Guimarães V M, de Rezende S T, Moreira M A, de Barros E G, Felix C R
Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, 70.910-900, Brazil.
Phytochemistry. 2001 Sep;58(1):67-73. doi: 10.1016/s0031-9422(01)00165-0.
Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. Enzymic hydrolysis of the RO is accomplished by alpha-galactosidase. While the content of RO decreases during seed germination, the activity of alpha-galactosidase increases substantially. Two alpha-galactosidases were isolated from germinating seeds by partition in an aqueous two-phase system followed by ion-exchange and affinity chromatography. One of the enzyme preparations (P1) showed a single protein with M(r) of 33 kDa, and the second (P2) had two proteins with M(r) of 31 and 33 kDa. Maximal activities against the synthetic substrate rho-nitrophenyl-alpha-D-galactopyranoside (rhoNPGal) were detected at pH 5.0-5.5 and 45-50 degrees C. Both enzymes were fairly stable at 40 degrees C, but lost most of their activities after 30 min at 50 degrees C. The K(m) values for hydrolysis of rhoNPGal by the P1 and P2 enzymes were 1.55 and 0.76 mM, respectively. The K(m) values determined for hydrolysis of raffinose and melibiose by the P2 enzyme were 5.53 and 5.34 mM, respectively and galactose was a competitive inhibitor (K(i)=0.65 mM). To different extents, both enzymes were sensitive to inhibition by galactose, melibiose, CuSO(4), and SDS. Sucrose and beta-mercaptoethanol showed discrete inhibitory effects on both enzymes.
棉子糖低聚糖（RO）是摄入大豆制品后导致肠胃胀气的主要因素。从种子或豆浆中去除RO对大豆类食品的接受度会产生积极影响。RO的酶促水解是通过α-半乳糖苷酶来完成的。虽然在种子萌发过程中RO的含量会降低，但α-半乳糖苷酶的活性会大幅增加。通过在水两相系统中分配，随后进行离子交换和亲和色谱，从萌发的种子中分离出了两种α-半乳糖苷酶。其中一种酶制剂（P1）显示为一种分子量为33 kDa的单一蛋白质，另一种（P2）有两种分子量分别为31 kDa和33 kDa的蛋白质。在pH 5.0 - 5.5和45 - 50℃时检测到对合成底物对硝基苯基-α-D-吡喃半乳糖苷（ρNPGal）的最大活性。两种酶在40℃时都相当稳定，但在50℃ 30分钟后大部分活性丧失。P1和P2酶水解ρNPGal的米氏常数（K(m)）分别为1.55和0.76 mM。P2酶水解棉子糖和蜜二糖的米氏常数分别为5.53和5.34 mM，半乳糖是竞争性抑制剂（抑制常数K(i)=0.65 mM）。两种酶在不同程度上对半乳糖、蜜二糖、硫酸铜（CuSO₄）和十二烷基硫酸钠（SDS）的抑制敏感。蔗糖和β-巯基乙醇对两种酶都有不同的抑制作用。
