CAJ | 학술논문

为认识和应用极端嗜热细菌腾冲嗜热厌氧杆菌(T.tengcongensis MB4~T)产生的葡聚糖磷酸化酶(Tte-GlgP)的性质,用电喷雾质谱法(ESI-MS)测定Tte-GlgP的底物谱.结果表明:纯化后的Tte-GIgP有较广的底物范围,能将可溶性淀粉、麦芽糊精、糖原、麦芽七糖、麦芽五糖和麦芽三糖底物转化为1-磷酸葡萄糖(G-1-P).用改进后的双酶法测定Tte-GlgP对上述底物的转化效率,以产生的G-1-P的量为指标(μmol/L),在相同的实验条件下,以麦芽七糖和麦芽五糖为底物时,转化效率相对高,分别为86.83μmol/L和85.79μmol/L;糊精和可溶性淀粉次之,分别为82.9μmol/L G-1-P和69.68μmol/L G-1-P;最低的分别为糖原和麦芽三糖,产生的G-1-P分别为45.81 μmol/L和43.60μmol/L.两种分析方法均证明麦芽寡糖、麦芽糖糊精和淀粉为Tte-GlgP的最适底物.以上述双酶法为酶活性测定方法,测得在体系pH8.0时,Tte-GlgP的活性最高;在最适pH值条件下以可溶性淀粉为底物时Tte-GlgP催化反应的最适温度为60℃;在60℃保温6h后,Tte-GlgP仍然有90%的活性残留,说明Tte-GlgP具有热稳定性.
위인식화응용겁단기열세균등충기열염양간균(T.tengcongensis MB4~T)산생적포취당린산화매(Tte-GlgP)적성질,용전분무질보법(ESI-MS)측정Tte-GlgP적저물보.결과표명:순화후적Tte-GIgP유교엄적저물범위,능장가용성정분、맥아호정、당원、맥아칠당、맥아오당화맥아삼당저물전화위1-린산포도당(G-1-P).용개진후적쌍매법측정Tte-GlgP대상술저물적전화효솔,이산생적G-1-P적량위지표(μmol/L),재상동적실험조건하,이맥아칠당화맥아오당위저물시,전화효솔상대고,분별위86.83μmol/L화85.79μmol/L;호정화가용성정분차지,분별위82.9μmol/L G-1-P화69.68μmol/L G-1-P;최저적분별위당원화맥아삼당,산생적G-1-P분별위45.81 μmol/L화43.60μmol/L.량충분석방법균증명맥아과당、맥아당호정화정분위Tte-GlgP적최괄저물.이상술쌍매법위매활성측정방법,측득재체계pH8.0시,Tte-GlgP적활성최고;재최괄pH치조건하이가용성정분위저물시Tte-GlgP최화반응적최괄온도위60℃;재60℃보온6h후,Tte-GlgP잉연유90%적활성잔류,설명Tte-GlgP구유열은정성.
In order to provide valuable evidences for the in-depth understanding and applicadon of glycogen phosphorylase from Thermoanaerobacter tengcongensis MB4~T (Tte-GlgP), the substrate spectrum of Tte-GlgP was determined by ESI-MS method. When soluble starch and maltoheptaose were respectively used as the substrates, the production of alpha-D-glucose-1-phosphate (G-1-P) was clearly revealed in the ESI-MS chromatogram. When glycogen, maltodextrin and maltotriose were subjected to the same reaction, the production of G-1-P was also clearly detected. These results suggested Tte-GlgP had a relative wide substrate spectrum. To evaluate the relative activity of Tte-GlgP towards these substrates, a modified coupled-enzyme method was used to measure the production of G-1-P in these reactions. When 0.25% soluble starch, glycogen, maltodextrin, maltoheptaose, maltopentaose, and maltotriose were used as substrate, respectively, it was established that the transformation efficiency represented by the production of G-1-P (μmol/L), was relatively higher for maltooligosaccharides (maltoheptaose and maltopentaose, with production of 86.83 μmol/L and 85.79 μmol/L G-1-P, respectively), maltodextrin (82.9 μmol/L G-1-P) and soluble starch (69.68 μmol/L G-1-P), but lower for glycogen and maltotriose (45.81 μmol/L and 43.60 μmol/L G-1-P, respectively) at the same reaction condition. These results were consistent with those of ESI-MS analysis. Both suggested that Tte-GlgP could transform a wide variety of glucans into G-1-P, with maltooligosaccharides, maltodextrin and starch as the optimum substrates. For the determination of optimum conditions for reaction of Tte-GlgP with soluble starch as the substrate, 50 mmol/L potassium phosphate buffers with pH ranging from 5.0 to 9.0 were used to prepare the reaction mixtures. The reactions were performed respectively at 50, 60 ℃ and 80 ℃ for 30 min, and the highest activity of Tte-GlgP was achieved at pH 8.0 in all these assays, suggesting that the optimum pH for Tte-GlgP was pH 8.0. Then with this optimum pH (8.0), the reactions were performed at different temperature from 37 ℃ to 80 ℃ for 30 min to detect the optimum temperature, which was revealed to be 60 ℃. To determine the thermostability, Tte-GlgP was incubated at different temperatures from 50 ℃ to 80℃ for 0-21 h, and the residual activity was measured by the modified coupled-enzyme method (0.25% soluble starch was used as substrate). Results suggested that there were still 90% and 64% residual activity after treatment for 6 h at 60 ℃ and 70 ℃, respectively. Thus the Tte-GlgP is indeed a thennostable enzyme as expected.