NaF对酸性海藻糖酶酶活测定的影响及作用机制探讨

Effects and Mechanisms of Sodium Fluoride on Assay of Acid Trehalase Activity

研究了氟化钠对酵母酸性海藻糖酶酶活力测定的影响。构建己糖转运蛋白基因缺失突变株TL-105(hxt14△)和TL-106(gal2△),分析氟化钠对菌株BY6-9α(亲本菌株)、TL-103(ath1△)、TL-104(agt1△)、TL-105(hxt14△)和TL-106(gal2△)酸性海藻糖酶酶活力、海藻糖酶分泌、海藻糖分泌、葡萄糖摄取和葡萄糖分泌的影响,结果表明,氟化钠对中性海藻糖酶、酸性海藻糖酶和海藻糖分泌均无影响,说明氟化钠对酸性海藻糖酶酶活力测定的影响与海藻糖酶和海藻糖分泌无关。在稳定期,柠檬酸法测得菌株TL-105(hxt14△)和TL-106(gal2△)的酸性海藻糖酶酶活力分别比亲本菌株提高了17.06%和300.23%,而柠檬酸氟化钠法测得菌株BY6-9α、TL-105(hxt14△)和TL-106(gal2△)的酸性海藻糖酶酶活力却相差不大,同时,在对数期,菌株BY6-9α和TL-103(ath1△)在酸性海藻糖酶酶活力测定前(氟化钠30℃诱导30 min)溶液中葡萄糖含量分别达到108.53±1.39和30.53±1.02 mg/L,这说明氟化钠能显著影响菌株对胞外葡萄糖的摄取并导致胞内葡萄糖的分泌,从而使测得的酸性海藻糖酶酶活力较高。

The effect of sodium fluoride on the determination of acid trehalase activity in baker,s yeast was investigated in this study. The recombinant baker＇s yeast stains TL-lO5（hxt14A） and TL-IO6（gal2A） with deleted genes encoding the hexose transporters were constructed. The acid trehalase activity, the secretion of neutral trehalase and acid trehalase, the secretion of glucose and trehalose and the uptake of extracellular glucose of BY6-9tt （parental strain）, TL-lO3（athlA）, TL-lO4（agtlA）, TL-lO5（hxt14A） and TL-lO6（gal2A） were measured. It was found that sodium fluoride had less effect on the secretion of neutral trehalase, acid trehalase and trehalose, although it had obvious effect on the assay of acid trehalase activity. Thus, sodium fluoride was not related to the secretion of intracellular trehalase or trehalose in the assay of acid trehalase activity. The acid trehalase activity, examined by citrate procedure, of strains TL-lO5（hxt14A） and TL-IO6（gal2A） at the stationary phase was 17.06% and 300.23% higher than those of the parental strain, respectively. However, the acid trehalase activities of the three strains examined by citrate and sodium fluoride method showed less difference. Furthermore, the extracellular glucose concentration of strains BY6-9u and TL-103（ath1A） at the exponential phase, which was incubated at 30 ~C for 30 min with sodium fluoride before the acid trehalase activity was assayed, was 108.53~1.39 and 30.53＋1.02 mg/L in the reaction mixture, respectively. These results indicated that sodium fluoride significantly affected the uptake of extracellular glucose and led to the secretion of intracellular glucose. Hence, the acid trehalase activity of the strains treated with sodium fluoride was higher than those without sodium fluoride treatment.