CN110607336B - 酶催化制备半胱氨酸寡肽及其膜敷料的制备方法 - Google Patents
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Abstract
本发明涉及一种酶催化制备半胱氨酸寡肽及其膜敷料的制备方法,包括以下步骤:在磷酸氢二钠‑柠檬酸缓冲液中加入L‑半胱氨酸甲酯盐酸盐、全氟叔丁醇和蛋白酶搅拌反应;将反应混合物经超滤膜过滤和浓缩;将经过超滤膜浓缩后的滤出液真空蒸馏、冷冻干燥后得到半胱氨酸寡肽。将半胱氨酸寡肽、二缩水甘油基乙醚与磺胺嘧啶银混合,涂布成膜,得到半胱氨酸寡肽膜敷料。本发明的方法具有反应条件温和、环境更友好、副反应少等优点,而且避免了繁琐的保护和去保护步骤。采用本发明的方法所制备的膜敷料以半胱氨酸寡肽为基质,抗菌性、生物相容性与可降解性等性能优秀。
Description
技术领域
本发明涉及一种酶催化制备半胱氨酸寡肽及其膜敷料的制备方法,本发明属于半胱氨酸寡肽的制备技术领域。
背景技术
皮肤是人体最大的器官,可以维持人体内环境稳定和阻止外界有害物质的入侵。然而烧烫伤等意外创伤或疾病造成皮肤损伤后,往往引发许多局部或全身反应,大面积的皮肤损伤甚至会对生命造成威胁。此时应及时为创面提供一个性能优良的创面覆盖物,起到创面保护作用,同时促进伤口的愈合,重建和恢复皮肤功能。天然生物高分子材料良好的生物相容性和优异的生物学性能早已得到人们的关注和认可,成为多种医用伤口敷料的极佳来源。生物敷料的研究不仅局限于覆盖和保护创面的作用,更开始向促进伤口愈合速度、提高伤口愈合质量方向发展。氨基酸或蛋白质类敷料的性能优异,不仅具有良好的生物相容性和生物降解性,其特殊结构和理化性能利于伤口愈合,具有吸收创面渗出液,防止感染,透气性、透水性等功能。
人工聚肽是以天然氨基酸为单体进行聚合反应,通过酰胺键而形成的聚氨基化合物,性质与天然肽、聚氨基酸和蛋白质类似。由人工聚肽形成的材料具有良好的生物亲和性和可降解性。聚肽或其改性低聚物表现出自组装、液晶行为等独特结构性质,具有较强可塑性,在生物、医药和天然高分子材料领域具有很大的应用潜力。半胱氨酸聚肽的研究在国外特别是在日本已比较成熟,我国刚刚起步。它具有很好的杀菌能力和热稳定性。在日本,半胱氨酸聚肽已被批准作为防腐剂添加于食品中,广泛用于方便米饭、湿熟面条、熟菜、海产品、酱类、酱油、鱼片和饼干的保鲜防腐中。半胱氨酸具有抗氧化性能,防止老化和有促进受损黏膜修复的作用。它同时是一种氨基酸类解毒药,它参与细胞的还原过程和肝脏内的磷脂代谢,有保护肝细胞不受损害,促进肝脏功能恢复和旺盛的药理效应。磺胺嘧啶银是磺胺类抗菌药,用于预防和治疗轻度烧烫伤继发创面感染。在半胱氨酸聚肽膜中混合磺胺嘧啶银可以发挥两者优点,提高敷料的抗菌、修复功能。
发明内容
本发明的目的之一是克服现有技术中存在的不足,提供一种酶催化制备半胱氨酸寡肽的制备方法,该制备方法具有反应条件温和、环境更友好、副反应少等优点,而且避免了繁琐的保护和去保护步骤。
本发明的另一目的是提供一种半胱氨酸寡肽膜敷料的制备方法。
按照本发明提供的技术方案,所述酶催化制备半胱氨酸寡肽的方法包括以下步骤:
a1、在25~50 mL磷酸氢二钠-柠檬酸缓冲液中加入20~40 g 的L-半胱氨酸甲酯盐酸盐、全氟叔丁醇和50~100U蛋白酶,反应混合物在20~50℃下搅拌反应6~12小时;其中,磷酸氢二钠-柠檬酸缓冲液中含有0.1~0.2M的磷酸氢二钠-柠檬酸,磷酸氢二钠-柠檬酸缓冲液的pH值为6.0~8.5,全氟叔丁醇的体积为磷酸氢二钠-柠檬酸缓冲液的体积的15%~40%;
b1、反应结束后,将反应混合物先经截留分子量4000~6000的超滤膜过滤,滤出液再经截留分子量150~250的超滤膜浓缩;
