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    • 4. 发明授权
    • Method of producing a product with crosslinked crystals of thermolysin
    • 用嗜热溶菌蛋白交联晶体生产产品的方法
    • US5801022A
    • 1998-09-01
    • US474968
    • 1995-06-07
    • Manuel A. NaviaNancy L. St. Clair
    • Manuel A. NaviaNancy L. St. Clair
    • A61K38/43A61K38/46C07K5/072C12M1/40C12N9/14C12N9/20C12N9/36C12N9/54C12N9/66C12N9/80C12N9/82C12N11/00C12Q1/00C12Q1/37G01N33/531C12P13/22C07K17/00C12P13/20G01N33/543
    • C12N9/2462C07K5/0613C12N11/00C12N9/14C12N9/20C12N9/54C12N9/6448C12N9/80C12N9/82C12Q1/00C12Q1/003C12Q1/37G01N33/531Y10S530/81
    • A protein such as an enzyme or antibody is immobilized by crosslinking crystals of the protein with a multifunctional crosslinking agent such as glutaraldehyde, and if desired lyophilizing the crosslinked crystals for storage. Crosslinking of the protein crystals provides stabilization for use under harsh conditions and for lyophilizing. The crystals crosslinked may be microcrystals having a cross-section of 10.sub.-1 mm or less. Crosslinked thermolysin, esterase, elastase, asparaginase and lysozyme crystals and crosslinked crystals of lipase from Geotrichum candidum and Candida cylindracea and of porcine origin can be used to convert a substrate to a product. Crosslinked thermolysin crystals are prepared that retain at least 96% of their initial activity after incubation for 4 days in the presence of a concentration of Pronase.TM. such as a thermolysin:Pronase.TM. ratio of 1:40 that causes the soluble uncrosslinked form of thermolysin that is crystallized to form the crystals that are crosslinked to lose at least 99% of its initial activity after incubation for 90 minutes under the same conditions. Crosslinked thermolysin crystals can be used to produce aspartame by combining the crystals with N-(benzyloxycarbonyl)-L-aspartic acid and L-phenylalanine methyl ester in a mixed aqueous/organic solvent such as a water-ethyl acetate mixture, and maintaining the combination under conditions to cause a condensation reaction to produce N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester, and removing the benzyloxycarbonyl group to obtain aspartame. Crosslinked antibody crystals have uses as an immunospecific reagent such as for detection of a substance in a sample, and for therapeutic purposes.
    • 通过用多官能交联剂如戊二醛交联蛋白质的晶体来固定蛋白质如酶或抗体,如果需要,将交联的晶体冻干以进行保存。 蛋白质晶体的交联提供在恶劣条件下使用和冻干的稳定性。 交联的晶体可以是横截面为10-1mm或更小的微晶。 交联的嗜热菌蛋白酶,酯酶,弹性蛋白酶,天冬酰胺酶和溶菌酶晶体,以及来自地热赤霉素和念珠菌和猪来源的脂肪酶的交联晶体可用于将底物转化成产物。 制备交联的嗜热菌解蛋白晶体,其在浓度为Pronase TM的浓度的例如1:40的嗜热菌蛋白酶:Pronase TM比率存在下孵育4天后保持其初始活性的至少96%,其导致可溶性未交联形式的嗜热菌蛋白酶, 结晶以形成交联的晶体,在相同条件下孵育90分钟后,其至少99%的初始活性损失。 通过将结晶与N-(苄氧基羰基)-L-天冬氨酸和L-苯丙氨酸甲酯在混合的水/有机溶剂如水 - 乙酸乙酯混合物中混合,可以使用交联的嗜热溶蛋白晶体制备天冬甜素,并保持组合 在引起缩合反应以产生N-(苄氧基羰基)-L-天冬氨酰基-L-苯丙氨酸甲酯的条件下,并除去苄氧羰基以得到阿斯巴甜。 交联抗体晶体具有作为免疫特异性试剂的用途,例如用于检测样品中的物质以及用于治疗目的。