金黄色葡萄球菌(Staphylococcusaureus ASIA4)葡萄激酶的制备及特性研究

N. H. Alzahrani, Fareed Shawky El-Shenawy
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摘要

从不同临床标本中分离到6株临床金黄色葡萄球菌。从尿路感染患者尿液样本中分离ASIA1和ASIA2;ASIA3从Assiut大学医院烧伤脓肿患者拭子样本中分离,ASIA4、ASIA5和ASIA6从南埃及癌症研究所不同癌症患者的血液样本中分离。所有分离菌株在血琼脂、加热血浆琼脂、酪蛋白琼脂和脱脂牛奶琼脂上产生不同直径的水解晕带的能力不同,且溶块率不同。金黄色葡萄球菌ASIA3、ASIA4和ASIA6在色氨酸豆汤上产生的葡萄激酶分别为4.83、5.98和2.08 U/mL,在酪蛋白水解酵母浸膏上产生的葡萄激酶分别为1.95、2.08和1.70 U/mL。另一方面,金黄色葡萄球菌ASIA1、ASIA2和ASIA5在CYEB上的产质为2.20、2.93和3.65 U/mL,而在TSB上的产质为2.10、1.88和3.41 U/mL。在以5.0 g蔗糖为碳源,10.0 g大豆为氮源,5.0 g NaCl, 5.0 g K2HPO4 5.0 g, pH 7.0为培养基,35℃孵育24 h的优化培养基上,金黄色葡萄球菌ASIA4的葡萄激酶产率提高了7.64倍,从2.08 U/mL提高到15.88 U/mL。最佳酶促反应条件为:反应时间25 min,酪蛋白为底物,反应pH 8.0,反应温度40℃。在温度15 ~ 45℃,pH 6.0 ~ 9.0范围内,EDTA对葡萄激酶活性的抑制作用为3.0% ~ 32.2%,在30.0 ~ 90.0 mM范围内,EDTA对葡萄激酶活性的抑制作用为3.0% ~ 32.2%,MgCl2在30 mM浓度下可使酶活性提高4%,在较高浓度下略有下降,而NaCl在低于90 mM的浓度下是有效的葡萄激酶激活剂。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Production and Characterization of Staphylokinase Enzyme from Staphylococcusaureus ASIA4
Six clinical Staphylococcus aureus strains isolated from different clinical samples. Isolates ASIA1 and ASIA2 isolated from urine samples of urinary tract infected patients; ASIA3 isolated from swab samples of burn abscess patients at Assiut University hospital as well as ASIA4, ASIA5 and ASIA6 obtained from blood samples of different cancer patients at South Egypt Cancer Institute. All isolates showed varied abilities to produce halo zones of hydrolysis with different diameters on blood agar, heated plasma agar, casein agar and skim milk agar plates along with different clot lyses percent. Staphylococcus aureus ASIA3, ASIA4 and ASIA6 produced 4.83, 5.98 and 2.08 U/mL of staphylokinase on tryptone soy broth reduced to 1.95, 2.08 and 1.70 U/mL on casein hydrolysate yeast extract broth,respectively. On the other hand, Staphylococcus aureus ASIA1, ASIA2 and ASIA5 gave 2.20, 2.93 and 3.65 U/mL on CYEB compared to 2.10, 1.88 and 3.41 U/mL on TSB as production medium. The staphylokinase yielded from the hyperactive producer Staphylococcus aureus ASIA4 was increased for 7.64-fold (from 2.08 U/mL to 15.88 U/mL) on the optimized fermentation medium composed of 5.0 g sucrose as carbon source, 10.0 g soy bean as nitrogen source, 5.0 g NaCl, K2HPO4 5.0 g and pH 7.0 that inoculated with isolate ASIA4 and incubated for 24 h at 35°C. Moreover, Staphylokinase activity reached its peak at the optimal enzymatic reaction conditions which were reaction time 25 min, casein as substrate, reaction pH 8.0, reaction temperature 40°C. In addition it retained 100% of its activity at temperature ranged between 15 and 45°C and pH ranged from pH 6.0 to 9.0 EDTA inhibited the enzyme activity by 3.0% to 32.2% with increasing its values from 30.0 to 90.0 mM. MgCl2 at a concentration of 30 mM increased the enzyme activity by 4% and then slightly decreased at higher concentrations but NaCl was potent staphylokinase activator at concentrations lower than 90 mM.
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