c1、将经过超滤膜浓缩后的滤出液在25~35℃下真空蒸馏去除溶剂,残余液经冷冻干燥后得到半胱氨酸寡肽。
步骤a1中,所述蛋白酶为菠萝蛋白酶、木瓜蛋白酶、胰蛋白酶、中性蛋白酶或者碱性蛋白酶中的一种。
步骤a1中,所述搅拌速度为200~400 rpm。
步骤c1中,所述真空蒸馏时的真空度控制在0~10Kpa。
步骤c1中,所述冷冻干燥的温度控制在-45~-20℃,冷冻干燥的时间控制在5~10小时。
使用半胱氨酸寡肽制备膜敷料的方法包括以下步骤:
a2、在15~25 mL去离子水中加入20~30 g 的半胱氨酸寡肽、二缩水甘油基乙醚与磺胺嘧啶银,半胱氨酸寡肽、二缩水甘油基乙醚与磺胺嘧啶银的重量比为 (10~30):(1~5):1;
b2、将混合物平涂于平板上形成厚度为0.08~0.12 cm的膜料,将膜料连同平板置于0~-20℃下冷藏4~10小时后取出;
c2、在室温下解冻后用蒸馏水中浸洗2~4次,去除未反应二缩水甘油基乙醚,得到半胱氨酸寡肽膜敷料。
本发明采用酶催化制备半胱氨酸寡肽的方法具有反应条件温和、环境更友好、副反应少等优点,而且避免了繁琐的保护和去保护步骤。
采用本发明的方法所制备的膜敷料以半胱氨酸寡肽为基质,抗菌性、生物相容性与可降解性等性能优秀。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
1.酶催化合成合成半胱氨酸寡肽
a1、在25 mL、0.1M、pH6.0的磷酸氢二钠-柠檬酸缓冲液(南昌雨露实验器材有限公司生产)中加入20 g 的L-半胱氨酸甲酯盐酸盐(国药集团化学试剂有限公司)、全氟叔丁醇(国药集团化学试剂有限公司)和50 U菠萝蛋白酶(上海沃凯生物技术有限公司),磷酸氢二钠-柠檬酸缓冲液的pH值为6.0,全氟叔丁醇的体积为磷酸氢二钠-柠檬酸缓冲液的体积的15 %,反应混合物在20℃下搅拌(300 rpm)反应12小时;
b1、反应结束后,将反应混合物经截留分子量5000的超滤膜过滤,滤出液经截留分子量200的超滤膜浓缩后;
c1、在30℃下真空蒸馏去除溶剂,真空蒸馏时的真空度控制在10pa残余液在冷冻干燥后得到半胱氨酸寡肽,冷冻干燥的温度控制在-30℃,冷冻干燥的时间控制在8小时;
2.半胱氨酸寡肽膜敷料的制备
a2、在20 mL去离子水中加入20 g 的半胱氨酸寡肽、二缩水甘油基乙醚(湖北恒景瑞化工有限公司)、磺胺嘧啶银(上海阿拉丁生化科技股份有限公司),半胱氨酸寡肽、二缩水甘油基乙醚、磺胺嘧啶银的重量比为10:1:1;
b2、将混合物平涂于玻璃平板(每5ml溶液约涂成厚度0.1 cm,面积5.0 cm×5.0cm),置于0℃下冷藏10小时后取出;
c2、室温下解冻后用蒸馏水浸洗3次,去除未反应二缩水甘油基乙醚,得到半胱氨酸寡肽膜敷料。
实施例2
1. 酶催化合成合成半胱氨酸寡肽
a1、在35 mL、0.15M、pH7.0的磷酸氢二钠-柠檬酸缓冲液(南昌雨露实验器材有限公司生产)中加入30 g 的L-半胱氨酸甲酯盐酸盐(国药集团化学试剂有限公司)、全氟叔丁醇(国药集团化学试剂有限公司)和70 U胰蛋白酶(上海沃凯生物技术有限公司),磷酸氢二钠-柠檬酸缓冲液的pH值为8.5,全氟叔丁醇的体积分数为35 %磷酸缓冲液,反应混合物在30℃下搅拌(200 rpm)反应10小时;
b1、反应结束后,将反应混合物经截留分子量5000的超滤膜过滤,滤出液经截留分子量200的超滤膜浓缩;
c1、在30℃下真空蒸馏去除溶剂,真空蒸馏时的真空度控制在5pa,残余液在冷冻干燥后得到半胱氨酸寡肽,冷冻干燥的温度控制在--20℃,冷冻干燥的时间控制在10小时。。
2. 半胱氨酸寡肽膜敷料的制备
a2、在20 mL去离子水中加入25 g 的半胱氨酸寡肽、二缩水甘油基乙醚(湖北恒景瑞化工有限公司)、磺胺嘧啶银(上海阿拉丁生化科技股份有限公司)。半胱氨酸寡肽、二缩水甘油基乙醚、磺胺嘧啶银的重量比为20:3:1;
