶活性的影响。结果酶活性在pH 6.0~6.5最强,且在pH 5~7范围内较稳定;在25~65℃范围内随着作用温度的升高酶的活性增强,但温度太高则变性失活;Na+、K+对其活性有轻微激活作用,Mn2+、Mg2+对溶菌酶活性无明显影响,Co2+、Ca2+、Cu2+、Fe2+、Zn2+使溶菌酶的活性下降;吐温20、吐温80、甘油溶液对酶活有抑制作用;EDTA-2Na在0.000 5%~0.300 0%浓度范围内对溶菌酶活性具有激活作用,浓度继续增加反而有抑制活性作用。结论初步试验结果表明溶菌酶可以制成合适的液体制剂。     

重组人干扰素β1b活性标准品的稳定性研究

目的 评价重组人干扰素β1b(recombinant human interferon β1b,rhIFN-β1b)活性标准品在不同贮存温度条件下的稳定性.方法 检测在不同贮存温度(37 ℃、室温、4和-20 ℃)条件下放置不同时间段的rhIFN-β1b活性标准品的外观、溶解性、水分、pH值及生物学活性.结果 活性标准品分别在4种温度条件下放置90周生物学活性都没有降低的趋势,并且没有明显差别,同时水分、pH值等都符合规定.结论 说明辅料配方合理,可有效保护rhIFN-β1b,减少其降解,活性标准品稳定性良好.     

Stabilization of Lysozyme by Benzyl Alcohol: Surface Tension and Thermodynamic Parameters

The aim of the study was to understand the effect of benzyl alcohol on biological activity, aggregation behavior, denaturant and heat-induced unfolding of lysozyme. Compatibility studies of lysozyme carried out with a number of anti-microbial preservatives, indicated benzyl alcohol to be the best suppressor of protein aggregation against heat stress. The effect of this preservative was checked at various pH values ranging from 4.0 to 9.0. In spite of reducing the thermal denaturation temperature (T_m) at all pH values, benzyl alcohol had a stabilizing effect on lysozyme in terms of retaining the biological activity when the enzyme was incubated at 75°C. The reduction in T_m with increasing benzyl alcohol concentration was correlated with decreasing surface tension of surrounding medium. A detailed thermodynamic study of lysozyme in the presence of benzyl alcohol was carried out at pH 6.2. Change in Gibb’s free energy of thermal unfolding at 25°C was found to remain constant in the presence of benzyl alcohol, indicating no interaction of benzyl alcohol with the native protein at room temperature. Both the enthalpy and entropy change at mid point of thermal unfolding were found to increase in the presence of benzyl alcohol indicating the stabilization of partially unfolded state.     

The toxicity study on marine low-temperature lysozyme.

Marine low-temperature lysozyme is purified from a marine bacterium. The lysozyme can keep high activity at low-temperature and has broad-spectrum antibiotic reaction. This study was undertaken to investigate the major characteristics, acute and subchronic toxicity of marine low-temperature lysozyme. The relative molecular weight of this lysozyme was determined as approximate 16 kD; its optimum pH value and temperature towards Micrococcus lysodleikticus were pH 6.5 and 35 degrees C, respectively. The lysozyme activity was slightly enhanced by Zn(2+) and Cu(2+) and slightly inhibited by Mn(2+) and Ag(+). The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%), KH(2)PO(4) (1.0%), etc. In experiments on acute toxicity, the drug was injected through the tail vein of mice, and intoxication symptoms and date of death were recorded. The 50% lethal dose (LD(50)) of Marine low-temperature lysozyme and 95%, 99% confidence interval (CI) was calculated. The subchronic study was designed to determine whether effects progressed with repeated Marine low-temperature lysozyme exposure. Wistar rats were tested by daily intragastric administration of Marine low-temperature lysozyme at the doses of 1.0; 0.5; 0.25 g/kg bw for 90 days. The LD(50) value of lysozyme was 4530 mg/kg bw; 90 days of Marine low-temperature lysozyme treatment at three doses, and there is no significant difference on blood biochemistry and organ index in drug treatment groups compared to saline treatment group. There is no affirmative pathologic change of all the observed organs in this study. The present results suggest that Marine low-temperature lysozyme can be safely used at the dose of experiment applied.     

