Molecular Engineering Of L-amino Acid Deaminase And Construction Of Self-assembly Of The Multi-enzyme System For D-amino Acid Synthesis

D-amino acids are used as intermediates in the synthesis of drugs,food additives and active ingredients of cosmetics.D-amino acids are widely applied in food,pharmacy,agriculture and cosmetics.The synthetic method of D-amino acids are mainly chemical synthesis and biosynthesis.Bioenzymatic synthesis of D-amino acids has the advantages of mild reaction condition,high catalytic efficiency,energy saving and environmental protection.As a biosynthetic method,multi-enzyme cascade reactions can improve the catalytic efficiency and avoid the accumulation of intermediates,so it is widely used in the synthesis of D-amino acids.Our group has previously constructed a multi-enzyme system comprised of L-amino acid deaminase from Proteus Mirabilis（PmLAAD）,diaminoheptanoate dehydrogenase from Symbiobacterium thermophilum（StDAPDH）and formate dehydrogenase（BsFDH）from Burkholderia stabilis to produce D-phenyalanine（D-Phe）from L-phenylalanine（L-Phe）.However,this multi-enzyme system still has some shortcomings,such as limited substrate spectrum of the key enzyme PmLAAD（mainly catalyzing L-Phe）,low catalytic activity of the key enzyme StDAPDH and low mass transfer efficiency of the cascade system composed of free enzymes.Our study aimed at improving this multi-enzyme system from two aspects:molecular engineering of the key enzyme and construction of self-assembly catalytic system.The main results are listed as follows:（1）The semi-rational design strategy was employed to conduct molecular engineering on PmLAAD.The substrate spectrum was broadened through site-directed saturation mutagenesis of E417,a key residue around the substrate binding site.Expansion of the substrate channel with mutations of E417（E417L,E417A）improved activity toward the bulky substrate L-Trp,and mutation of E417 to basic amino acids（E417K,E417H,E417R）enhanced the universal activity toward various L-amino acid substrates.The activity of the variant PmLAAD^E417Ktoward seven substrates L-Ala,L-Asp,L-Ile,L-Leu,L-Phe,L-Trp,and L-Val was 3.77-fold,1.41-fold,1.34-fold,1.29-fold,1.05-fold,6.65-fold,and 1.68-fold higher,respectively.The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions.（2）A self-assembled multi-enzyme system was constructed by using affinity peptides based on hydrogen bond interaction.Aiming at the assembly of StDAPDH and BsFDH in the key step of D-amino acid synthesis,the self-assembly system of StDAPDH and BsFDH was constructed by using a pair of hydrogen bonding based affinity peptide RIAD/RIDD.Through attaching short peptides to the C-and N-terminal of the enzyme respectively,St-AD（StDAPDH linked with RIAD short peptide at C-terminal）,St-DD（StDAPDH linked with RIDD at C-terminal）,Bs-AD（BsFDH linked with RIAD at C-terminal）,Bs-DD（BsFDH linking RIDD at C-terminal）,AD-St（StDAPDH linked with RIAD short peptide at N-terminal）,DD-St（StDAPDH linked with RIDD short peptide at N-terminal）,AD-Bs（BsFDH linked with RIAD at N-terminal）and DD-Bs（BsFDH linked with RIDD at N-terminal）was obtained.Different assembly combinations was tried:St AD-（BsDD）₂,BsAD-（StDD）₂,AD-Bs-（St-DD）₂,AD-St-（Bs-DD）₂,AD-St-（DD-Bs）₂,St-AD-（DD-Bs）₂,AD-Bs-（DD-St）₂ and Bs-AD-（DD-St）₂.The homology modeling and structural analysis showed that RIAD（or RIDD）peptide was mostly embedded in the homomultimeric structure of the enzymes,which significantly affected the self-assembly of RIAD/RIDD tagged enzymes,indicating that the efficiency and applicability of RIAD/RIDD self-assembly was limited by the polymeric structure of the target enzymes.（3）The self-assembly of StDAPDH and BsFDH was constructed by using a pair of short peptides SpyT/SpyC that can spontaneously form isomeric peptide bonds.SpyT or SpyC was linked to the N-terminal of the enzyme to construct the self-assembly system（SpyT-St）-（SpyC-Bs）and（SpyC-St）-（SpyT-Bs）,among which only assembly of（SpyT-St）-（SpyC-Bs）was successfully obtained.The effects of temperature,incubation time and protein ratio on self-assembly efficiency were investigated and the highest assembly efficiency was achieved when the protein concentration ratio of SpyT-St and SpyC-Bswas 1:1 and the optimal temperature and incubation time was 30℃ and 30 min,respectively.The catalytic efficiency of（SpyT-St）-（SpyC-Bs）assembly system was found to be 1.3 fold of that of free enzyme to produce D-Phe from L-Phe.