Acetylesterase Research Articles

Crop residues used as food drive enzyme activation and enzymatic stoichiometry in casts of the earthworm Aporrectodea caliginosa (Savigny, 1826)

Earthworms are drivers of important soil processes such as the nutrient cycle. Earthworms provide soil microbiota with mucus, temperature, and organic resources in their guts, enhancing organic matter decomposition. However, very little is known about the effect of different crop rotations on soil microbiota activation mediated by earthworm activity. We aimed to evaluate the effect of different crop rotations on enzymatic activity in A. caliginosa casts and on enzymatic stoichiometry patterns related to C, N, and P acquisition activities. We conducted a microcosm study using different crop residues as food supply: soybean (S), soybean plus maize (SM), vetch, maize, and soybean (VMS), and with no residue addition (NR). We measured α-glucosidase (AGLU), β-glucosidase (BGLU), cellobiohydrolase (CEL), acetylesterase (ACE), N-acetyl-glucosaminidase (NAG), and phosmonoesterase (PME) activities in A. caliginosa casts, immediately before starting the experiment for evaluating the initial condition (IC) and after the experiment in each residue treatment. SM and VMS residues enhanced BGLU, CEL, and NAG activity regarding S, NR, and IC treatments. S had similar or lower activity than NR and IC for all the analyzed enzymes. There was a positive, strong, and significant relationship among C, N, and P acquiring enzymes regardless of the treatment. We found lower ratios for C and N acquisition related to P acquisition than for C: N acquisition activity ratios. This result suggests P as the limiting nutrient for earthworm microbiomes. Our results indicate that soybean residue is unsuitable for A. caliginosa food requirements and that diversifying crop residues and the inclusion of maize as food enhance enzymatic activity mediated by earthworms and thus organic matter decomposition. We suggest differential activation and likely changes in microbiota composition as mechanisms responsible for higher enzymatic activation when earthworms are fed with different combination of crop residues.

Reducing enteric methane production from buffalo (Bubalus bubalis) by garlic oil supplementation in in vitro rumen fermentation system

Enteric methane production contributes significantly to the greenhouse gas emission globally. Although, buffaloes are integral part of livestock production in Asian countries, contributing milk, meat and draft power, the contribution of enteric methane to environmental pollution attracts attention. The present study investigated the efficacy of garlic (Allium sativum) oil in reducing enteric methane production from buffaloes (Bubalus bubalis) by in vitro rumen fermentation. Garlic oil (GOL) was tested at four concentrations [0 (Control), 33.33 µl (GOL-1), 83.33 µl (GOL-2) and 166.66 µl (GOL-3) per litre of buffered rumen fluid] in 100-ml graduated glass syringes and incubated at 39℃ for 24 h for in vitro rumen fermentation study. Supplementation of GOL-1 increased (p < 0.05) total gas production in comparison with GOL-3; however, it remained comparable (p > 0.05) with control and GOL-2. Graded doses of garlic oil inclusions reduced (p < 0.001) methane concentration (%) in total gas and total methane production (ml/g DM), irrespective of concentrations. The feed degradability, volatile fatty acids and microbial biomass production (MBP) were not affected (p > 0.05) by GOL-1, but these tended to decrease in GOL-2 with marked reduction (p < 0.01) in GOL-3. The decrease (p < 0.01) in NH3–N concentration in fermentation fluid in the presence of garlic oil, irrespective of concentration, suggests reduced deamination by inhibiting rumen proteolytic bacterial population. The activities of ruminal fibrolytic enzymes (CMCase, xylanase, β-glucosidase, acetyl esterase) were not affected by lower dose (GOL-1) of garlic oil; however, reduction (p < 0.05) of these enzymes activity in rumen liquor was evident at higher doses (GOL-2 and GOL-3) of supplementation. This study shows positive impact of garlic oil supplementation at low dose (33.33 µl/l of rumen fluid) in reducing enteric methane production, thereby, abatement of environmental pollution without affecting feed digestibility.

