PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on ß-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Analysis of differential expression proteins reveals the key pathway in response to heat stress in Alicyclobacillus acidoterrestris DSM 3922T.

For the purpose of investigating the heat resistance mechanism of Alicyclobacillus acidoterrestris, label-free quantification was used to reveal some cellular changes in A. acidoterrestris during heat stress. Totally, 545 differential expression proteins were respectively identified at heat stress of 65 °C for 5 min, of which 258 proteins were up-regulated and 287 proteins were down-regulated. These significantly changed proteins were mapped to 100 pathways and some of them were mostly related to protection or repair of macromolecules such as proteins and DNA, cell wall formation, which indicated that these proteins might play crucial roles in response to heat stress. The KEGG pathway analysis combined with protein functional analysis and further validation at mRNA level suggested that A. acidoterrestris sensed the temperature rise in environment through alterations in the secondary structure of DNA and RNA molecules. The biosynthesis of antibiotics pathway and the ribosomes might be involved in signal transduction in heat stress and further trigger a large number of proteins playing a critical role in the regulation of heat stress in A. acidoterrestris. The study firstly demonstrated the global physiological response to heat stress and the results provided a better understanding of thermal adaption mechanism of A. acidoterrestris.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grew while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.

Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis.

UNLABELLED: Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic ß-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic ß-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCE: Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic ß-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.

Degradation of phenolic compounds by the lignocellulose deconstructing thermoacidophilic bacterium Alicyclobacillus Acidocaldarius.

Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium, has a repertoire of thermo- and acid-stable enzymes that deconstruct lignocellulosic compounds. The work presented here describes the ability of A. acidocaldarius to reduce the concentration of the phenolic compounds: phenol, ferulic acid, ρ-coumaric acid and sinapinic acid during growth conditions. The extent and rate of the removal of these compounds were significantly increased by the presence of micro-molar copper concentrations, suggesting activity by copper oxidases that have been identified in the genome of A. acidocaldarius. Substrate removal kinetics was first order for phenol, ferulic acid, ρ-coumaric acid and sinapinic acid in the presence of 50 µM copper sulfate. In addition, laccase enzyme assays of cellular protein fractions suggested significant activity on a lignin analog between the temperatures of 45 and 90 °C. This work shows the potential for A. acidocaldarius to degrade phenolic compounds, demonstrating potential relevance to biofuel production and other industrial processes.

Culture dependent and independent genomic identification of Alicyclobacillus species in contaminated commercial fruit juices.

Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming, bacteria species which occasionally cause spoilage of heat-processed fruit juices by producing guaiacol taint. In this study, Alicyclobacillus contamination of commercial fruit juices in West Africa was investigated using culture-dependent and -independent approaches. Firstly, a total of 225 fruit juice products from Ghana (n = 39) and Nigeria (n = 186) were enriched with yeast-starch-glucose (YSG) broth (pH 3.7) following heat shock at 80 °C for 10 min. Alicyclobacillus was detected in 11.6% (26) of samples. Isolates were identified to the genus taxonomic level by genus-specific PCR which targeted the squalene-hopene-cyclase (shc) gene followed by analysis of the almost-complete 16S ribosomal RNA (rRNA) gene sequences that identified 16 Alicyclobacillus acidoterrestris, 7 Alicyclobacillus acidocaldarius and 3 Alicyclobacillus genomic species 1 (Alicyclobacillus sp. 1). Whole-genome fingerprinting using PCR-RAPD primers Ba-10, F-61 and F-64 grouped the 16 A. acidoterrestris isolates into two genetic clusters. Furthermore, high performance liquid chromatographic (HPLC) analyses revealed the activity of vanillic-acid decarboxylase (vdc) in all A. acidoterrestris isolates due to guaiacol production from vanillic-acid. Lastly, species-specific PCR-DGGE targeting the 16S rRNA gene clearly discriminated between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius group. Information provided by this study is fundamental to the development of effective strategies for the improvement of quality and shelf-life of processed tropical fruit juices in W. Africa.