Biodegradable and biocompatible thermosensitive polymer based injectable implant for controlled release of protein

This study was aimed at developing a suitable controlled release system for proteins by modifying the structure of thermosensitive copolymer monomethoxy poly(ethylene-glycol)-co-poly(D,L-lactide-co-glycolide)-co-monomethoxy poly(ethylene-glycol) (mPEG-PLGA-mPEG). Eleven mPEG-PLGA-mPEG copolymers were synthesized and characterized by (1)H NMR and gel permeation chromatography (GPC). Thermosensitivity of the copolymers was tested using the tube inversion method. Four of the eleven synthesized copolymers were dissolved in water as injectable solutions at room temperature which turned into gels abruptly at body temperature (37 degrees C), indicating the potential use as in vivo drug delivery system. Lysozyme was used as a model protein to study in vitro release characteristics of the copolymer based delivery system. The copolymer based formulations released lysozyme (quantified by micro-BCA protein assay) over 10-30 days, depending on copolymer structure. The released lysozyme was confirmed to conserve its structural stability by differential scanning calorimetry (DSC) and circular dichroism (CD), and biological activity by specific enzyme activity assay. Furthermore, the copolymer based formulations showed excellent biocompatibility as tested by MTT assay and in vivo histological evaluation. Therefore, the copolymers controlled the in vitro release of lysozyme while conserving protein stability and biological activity, indicating that it is an appropriate delivery system for long term controlled release of proteins.     

THE PH DEPENDENCE OF BINDING OF α, α‘-DIBROMO-P-XYLENESULFONIC ACID TO LYSOZYME

The chemical modification of lysozyme (I) has been accomplished with α,α'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28°C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-³⁵S were bound per molecule of I at both pH 9 and pH 8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intramolecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

Batch purification of high-purity lysozyme from egg white and characterization of the enzyme modified by PEGylation

PEGylation is one of the most promising and extensively studied strategies for improving the pharmacological properties of proteins as well as their physical and thermal stability. Purified lysozyme obtained from hen egg white by batch mode was modified by PEGylation with methoxypolyethyleneglycol succinimidyl succinato (mPEG-SS, MW 5000). The conjugates produced retained full enzyme activity with the substrate glycol chitosan, independent of degree of enzyme modification, although lysozyme activity with the substrate Micrococcus lysodeikticus was altered according to the degree of modification. The conjugate with a low degree of modification by mPEG-SS retained 67% of its enzyme activity with the M. lysodeikticus substrate. The mPEG-SS was also shown to be a highly reactive polymer. The effects of pH and temperature on PEGylated lysozymes indicated that the conjugate was active over a wide pH range and was stable up to 50°C. This conjugate also showed resistance to proteolytic degradation, remained stable in human serum, and displayed greater antimicrobial activity than native lysozyme against Gram-negative bacteria.     

Poly Lactic Acid Based Injectable Delivery Systems for Controlled Release of a Model Protein,Lysozyme

The objective of this study was to evaluate the critical formulation parameters (i.e., polymer concentration, polymer molecular weight, and solvent nature) affecting the controlled delivery of a model protein, lysozyme, from injectable polymeric implants. The conformational stability and biological activity of the released lysozyme were also investigated. Three formulations containing 10%, 20%, and 30% (w/v) poly lactic acid (PLA) in triacetin were investigated. It was found that increasing polymer concentration in the formulations led to a lower burst effect and a slower release rate. Formulation with a high molecular weight polymer showed a greater burst effect as compared to those containing low molecular weight. Conformational stability and biological activity of released samples were studied by differential scanning calorimeter and enzyme activity assay, respectively. The released samples had significantly (P < 0.05) greater conformational stability and biological activity in comparison to the control (lysozyme in buffer solution kept at same conditions). Increasing polymer concentration increased both the conformational stability and the biological activity of released lysozyme. In conclusion, phase sensitive polymer-based delivery systems were able to deliver a model protein, lysozyme, in a conformationally stable and biologically active form at a controlled rate over an extended period.     

