College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, P.R. China, 430070; Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan 173234, Himachal Pradesh, India.
College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, P.R. China, 430070.
Int J Biol Macromol. 2018 Oct 1;117:683-690. doi: 10.1016/j.ijbiomac.2018.04.106. Epub 2018 Apr 22.
A thermo-stable purified serine hydroxymethyltransferase (SHMT; 418 AA) was used for the carrier free immobilization using pectin as a coach molecule and formaldehyde as a cross-linker. The purified protein was cross linked with formaldehyde in the presence of pectin to form stable and active aggregates. The cross-linked enzyme aggregates [CLEAs] of SHMT showed improved catalytic properties and reusability. The SHMT-CLEAs showed a noteworthy change in the thermo-stability and activity compared to its free counterpart. The optimum activity for free SHMT was reported at 55 °C and pH 7.5 which SHMT CLEAs showed maximum activity at 60 °C and pH 8.0. Similarly, the CLEAs were noticed to increase the thermo-stability in comparison to free enzyme. The divalent salt ion Ca and Ba were found to enhance the activity at 1 and 5 mM of concentrations while Ni, Co and Zn strongly inhibited the activity of both free as well as CLEAs. The Vmax and km values for free SHMT were recorded to be 1.21 μM s and 272 μM while for CLEAs V 1.42 μM s and k 248.6 μM was recorded. Thus, a 120% increase in the V was recorded for SHMT-CLEAs. The CLEAs were also found to be more stable at pH 6.5 and 8.5 pHs and retained 50% of its original activity for 180 and 200 min respectively. The CLEAs also retained 72% of its activity after 12 repetitive cycles of d-phenylserine hydrolysis. Also, the synthesized CLEAs retained more than 60% of its original activity after 10 days of incubation at 25 °C in comparison to free enzyme which loses more than 90% of its residual activity. Thus, with improved thermostability and activity the CLEAs of SHMT can be used repetitively at industrial scale for the synthesis of commercially important amino acids.
使用果胶作为引导分子和甲醛作为交联剂，对热稳定的纯化丝氨酸羟甲基转移酶 (SHMT；418AA) 进行无载体固定化。在果胶存在下，将纯化的蛋白质与甲醛交联，形成稳定且有活性的聚集体。SHMT 的交联酶聚集体 [CLEAs] 表现出改善的催化性质和可重复使用性。与游离酶相比，SHMT-CLEAs 的热稳定性和活性有显著变化。游离 SHMT 的最佳活性在 55°C 和 pH7.5 下报道，而 SHMT CLEAs 在 60°C 和 pH8.0 下表现出最大活性。同样，与游离酶相比，CLEAs 被发现增加了热稳定性。二价盐离子 Ca 和 Ba 在 1 和 5mM 浓度下发现可以提高活性，而 Ni、Co 和 Zn 强烈抑制游离酶和 CLEAs 的活性。游离 SHMT 的 Vmax 和 km 值分别记录为 1.21μM·s 和 272μM，而 CLEAs 的 V 为 1.42μM·s 和 k 为 248.6μM。因此，SHMT-CLEAs 的 V 增加了 120%。还发现 CLEAs 在 pH6.5 和 8.5 时更稳定，分别在 180 和 200min 时保留其原始活性的 50%。CLEAs 在重复 12 次 d-苯丙氨酸水解循环后仍保留其 72%的活性。此外，与游离酶相比，在 25°C 孵育 10 天后，合成的 CLEAs保留了超过 60%的原始活性，而游离酶失去了超过 90%的剩余活性。因此，通过提高热稳定性和活性，SHMT 的 CLEAs 可以在工业规模上重复用于合成具有商业重要性的氨基酸。