Extraction, partial purification and characterisation of vanillic acid decarboxylase from Alicyclobacillus acidoterrestris DSM 3923.

BACKGROUND: Vanillic acid decarboxylase (VAD) is the key enzyme responsible for guaiacol production in Alicyclobacillus acidoterrestris; however, information related to this enzyme is currently unavailable. The aim of this study is to characterise the VAD from A. acidoterrestris. RESULTS: Specific activity of VAD in vanillic acid-induced A. acidoterrestris DSM 3923 cells was highest in the early stage of the log phase, and almost undetectable in the stationary and death phases. Of the four techniques used to extract VAD, sonication was found to be the most effective and recovered 3.23 U mg(-1) of VAD. Through optimisation of the crucial parameters for sonication, the recovery of VAD had more than doubled (6.81 U mg(-1) ). The crude enzyme extract was purified by ammonium sulfate precipitation and a 9.87-fold purification was obtained. The partially purified VAD exhibited optimum activity at pH 6.0-6.5, 45°C and was stable at pH 5.0-7.5, 20-45°C. The Km and Vmax values of the VAD were 0.53 mmol L(-1) and 96 U mg(-1) protein, respectively. VAD activity was stimulated by Co(2+) and Mn(2+) , but was inhibited by Ni(2+) , Cu(2+) , Ba(2+) and Fe(3+) . Cinnamic acid, ferulic acid, resveratrol, quercetin and rutin at the concentration of 1 mmol L(-1) could completely inhibit the activity of VAD. CONCLUSION: The present study provides the first report on the characteristics of the VAD from A. acidoterrestris, which will contribute to the development of more effective control methods to minimise A. acidoterrestris-related spoilage in fruit juices. © 2015 Society of Chemical Industry.

Spore inactivation and DPA release in Alicyclobacillus acidoterrestris under different stress conditions.

This paper reports on the inactivation of spores of 5 strains of Alicyclobacillus acidoterrestris under different stress conditions (acidic and alkaline pH, high temperature, addition of lysozyme, hydrogen peroxide and p-coumaric acid). The research was divided into two different steps; first, each stress was studied alone, thus pointing out a partial uncoupling between spore inactivation and DPA release, as H2O2 reduced spore level below the detection but it did not cause the release of DPA. A partial correlation was found only for acidic and alkaline pH. 2nd step was focused on the combination of pH, temperature and H2O2 through a factorial design; experiments were performed on both fresh and 4 month-old spores and pinpointed a different trend for DPA release as a function of spore age.

A G-quadruplex DNAzyme-based colorimetric method for facile detection of Alicyclobacillus acidoterrestris.

The rapid and sensitive detection of Alicyclobacillus acidoterrestris (AA) has become very important due to the frequent occurrence of fruit juice spoilage by AA. In the present study, using guaiacol, both as the metabolic product of AA related to its concentration and as a green colorimetric substrate of G-quadruplex DNAzyme, a novel G-quadruplex DNAzyme-based colorimetric method for a rapid detection of AA has been developed for the first time. Under optimal conditions, AA has been successfully detected in the concentration range of 10(2)-10(5) cfu mL(-1) with a detection limit of 85 cfu mL(-1). The recoveries ranging from 71.8% to 115.7% with relative standard deviation from 1.2% to 6.6% in spiked apple and orange juice samples were obtained. Results demonstrate that the sensitivity and precision of the developed method is comparable with most other analytical methods and is prominently rapid than them. We believe that the work provides a novel and effective approach and is beneficial for monitoring and reducing the risk of AA contaminations during the process of fruit juice production.

Patulin reduction in apple juice by inactivated Alicyclobacillus spp.

