Synthesis and characterization of cross linked enzyme aggregates of serine hydroxyl methyltransferase from Idiomerina leihiensis.

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.