Controlled release of a model protein lysozyme from phase sensitive smart polymer systems

The purpose of this study was to investigate the suitability of phase sensitive smart polymer-based protein formulations in order to deliver a model protein, lysozyme, in a conformationally stable and biologically active form at a controlled rate over extended period of time. Four different formulations, using D,L-poly(lactide) (D,L-PLA) and a solvent mixture of different ratios of benzyl benzoate (BB) and benzyl alcohol (BA), were prepared. Conformational stability and biological activity of lysozyme were studied by differential scanning calorimeter and enzyme activity assay, respectively. We found a significant (P < 0.05) increase in burst and rate of release of incorporated lysozyme from formulations containing greater proportion of BA. In order to increase the conformational stability and biological activity of lysozyme, we incorporated mannitol as stabilizer into formulations. Mannitol increased the conformational and biological activity of lysozyme in comparison to the control formulation prepared without mannitol. In conclusion, phase sensitive smart polymer-based delivery systems were able to deliver a model protein, lysozyme, in a conformationally stable and biologically active form at a controlled rate over extended period of time.     

Poly lactic acid based injectable delivery systems for controlled release of a model protein, lysozyme

The objective of this study was to evaluate the critical formulation parameters (i.e., polymer concentration, polymer molecular weight, and solvent nature) affecting the controlled delivery of a model protein, lysozyme, from injectable polymeric implants. The conformational stability and biological activity of the released lysozyme were also investigated. Three formulations containing 10%, 20%, and 30% (w/v) poly lactic acid (PLA) in triacetin were investigated. It was found that increasing polymer concentration in the formulations led to a lower burst effect and a slower release rate. Formulation with a high molecular weight polymer showed a greater burst effect as compared to those containing low molecular weight. Conformational stability and biological activity of released samples were studied by differential scanning calorimeter and enzyme activity assay, respectively. The released samples had significantly (P < 0.05) greater conformational stability and biological activity in comparison to the control (lysozyme in buffer solution kept at same conditions). Increasing polymer concentration increased both the conformational stability and the biological activity of released lysozyme. In conclusion, phase sensitive polymer-based delivery systems were able to deliver a model protein, lysozyme, in a conformationally stable and biologically active form at a controlled rate over an extended period.     

Supercritical fluid processing of proteins: lysozyme precipitation from aqueous solution

Aqueous solutions of hen egg lysozyme (3% w/v) were dispersed and precipitated by a homogenous mixture of supercritical carbon dioxide-ethanol using the Solution Enhanced Dispersion by Supercritical fluid (SEDS) process. The effects of different working conditions, such as temperature, pressure and the flow rates of the solution and ethanol, on the particle-formation process were studied. The morphology, particle size and size distribution and biological activity of the protein were determined. The precipitates were examined with high-sensitivity differential scanning calorimetry (HSDSC) and high-performance cation-exchange chromatography. Particle size measurements showed the precipitates to be aggregates with primary particles of size 1-5 microm. The similarity of HSDSC data for unprocessed and processed samples indicated that the different physical forces that stabilise the native form of lysozyme are unchanged after SEDS processing. From FT-Raman spectroscopic studies secondary structural changes were observed in certain SEDS-produced lysozyme, with most processed samples displaying a slightly more disordered secondary structure than the unprocessed sample. However, SEDS samples produced at 200 bar and 40 degrees C exhibited negligible disturbance. Thus the SEDS process utilising aqueous solution was able to bring about size reduction of lysozyme with minimal loss of biological activity.