UNLABELLED: This study aimed to investigate the reduction of patulin (PAT) in apple juice by 12 inactivated Alicyclobacillus strains. The reduction rate of PAT by each strain was determined by high-performance liquid chromatography (HPLC). The results indicated that the removal of PAT was strain specific. Alicyclobacillus acidoterrestris 92 and A. acidoterrestris 96 were the most effective ones among the 12 tested strains in the removal of PAT. Therefore, these two strains were selected to study the effects of incubation time, initial PAT concentration and bacteria powder amount on PAT removal abilities of Alicyclobacillus. The highest PAT reduction rates of 88·8 and 81·6% were achieved after 24-h incubation with initial PAT concentration of 100 µg l(-1) and bacteria powder amount of 40 g l(-1) , respectively. Moreover, it was found that the treatment by these 12 inactivated Alicyclobacillus strains had no negative effect on the quality parameters of apple juice. Similar assays were performed in supermarket apple juice, where inactivated Alicyclobacillus cells could efficiently reduce PAT content. Taken together, these data suggest the possible application of this strategy as a means to detoxify PAT-contaminated juices. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivated Alicyclobacillus cells can efficiently reduce patulin concentration in apple juice. It provides a theoretical foundation for recycling of Alicyclobacillus cells from spoiled apple juice to reduce the source of pollution and the cost of juice industry. This is the first report on the use of Alicyclobacillus to remove patulin from apple juice.

Influence of physicochemical characteristics on the growth and guaiacol production of Alicyclobacillus acidoterrestris in fruit juices.

Alicyclobacillus acidoterrestris is a bacterium known for causing spoilage in the taste and odour of fruit juices due to its thermoacidophilic nature. Its spoilage is attributed to the formation of guaiacol, which requires the presence of suitable precursors in the juices that A. acidoterrestris can metabolize. Therefore, A. acidoterrestris could exhibit different behaviour depending on the physicochemical characteristics the juice. In this study, we aimed to evaluate the behaviour of five A. acidoterrestris strains in seven different fruit juices by monitoring total cell and spore populations and quantifying guaiacol production. Also, physicochemical and phenolic profile, focusing on antimicrobials and guaiacol precursors, were analysed to better understand differences. Results showed growth in orange, apple, and plum juices for all the tested strains, with total cell populations reaching approximately 7 log cfu/mL, except for plum juice. In persimmon juice, growth was only observed in 3 out of 5 strains, for both total cells and spores. In contrast, all strains were inhibited in peach, black grape, and strawberry juices, maintaining a consistent population around 4 log cfu/mL. A strong negative correlation was observed between bacterial population and compounds such as kaempferol (for strains R3, R111, and P1), cyanidin chloride (for strains R111 and P1), and p-coumaric acid (for strain 7094 T). Regarding guaiacol production, orange and persimmon juices exhibited the highest guaiacol levels, with strain P1 (362.3 ± 12.6 ng/mL) and strain EC1 (325.1 ± 1.4 ng/mL) as the top producers, respectively. Plum, black grape, and strawberry juices showed similar guaiacol concentrations (16.9 ± 2.8 to 105.0 ± 33.7 ng/mL). Vanillin was showed positive correlations with guaiacol production in almost all strains (7094 T, R3, R111, and P1), with correlation coefficients of 0.97, 0.99, 0.82, and 0.87, respectively. We have reported different behaviour of A. acidoterrestris strains depending on juice type. Despite growth inhibition observed in some juices, enough guaiacol quantities to spoil the juice can be produced. This highlights the necessity of exploring strategies to prevent guaiacol production, even under growth restriction.

High Salt-Resistant Urethanase Degrades Ethyl Carbamate in Soy Sauce.

Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum (ApUH) was successfully discovered using a database search. ApUH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity (Km, 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. ApUH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.

Metabolic response of Alicycliphilus denitrificans strain BC toward electron acceptor variation.

Alicycliphilus denitrificans is a versatile, ubiquitous, facultative anaerobic bacterium. Alicycliphilus denitrificans strain BC can use chlorate, nitrate, and oxygen as electron acceptor for growth. Cells display a prolonged lag-phase when transferred from nitrate to chlorate and vice versa. Furthermore, cells adapted to aerobic growth do not easily use nitrate or chlorate as electron acceptor. We further investigated these responses of strain BC by differential proteomics, transcript analysis, and enzyme activity assays. In nitrate-adapted cells transferred to chlorate and vice versa, appropriate electron acceptor reduction pathways need to be activated. In oxygen-adapted cells, adaptation to the use of chlorate or nitrate is likely difficult due to the poorly active nitrate reduction pathway and low active chlorate reduction pathway. We deduce that the Nar-type nitrate reductase of strain BC also reduces chlorate, which may result in toxic levels of chlorite if cells are transferred to chlorate. Furthermore, the activities of nitrate reductase and nitrite reductase appear to be not balanced when oxygen-adapted cells are shifted to nitrate as electron acceptor, leading to the production of a toxic amount of nitrite. These data suggest that strain BC encounters metabolic challenges in environments with fluctuations in the availability of electron acceptors. All MS data have been deposited in the ProteomeXchange with identifier PXD000258.