Exploring Feeding Potential of Stovers from Novel Sorghum (Sorghum bicolor L.) Cultivars by In vitro Fermentation Pattern, Gas Production, Microbial Abundance and Ruminal Enzyme Production in Buffalo

Background: Crop residues play a central role in ruminant’s diet in developing countries. Due to low in nutritional quality, there is limitation in ruminant production through feeding of these residues. Therefore, production of quality crop residues through plant breeding programme without conceding grain yield is of prime importance. The present experiment envisaged the feeding value of stovers from three different novel sorghum (Sorghum bicolor L.) cultivars by in vitro fermentation pattern, gas production, microbial abundance and ruminal enzyme production in buffalo. Methods: Stovers from three different genotypes of sorghum cultivars viz. normal sorghum (CSV-27), brown midrib (bmr) sorghum (SPV-2018) and sweet sorghum (CSH 22SS) were analyzed for proximate principles and fibre fractions. Each stover sample was incubated (200 ± 5 mg) with 30 ml buffered rumen fluid in 100 ml calibrated glass syringes at 39ºC for 24 h following in vitro gas production system using rumen liquor from Murrah buffaloes. The gas production in each syringe was recorded during incubation at 4, 8, 12, 18 and 24 h intervals. Incubations were terminated at 24 h and methane concentration in the head space gas from syringes incubated with each stover sample was analyzed. Supernatant of each syringe contents was analyzed for volatile fatty acids (VFA) estimation. Truly degradable dry matter (TDDM) was determined and microbial biomass production (MBP) and portioning factor (PF) was calculated. Ruminal fibrolytic enzyme production and microbial abundance in each syringe content was estimated. Result: The stover of bmr sorghum showed highest organic matter, followed by normal sorghum and lowest in sweet sorghum. The neural detergent fibre (NDF) ranges between 69.22 to 74.65%, with highest in bmr sorghum and lowest in sweet sorghum. The stover sample of bmr sorghum contained lowest (P less than 0.05) acid detergent fibre (ADF) among the three cultivars examined, which resulted with highest (P less than 0.05) hemicellulose (37.72%) content. Lowest acid detergent lignin (ADL) was found in stovers of bmr cultivar (1.27%) and highest in sweet sorghum (8.36%). The fermentation pattern of bmr sorghum stovers exhibited higher (P less than 0.05) total gas production, dry matter degradability, VFA production, ruminal enzymes (CMCase, xylanase, acetyl esterase) and abundance of total ruminal bacterial population than normal and sweet sorghum stovers. Therefore, this study establishes the enhanced feeding value of stovers from bmr sorghum cultivar compared to normal and sweet sorghum cultivars for ruminant production.

A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases

The gut microbiota plays a central role in human health by enzymatically degrading dietary fiber and concomitantly excreting short chain fatty acids that are associated with manifold health benefits. The polysaccharide xylan is abundant in dietary fiber but noncarbohydrate decorations hinder efficient cleavage by glycoside hydrolases (GHs) and need to be addressed by carbohydrate esterases (CEs). Enzymes from carbohydrate esterase families 1 and 6 (CE1 and 6) perform key roles in xylan degradation by removing feruloyl and acetate decorations, yet little is known about these enzyme families especially with regard to their diversity in activity. Bacteroidetes bacteria are dominant members of the microbiota and often encode their carbohydrate-active enzymes in multigene polysaccharide utilization loci (PULs). Here we present the characterization of three CEs found in a PUL encoded by the gut Bacteroidete Dysgonomonas mossii. We demonstrate that the CEs are functionally distinct, with one highly efficient CE6 acetyl esterase and two CE1 enzymes with feruloyl esterase activities. One multidomain CE1 enzyme contains two CE1 domains: an N-terminal domain feruloyl esterase, and a C-terminal domain with minimal activity on model substrates. We present the structure of the C-terminal CE1 domain with the carbohydrate-binding module that bridges the two CE1 domains, as well as a complex of the same protein fragment with methyl ferulate. The investment of D. mossii in producing multiple CEs suggests that improved accessibility of xylan for GHs and cleavage of covalent polysaccharide-polysaccharide and lignin-polysaccharide bonds are important enzyme activities in the gut environment.

