(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Firmicutes: NE > Bacilli: NE > Bacillales: NE > Paenibacillaceae: NE > Aneurinibacillus group: NE > Aneurinibacillus: NE > Aneurinibacillus thermoaerophilus: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MQKERKNQYPIVLVHGFAGWGRDEMLGVKYWGGMHDIQEDLKQYGYETHT AVVGPFSSNWDRACELYAQLVGGTVDYGAAHAEKYGHDRFGRTYPGLLKN WDGEHKIHLIGHSMGGQTVRVLTQLLKEGSQEEREYAKKHGVQLSPLFEG GKSWVHSVTTIATPNDGTTLADVVTQLIPAAQQIMGLAAAVSGNTNVPVY DFKLDQWGLKRKAGESFVHYADRVWNSGIWTNTKDISAWDLKPEGAKELN NWVKAQPDVYYFSYSGEATFRSLITGHHLPDLTMNKLITPFGIFLGCYGS DEKWWQNDGIVNTISMNGPKLGSTDEIVPYDGTPKIGKWNDMGIQENWDH ADYIGLSLSYVLGIEKIEDFYRGVADMLGSLSVR
Biocatalysis has emerged as an important tool in synthetic organic chemistry enabling the chemical industry to execute reactions with high regio- or enantioselectivity and under usually mild reaction conditions while avoiding toxic waste. Target substrates and products of reactions catalyzed by carboxylic ester hydrolases are often poorly water soluble and require organic solvents, whereas enzymes are evolved by nature to be active in cells, i.e., in aqueous rather than organic solvents. Therefore, biocatalysts that withstand organic solvents are urgently needed. Current strategies to identify such enzymes rely on laborious tests carried out by incubation in different organic solvents and determination of residual activity. Here, we describe a simple assay useful for screening large libraries of carboxylic ester hydrolases for resistance and activity in water-miscible organic solvents. We have screened a set of 26 enzymes, most of them identified in this study, with four different water-miscible organic solvents. The triglyceride tributyrin was used as a substrate, and fatty acids released by enzymatic hydrolysis were detected by a pH shift indicated by the indicator dye nitrazine yellow. With this strategy, we succeeded in identifying a novel highly organic-solvent-tolerant esterase from Pseudomonas aestusnigri In addition, the newly identified enzymes were tested with sterically demanding substrates, which are common in pharmaceutical intermediates, and two enzymes from Alcanivorax borkumensis were identified which outcompeted the gold standard ester hydrolase CalB from Candida antarctica IMPORTANCE Major challenges hampering biotechnological applications of esterases include the requirement to accept nonnatural and chemically demanding substrates and the tolerance of the enzymes toward organic solvents which are often required to solubilize such substrates. We describe here a high-throughput screening strategy to identify novel organic-solvent-tolerant carboxylic ester hydrolases (CEs). Among these enzymes, CEs active against water-insoluble bulky substrates were identified. Our results thus contribute to fostering the identification and biotechnological application of CEs.
Title: Characterization of a Novel Alkalophilic Lipase From Aneurinibacillus thermoaerophilus: Lid Heterogeneity and Assignment to Family I.5 Zottig X, Meddeb-Mouelhi F, Charbonneau DM, Beauregard M Ref: Protein J, 36:478, 2017 : PubMed
Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase alpha/beta hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 degrees C under neutral pH, but its maximal activity was detected at pH 10 and 45 degrees C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 degrees C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure-function relationships in lipases.
Title: A new thermostable and organic solvent-tolerant lipase from Aneurinibacillus thermoaerophilus strain HZ. Masomian M, Rahman RNZRA, Salleh AB, Basri M Ref: Process Biochemistry, 48:169, 2013 : PubMed
A thermostable and organic solvent-tolerant lipase produced by Aneurinibacillus thermoaerophilus strain HZ was purified and characterised. The lipase was purified to apparent homogeneity with two steps: anion exchange chromatography on Q-Sepharose and gel filtration on Sephadex-G75. A final specific activity of 43.5 U/mg was obtained with an overall recovery of 19.7% and 15.6 purification fold. The molecular mass of the HZ lipase was estimated to be 50 kDa. The optimum pH for the activity of the purified HZ lipase was 7.0. The stability showed a broad range of pH values between pH 4.0 and 9.0 at 30 C. The purified HZ lipase exhibited an optimum temperature of 65 C with a half-life of 3 h and 10 min at 65 C. The activity of the purified HZ lipase was stimulated in the presence of Ca2+. Organic solvents such as dimethyl sulfoxide (DMSO), methanol, n-tetradecane and n-hexadecane enhanced the lipase activity. Studies on the effect of oil showed that the lipase preferred natural oil, such as sunflower oil, over synthetic substrates.