Synthesis of heterocyclic terpenoids by promiscuous squalene-hopene cyclases.

PROMISCUOUS ENZYMES: The substrate promiscuity of squalene-hopene cyclases has been explored and applied in the enzyme-catalyzed synthesis of heterocyclic terpenoids. Features of this work include cyclization reactions without pyrophosphate activation, and stereospecific ring closure of substrates of varying chain length and terminal nucleophile. This provides a biocatalytic alternative to traditional chemical catalysts.

Unveiling the complete genome sequence of Alicyclobacillus acidoterrestris DSM 3922T, a taint-producing strain.

Several species from the Alicyclobacillus genus have received much attention from the food and beverages industries. Their presence has been co-related with spoilage events of acidic food matrices, namely fruit juices and other fruit-based products, the majority attributed to Alicyclobacillus acidoterrestris. In this work, a combination of short and long reads enabled the assembly of the complete genome of A. acidoterrestris DSM 3922T, perfecting the draft genome already available (AURB00000000), and revealing the presence of one chromosome (4,222,202 bp; GC content 52.3%) as well as one plasmid (124,737 bp; GC content 46.6%). From the 4,288 genes identified, 4,004 sequences were attributed to coding sequences with proteins, with more than 80% being functionally annotated. This allowed the identification of metabolic pathways and networks and the interpretation of high-level functions with significant reliability. Furthermore, the additional genes of interest related to spore germination, off-flavor production, namely the vdc cluster, and CRISPR arrays, were identified. More importantly, this is the first complete and closed genome sequence for a taint-producing Alicyclobacillus species and thus represents a valuable reference for further comparative and functional genomic studies.

Pivalic acid acts as a starter unit in a fatty acid and antibiotic biosynthetic pathway in Alicyclobacillus, Rhodococcus and Streptomyces.

A biosynthetic pathway using pivalic acid as a starter unit was found in three bacterial species, Alicyclobacillus acidoterrestris, Rhodococcus erythropolis and Streptomyces avermitilis. When deuterium-labelled pivalic acid was added to A. acidoterrestris and R. erythropolis nutrient media it was incorporated into fatty acids to give rise to tert-butyl fatty acids (t-FAs). In addition, in R. erythropolis, pivalic acid was transformed into two starter units, i.e. isobutyric and 2-methylbutyric acid, which served as precursors of corresponding iso-even FAs and anteiso-FAs. In S. avermitilis the biosynthesis also yielded all three branched FAs; apart from this pathway, both pivalic and 2-methylbutyric acids were incorporated into the antibiotic avermectin.

Isolation and identification of thermo-acidophilic bacteria from orchards in china.

Eight strains of thermo-acidophilic bacteria have been isolated from apple orchards in Shaanxi Province, China. The isolated strains were identified at the species level by comparing 16S rRNA gene sequences. It was found that all strains could be assigned to two genera. The strain YL-5 belonged to Alicyclobacillus, and other isolates belonged to Bacillus. The enzymatic patterns by the API ZYM system showed very significant differences between 12 strains of Alicyclobacillus and 8 strains of Bacillus. The ability of guaiacol production varied among different strains.

Characterization of EstZY: A new acetylesterase with 7-aminocephalosporanic acid deacetylase activity from Alicyclobacillus tengchongensis.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic ß-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC2 to p-NPC8. Maximum enzyme activity was recorded at pH 9.0 and 50 °C, where Km and Vmax were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 µM min-1, respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.