Quantitative Proteome Profiling Provides Insight into the Proteins Associated with β-Glucan Accumulation in Hull-less Barley Grains.

The hull-less barley (Qingke) is widely planted as a staple food crop in the Tibetan area, China, and the grains contains high content of β-glucan (BG). The mechanisms of BG synthesis and accumulation in qingke has not been studied at the protein level. This study characterized the proteins associated with BG synthesis and accumulation during qingke seed development. The proteome profiles of qingke seeds taken at 20, 30, and 40 days after flowering were compared using the TMT-based quantitative proteomics. A total of 4283 proteins were identified, with 759 being differentially expressed (DEPs) throughout seed development. Comparisons of protein expression pattern, functions, and pathway enrichment tests highlight cell wall modification, carbon and energy metabolism, polysaccharide metabolism, post-transcriptional modifications, and vesicular transport as critical biological processes related to qingke BG accumulation. Furthermore, induction of starch synthase, starch branching enzyme, pectin acetyl esterases, beta-glucosidases, beta-amylases, 1,4-beta-xylan, xyloglucan, α-amylase inhibitors, and glycosyltransferases underpinned BG synthesis. The results also indicated that the proteins involved in glycolytic, gluconeogenesis, and glyoxylate bypass pathways provided energy and reducing power for BG storage. Parallel reaction monitoring (PRM) and quantitative real-time PCR (qPCR) analyses confirmed the expression profile of the proteins obtained by TMT-based proteomics. The current results provided an insight into the mechanisms of BG synthesis and accumulation during qingke seed development.

Properties of a Newly Identified Acetyl Esterase and Its Degradation of Diisobutyl Phthalate

AbstractPhthalic acid esters (PAEs) are attracting growing attention because of environmental pollution and their harms to mammals, and diisobutyl phthalate (DiBP) in PAEs is still little studied. Biodegradation is a fast, efficient and inexpensive treatment preferentially used to deal with the pollution of PAEs. In this study, the recombinant esterase Est824 was obtained by heterologous expression and purification. The optimal reaction temperature and pH of the enzyme are 40 °C and 8.0 respectively. The enzyme reserves activity above 70 % in metal ion solutions at varying concentrations and thus shows high metal ion resistance. In high‐concentration salt solutions, the enzyme activity remains above 50 %, indicating that the enzyme has strong salt tolerance. The response surface method was used to optimize the fermentation conditions. With the Box‐Behnken design, the esterase yield was 1003.54 U/L. The DiBP degradation effect of the enzyme was detected by HPLC, and the degradation rate was 100 %. Therefore, the enzyme has good industrial application prospects and may be a potential candidate for the degradation of PAEs.

Multimodular fused acetyl\u2013feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

BackgroundPlant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated.ResultsTwo carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns.ConclusionWe demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan and xylan-rich biomass. Moreover, we demonstrated for the first time a synergistic effect between the two fused catalytic domains of a multidomain carbohydrate esterase.

Biochemical Alterations in the Leaves of Resistant and Susceptible Mungbean Genotypes Infected with Mungbean Yellow Mosaic India Virus

