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

Ashok Kumar; Gaobing Wu; Ziduo Liu

doi:10.1016/j.ijbiomac.2018.04.106

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

Int J Biol Macromol. 2018 Oct 1:117:683-690. doi: 10.1016/j.ijbiomac.2018.04.106. Epub 2018 Apr 22.

Authors

Ashok Kumar¹, Gaobing Wu², Ziduo Liu³

Affiliations

¹ 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.
² College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, P.R. China, 430070.
³ College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, P.R. China, 430070. Electronic address: lzd@mail.hzau.edu.cn.

PMID: 29694840
DOI: 10.1016/j.ijbiomac.2018.04.106

Abstract

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 Ca²⁺ and Ba²⁺ were found to enhance the activity at 1 and 5 mM of concentrations while Ni⁺, Co²⁺ and Zn²⁺ 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^-1 and 272 μM while for CLEAs V_max 1.42 μM s^-1 and k_m 248.6 μM was recorded. Thus, a 120% increase in the V_max 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.

Keywords: Cross-linked enzyme aggregates; Formaldehyde; Pectin; Reusability metal ions; SHMT.

MeSH terms

Alteromonadaceae / enzymology
Cross-Linking Reagents
Enzyme Stability*
Enzymes, Immobilized / chemistry*
Glycine Hydroxymethyltransferase / chemistry*
Hydrogen-Ion Concentration
Hydroxyl Radical / chemistry
Kinetics
Protein Aggregates*

Substances

Cross-Linking Reagents
Enzymes, Immobilized
Protein Aggregates
Hydroxyl Radical
Glycine Hydroxymethyltransferase