Professor Gao Renjun's team has made important progress in the design and synthesis of anionic liquid enzymes

The team of Professor Gao Renjun from the Key Laboratory of Molecular Enzymatic Engineering of the Ministry of Education, College of Life Sciences, Jilin University, together with the team of Professor Guo Yin of Aarhus University in Denmark, proposed a design strategy
for a new solvent-free liquid protein (enzyme).
The LipA of Bacillus subtilis lipase was first ionized and modified, and then crosslinked with ionic polymers to successfully synthesize liquid enzymes, so that room temperature enzymes had the catalytic properties of superthermophilic enzymes, which has great potential
for application in biocatalysis 。 The relevant research has been published in Advanced Recently.
Science.
(2022, 2202359).
https://doi.
org/10.
1002/advs.
202202359

Professor Gao Renjun's research group has been engaged in the research of thermophiles for a long time, but due to the limited sources of thermophiles, their selection and application are often limited
.
Solvent-free liquid proteins (also known as biofluids or protein liquids) are a new class of hybrid nanobiomaterials that have great potential
in the field of biocatalysis due to their attractive properties, including near-native structure and superthermal stability.
The charge is filled with the surface of the enzyme molecule by ionic modifier, and then a certain number of ionic polymers are crosslinked by electrostatic interaction, so that the polymer is stably wrapped on the surface of the enzyme to form a "shell" structure
with a certain thickness.
It is this "shell" that increases the volume of protein particles after removing the solvent, increases the protein molecular spacing, effectively prevents the interaction between enzyme molecules, and maintains the conformational stability of the enzyme, so that ordinary enzyme molecules can form ionic solvent-free liquid enzymes at normal pressure by heating and annealing and at high temperatures (Figure 1).

In this study, the room temperature enzyme was modified into a superthermophile enzyme through the modification of nanomaterials, so that enzymes from various sources have the potential to
become thermophiles.

Figure 1, Liquid enzyme synthesis pathway
.

In this study, the authors selected the LipA 8M mutant of Bacillus subtilis lipase as the research object, and used succinic anhydride to completely modify the amino group on the surface of the protein molecule (Lys residue side chain and N-terminal α-amino) to maximize the anion on the protein surface, and then crosslink with the cationic polymer (cS) to form an anionic solvent-free liquid protein [a8M][cS].

。 For contrast, the protein surface anion was modified with N,N'-dimethyl-1,3-propanediamine (DMPA) and crosslinked with anionic polymer (S) to form a cationic solvent-free liquid protein [c8M][S] (Figure 2).

Figure 2, the difference between anionic and cationic liquid enzymes

Various stages of liquid protein synthesis were characterized and identified
by various biophysical and chemical characterization methods such as SRCD, SAXS, DSC, ATR-FTIR, UV-vis, etc.
The results showed that in the study of lipase preparation of liquid protein, liquid lipase [a8M][cS] synthesized by anionic modification was higher than that of cationic liquid lipase [c8M][S]
in terms of structural integrity and activity.
In this study, myoglobin (Mb) was also used as the control protein for the synthesis
of the corresponding liquid protein.
In the study of liquid protein synthesis of myoglobin, anionic modification led to complete denaturation and unfolding of Mb, and the optical rotation of the protein was also changed, but the protein was refolded
after crosslinking with the cS polymer.
The α-helical level of anionic liquid protein [aMb][cS] is higher than that of [cMb][S], and the stability is stronger
.
In addition, the activity of anionic liquid enzymes is much higher than that of cationic liquid enzymes, and it still maintains more than 10% activity at 120°C (Figure 3), realizing the transition
from room temperature enzymes to superthermophilic enzymes.
Anionic solvent-free liquid proteins have more obvious advantages and stronger applicability, providing researchers with more options
for catalyzing reactions at high temperatures.

Figure 3, Comparison of circular dichism and enzyme activity of different liquid enzymes

This research work was supported
by grants from China Scholarship Council, the Novo Nordisk Foundation and the AUFF Innovation Fund.
The first thesis was completed by Jilin University, and the first author was Zhou Ye,
a doctoral student who graduated from the School of Life Sciences of Jilin University.
Professor Gao Renjun from the Key Laboratory of Molecular Enzymatic Engineering, Ministry of Education, College of Life Sciences, Jilin University, and Professor Guo Yun, School of Technical Sciences, Aarhus University, Denmark, are the co-corresponding authors
of the paper.

Full text link to paper: https://onlinelibrary.
wiley.
com/doi/10.
1002/advs.
202202359