Mungbean (Vigna radiata), an important leguminous crop, is highly susceptible to yellow mosaic disease (YMD) caused by Mungbean yellow mosaic India virus (MYMIV), resulting in high yield penalty. Previously, different varieties of mungbean have been reported possessing resistance/ tolerance against MYMIV. However, the molecular events occurring during compatible and incompatible interactions between mungbean and MYMIV are yet to be explored. Therefore, in this study using MYMIV-resistant (NM-2016), moderately resistant (NM-2011) and susceptible genotype (VC-1647C), alterations in various biochemical attributes due to MYMIV infection were analysed and compared with healthy non-inoculated control plants for understanding the resistance mechanism. After MYMIV inoculation, the level of total phenolic contents (TPC) and total soluble proteins (TSP) increased significantly in the susceptible genotype. However, the level of Malondialdehyde (MDA) and Ascorbate peroxidase (APX) remained same in all the genotypes. Level of superoxide dismutase (SOD) and catalase (CAT) decreased in the susceptible genotype but CAT level increased in the moderately resistant genotype. Protease level decreased significantly in all the genotypes while esterase level increased in moderately resistant and susceptible genotype. Peroxidase (POD) increased only in moderately resistant genotype and Total Oxidant Status (TOS) increased significantly in the susceptible genotype. Due to MYMIV infection the level of all plant pigments decreased in all the genotypes tested. The comparative proteome analysis using SDS-PAGE resolved 22 peptides with molecular weight from 12.5 to 163 kDa. Differential expression of protein phosphatase 2C (PP2C) and Cytochrome b6 (Photosynthesis) in resistant and moderately resistant genotypes; Pectin acetyl esterase and Resistant specific protein-1(4) in resistant genotype and up-regulation of superoxide dismutase [Cu-Zn] and RuBisco by MYMIV may have triggered signal transduction pathway and consequently induced a resistance response against MYMIV in V. radiata by activating PR proteins.

An Aminopyrrolidinyl Phosphonates—A New Class of Antibiotics: Facile Synthesis and Predicted Biological Activity

A novel class of aminopyrrolidinyl phosphonates was synthesized in 74% - 80% isolated yield by the addition of three-fold excess of primary amines to diethyl 4-chloro-1-butynylphosphonates. The reaction was carried out at room temperature and in the absence of solvent or catalyst to give solely compounds which showed predicted biological activity based on PASS program. Some of the synthesized derivatives of antibiotics exhibit properties for the treatment of stroke, the treatment of acute neurological disorders, and can also be acetyl esterase inhibitors.

Structural and functional analyses of the lipase CinB from Enterobacter asburiae

Lipases are widely present in various plants, animals and microorganisms, constituting a large category of enzymes. They have the ability to catalyze the cleavage of ester bonds. The lipase CinB from Enterobacter asburiae (E. asburiae) is an acetyl esterase. The primary amino acid sequence suggests that the EaCinB protein belongs to the α/β-hydrolase (ABH) superfamily of the esterase/lipase superfamily. However, its molecular functions have not yet been determined. Here, we report the crystal structure of E. asburiae CinB at a 1.45 Å resolution. EaCinB contains a signal peptide, cap domain and catalytic domain. The active site of EaCinB contains the catalytic triad (Ser180-His307-Asp277) on the catalytic domain. The oxyanion hole is composed of Gly106 and Gly107 within the conserved sequence motif HGGG (amino acid residues 106–109). The substrate is accessible between the α1 and α2 helices or the α1 helix and catalytic domain. Narrow substrate pockets are formed by the α2 helix of the cap domain. Site-directed mutagenesis showed that EaCinB-W208H exhibits a higher catalytic ability than EaCinB-WT by approximately nine times. Our results provide insight into the molecular function of EaCinB.

Rumen microflora, fermentation pattern and microbial enzyme activity in sheep fed paddy straw based complete feed fortified with probiotics

ABSTRACT The use of probiotics for farm animals has increased considerably over the last 25 years. The study was conducted to evaluate the effect of probiotics (Saccharomyces cerevisiae 2 × 1010 cfu/g + Lactobacillus acidophilus 6 × 109 cfu/g) on the rumen microflora, fermentation pattern and the enzyme activity. A 90 day feeding trial was conducted in the Corriedale sheep which were fed paddy straw based complete feed mixed with probiotics (Saccharomyces cerevisiae 2 × 1010 cfu/g + Lactobacillus acidophilus 6 × 109 cfu/g). The complete feed without probiotics served as a control. The real-time PCR allowed us to observe the changes in the number of rumen microbes. The mean values of total bacterial, fibrolytic bacteria, fungi and protozoa count irrespective of period were significantly (p < 0.01) higher in probiotic mix supplemented group than in non-supplemented group. With respect to fermentation pattern, Rumen pH, TVFA, total nitrogen and TCA perceptible nitrogen were significantly (p < 0.01) higher in probiotic supplemented group than non-supplemented group. The enzyme activity viz., carboxymethyl cellulose, xylanase, amylase, glucosidase, acetyl esterase and protease were significantly higher (p < 0.05) in probiotic mix supplemented group as compared to non-supplemented group. This study contributes to understand the positive impact of probiotics on rumen microbial count, rumen fermentation parameters and the enzyme activity.