b2、将混合物平涂于玻璃平板(每5ml溶液约涂成厚度0.1 cm,面积5.0 cm×5.0cm),置于-10℃下冷藏6小时后取出;
c2、室温下解冻后用蒸馏水浸洗3次,去除未反应二缩水甘油基乙醚,得到半胱氨酸寡肽膜敷料。
实施例3
1. 酶催化合成合成半胱氨酸寡肽
a1、在50 mL、0.2M、pH8.0的磷酸氢二钠-柠檬酸缓冲液(南昌雨露实验器材有限公司生产)中加入40 g 的L-半胱氨酸甲酯盐酸盐(国药集团化学试剂有限公司)、全氟叔丁醇(国药集团化学试剂有限公司)和100 U碱性蛋白酶(上海沃凯生物技术有限公司),磷酸氢二钠-柠檬酸缓冲液的pH值为7,全氟叔丁醇的体积分数为40 %磷酸缓冲液,反应混合物在50℃下搅拌(400 rpm)反应6小时;
b1、反应结束后,将反应混合物经截留分子量5000的超滤膜过滤,滤出液经截留分子量200的超滤膜浓缩;
c1、在30℃下真空蒸馏去除溶剂,真空蒸馏时的真空度控制在1pa,残余液在冷冻干燥后得到半胱氨酸寡肽,冷冻干燥的温度控制在-45℃,冷冻干燥的时间控制在5小时;
2. 半胱氨酸寡肽膜敷料的制备
a2、在20 mL去离子水中加入30 g 的半胱氨酸寡肽、二缩水甘油基乙醚(湖北恒景瑞化工有限公司)、磺胺嘧啶银(上海阿拉丁生化科技股份有限公司),半胱氨酸寡肽、二缩水甘油基乙醚与磺胺嘧啶银的重量比为30:5:1;
b2、将混合物平涂于玻璃平板(每5ml溶液约涂成厚度0.1 cm,面积5.0 cm×5.0cm),置于-20℃下冷藏4小时后取出;
c2、室温下解冻后用蒸馏水浸洗3次,去除未反应二缩水甘油基乙醚,得到半胱氨酸寡肽膜敷料。
本发明的优点是:本发明采用酶催化制备半胱氨酸寡肽的方法具有反应条件温和、环境更友好、副反应少等优点,而且避免了繁琐的保护和去保护步骤。
采用本发明的方法所制备的膜敷料以半胱氨酸寡肽为基质,抗菌性、生物相容性与可降解性等性能优秀。
Claims (4)
1.一种酶催化制备半胱氨酸寡肽膜敷料的方法,其特征是该方法包括以下步骤:
(1)半胱氨酸寡肽的制备
a1、在25~50mL磷酸氢二钠-柠檬酸缓冲液中加入20~40g的L-半胱氨酸甲酯盐酸盐、全氟叔丁醇和50~70U蛋白酶,反应混合物在20~30℃下搅拌反应6~12小时;其中,磷酸氢二钠-柠檬酸缓冲液中含有0.1~0.2M的磷酸氢二钠-柠檬酸,磷酸氢二钠-柠檬酸缓冲液的pH值为6.0~8.5,全氟叔丁醇的体积为磷酸氢二钠-柠檬酸缓冲液的体积的15%~40%;所述蛋白酶为菠萝蛋白酶或胰蛋白酶;
b1、反应结束后,将反应混合物先经截留分子量4000~6000的超滤膜过滤,滤出液再经截留分子量150~250的超滤膜浓缩;
c1、将经过超滤膜浓缩后的滤出液在25~35℃下真空蒸馏去除溶剂,残余液经冷冻干燥后得到半胱氨酸寡肽;
(2)半胱氨酸寡肽膜敷料的制备
a2、在15~25mL去离子水中加入20~30g的半胱氨酸寡肽、二缩水甘油基乙醚与磺胺嘧啶银,半胱氨酸寡肽、二缩水甘油基乙醚与磺胺嘧啶银的重量比为(10~30):(1~5):1;
b2、将混合物平涂于平板上形成厚度为0.08~0.12cm的膜料,将膜料连同平板置于0~-20℃下冷藏4~10小时后取出;
c2、在室温下解冻后用蒸馏水中浸洗2~4次,去除未反应二缩水甘油基乙醚,得到半胱氨酸寡肽膜敷料。
2.如权利要求1所述的方法,其特征是:步骤a1中,所述搅拌速度为200~400rpm。
3.如权利要求1所述的方法,其特征是:步骤c1中,所述真空蒸馏时的真空度控制在0~10pa。
4.如权利要求1所述的方法,其特征是:步骤c1中,所述冷冻干燥的温度控制在-45~-20℃,冷冻干燥的时间控制在5~10小时。
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