Structural And Functional Studies Of Aspergillus Niger GZUF36 Lipase

Lipase is one of the important industrial enzymes,and its sources,types,structures,and catalytic reactions are diverse,so it is widely used in the fields of food,medicine,and new energy development.Aspergillus niger lipase is one of the earliest lipases used.However,the crystal structure of Aspergillus niger lipase is unknown,and the structural basis of its properties is unclear.Therefore,in this paper,based on the Aspergillus niger GZUF36 strain,reverse transcription PCR was used to obtain an Aspergillus niger GZUF36 sn-1,3 selective extracellular lipase?EXANL1?gene,and EXANL1was expressed in E.coli and Pichia pastoris respectively.The isolation and purification and enzymatic properties of the expressed recombinase were deeply researched.Based on homology modeling and small-angle X-ray scattering,structural analysis of the recombinase expressed in Pichia pastoris was carried out,which provided references for the further crystal structure analysis and molecular modification of EXANL1.The main research contents and results are as follows:?1?Clone expression,purification and enzymatic characterization of EXANL1 in E.coliAccording to the Aspergillus niger lipase gene sequence reported by NCBI database?GenBank ID:KJ703109.1?,primers were designed,and reverse transcription PCR amplification obtained the coding protein sequence of EXANL1with a total length of 894 bp.Bioinformatics analysis showed the EXANL1 gene with other Aspergillus niger lipase gene sequence similarity is more than 90%,coding 297amino acids,57 bp signal peptide sequence.Predicted protein mature peptide?278amino acids?with the amino acid sequences of Aspergillus niger F044,Aspergillus niger CBE 332.1,Aspergillus niger CBS 513.88 and Aspergillus niger A733 lipase showed 99%homology,except that there were differences in amino acids at individual sites,with an isoelectric point of 4.76,a molecular weight of 30 kDa,and a potential glycosylation site is N59,N150,N269.Based on the multiple alignments of the amino acid sequence of EXANL1 with known structures,the catalytic triad of EXANL1?Ser173,Asp228,His285?was determined,the oxyanion hole formed amino acids S84,L155 and S154,and the lid region was ¹¹¹STVKNWIADLD¹²⁰.EXANL1 encoding mature peptide gene was cloned into E.coli expression vector pET28a?+?,transformed into E.coli BL21?DE3?,cultured at 20?,and 0.1 mM IPTG induced showed the highest activity expression.A nickel column was used to purify the recombinase with a 6x His tag at the N-terminus in one step.The measured enzymatic properties showed that the optimum temperature and optimum pH of recombinant EXANL1 were 35?and 4,respectively.After incubating at 55?for30 min,the remaining enzyme activity was only 51±3.97%.After incubating at pH3-10 for 48 h,the remaining enzyme maintained 90%.Comparing with the enzymatic properties of Aspergillus niger F044 lipase recombinantly expressed with E.coli,we found that even the differences in amino acids at individual sites will lead to larger differences in enzymatic properties.?2?Highly active expression of EXANL1 and its detailed enzymatic propertiesThe gene encoding the mature peptide was cloned and expressed in Pichia pastoris,and the expression was induced by methanol for 72 h.The enzyme activity?17.56±0.31 U/mL?equivalent to that of the wild enzyme was obtained from the fermentation supernatant.The recombinant EXANL1 was purified from the fermentation supernatant using a combination of ammonium sulfate precipitation and a nickel column.The recombinant EXANL1 was displayed as two bands with close molecular weight due to glycosylation modification.The measured enzymatic properties showed that the optimum temperature and optimum pH of recombinant EXANL1 were 40-45?and 4,respectively.After incubating at 55?for 1 h,the remaining enzyme activity was 80%.After incubating at pH 2-8 for 48 h,the remaining enzyme activity was also above 80%.The substrate specificity showed that the most suitable substrate for recombinant EXANL1 was p-nitrophenyl octyl ester,suggesting that recombinant EXANL1 prefers to hydrolyze medium and long-chain esters.In the tested 50%organic solvents,recombinant EXANL1 was relatively stable,especially in 50%n-hexane,the enzyme activity increased by 6%;the tested detergents?final concentration of 0.01%?all promoted the enzyme activity,of which Tween 20 increased the most,nearly 20%.All the metal ions tested did not activate the enzyme,but some inhibited the enzyme activity,especially Ca²⁺and Fe³⁺.The kinetic parameters showed the Vmax and Km value of recombinant EXANL1 was 10U/mL and 68 g/L,respectively.Due to the multiple insertions of amino acids and glycosylation modification at the N-terminus,the Pichia pastoris-expressed recombinase has an increased temperature and thermal stability compared with the prokaryotic recombinase.?3?Based on homology modeling and small-angle X-ray scattering?SAXS?,the structure of Pichia pastoris expressed recombinase was characterizedBased on the recombinase amino acid sequence expressed by Pichia pastoris?pEXANL1?,the Modeller 9.18 multi-template modeling was used to predict the three-dimensional structure of pEXANL1.The results showed that the overall structure of the model was compactly folded,in line with the structural characteristics of?/?hydrolase folding.By superimposing the closed conformation of the model pEXANL1 with the open conformation?PDB:5AP9?A?of the TLL complex containing triglyceride-like substrates,it was shown that the conformation of the lid would change when the substrate was bound,and the catalytic triad Ser162-His274-Asp217 was located catalytic cracks.Since X₁ in the Histidine motif X₁X₂X₃H was Val,pointing to the surface of the protein,pEXANL1 formed three substrate-binding pockets.The three substrate-binding pockets held the three chains of triglycerides,and the X₁ of the diglyceride lipase was Phe,which causes its lipase to have only two substrate-binding pockets,each containing two carboxylic acid chains,so it can only hydrolyze diglycerides and monoglycerides.Therefore,the Histidine motif determined the substrate specificity of lipase.Hydroxymethyl diethyl phosphate?DEP?showed interactions with Ser144,Ser145,Tyr28,His257,Trp88 and Asp91 through hydrophobic interactions and with the oxygen anion holes Ser82 and Leu145 through hydrogen bonds,firmly bonding to the active center.Tricaprylin showed interactions with Ser82?Gly81?Tyr28?Phe111?Pro177?Asp 91?Ser144?Ile204?Leu208?Val205?Leu258?Trp88?Val254 through hydrophobic interactions and with Ser82 and Leu145 through hydrogen bonds.SAXS analyzed the structure of Aspergillus niger lipase in a solution for the first time and showed that pEXANL1 was compact and fully folded in solution.SAXSMoW estimated molecular weight was 31.62 kDa,which was consistent with the theoretical molecular weight?31.5 kDa?.The radius of gyration calculated by Guinier plot was 2.00±0.50-2.24±0.21?nm?,the distance distribution function showed that the average maximum size was 8 nm.The ab initio calculated shape outline showed a compact structure like a big chicken leg,which was well superimposed with the pEXANL1 three-dimensional structure model.The three-dimensional structure model was also in good agreement with the experimental scattering data.