Identification and characterization of an acetyl esterase from Paenibacillus sp. XW-6-66 and its novel function in 7-aminocephalosporanic acid deacetylation.

To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation. The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the α/β hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40°C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5U/mg, and the Kcat/Km value was 2.04s-1mM-1. Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.

Cell wall hydrolases act in concert during aerenchyma development in sugarcane roots.

Cell wall disassembly occurs naturally in plants by the action of several glycosyl-hydrolases during different developmental processes such as lysigenous and constitutive aerenchyma formation in sugarcane roots. Wall degradation has been reported in aerenchyma development in different species, but little is known about the action of glycosyl-hydrolases in this process. In this work, gene expression, protein levels and enzymatic activity of cell wall hydrolases were assessed. Since aerenchyma formation is constitutive in sugarcane roots, they were assessed in segments corresponding to the first 5 cm from the root tip where aerenchyma develops. Our results indicate that the wall degradation starts with a partial attack on pectins (by acetyl esterases, endopolygalacturonases, β-galactosidases and α-arabinofuranosidases) followed by the action of β-glucan-/callose-hydrolysing enzymes. At the same time, there are modifications in arabinoxylan (by α-arabinofuranosidases), xyloglucan (by XTH), xyloglucan-cellulose interactions (by expansins) and partial hydrolysis of cellulose. Saccharification revealed that access to the cell wall varies among segments, consistent with an increase in recalcitrance and composite formation during aerenchyma development. Our findings corroborate the hypothesis that hydrolases are synchronically synthesized, leading to cell wall modifications that are modulated by the fine structure of cell wall polymers during aerenchyma formation in the cortex of sugarcane roots.

Arabinosyl Deacetylase Modulates the Arabinoxylan Acetylation Profile and Secondary Wall Formation.

Acetylation, a prevalent modification of cell-wall polymers, is a tightly controlled regulatory process that orchestrates plant growth and environmental adaptation. However, due to limited characterization of the enzymes involved, it is unclear how plants establish and dynamically regulate the acetylation pattern in response to growth requirements. In this study, we identified a rice (Oryza sativa) GDSL esterase that deacetylates the side chain of the major rice hemicellulose, arabinoxylan. Acetyl esterases involved in arabinoxylan modification were screened using enzymatic assays combined with mass spectrometry analysis. One candidate, DEACETYLASE ON ARABINOSYL SIDECHAIN OF XYLAN1 (DARX1), is specific for arabinosyl residues. Disruption of DARX1 via Tos17 insertion and CRISPR/Cas9 approaches resulted in the accumulation of acetates on the xylan arabinosyl side chains. Recombinant DARX1 abolished the excess acetyl groups on arabinoxylan-derived oligosaccharides of the darx1 mutants in vitro. Moreover, DARX1 is localized to the Golgi apparatus. Two-dimensional 13C-13C correlation spectroscopy and atomic force microscopy further revealed that the abnormal acetylation pattern observed in darx1 interrupts arabinoxylan conformation and cellulose microfibril orientation, resulting in compromised secondary wall patterning and reduced mechanical strength. This study provides insight into the mechanism controlling the acetylation pattern on arabinoxylan side chains and suggests a strategy to breed robust elite crops.

An Esterase-like Lyase Catalyzes Acetate Elimination in Spirotetronate/Spirotetramate Biosynthesis.

Spirotetronate and spirotetramate natural products include a multitude of compounds with potent antimicrobial and antitumor activities. Their biosynthesis incorporates many unusual biocatalytic steps, including regio- and stereo-specific modifications, cyclizations promoted by Diels-Alderases, and acetylation-elimination reactions. Here we focus on the acetate elimination catalyzed by AbyA5, implicated in the formation of the key Diels-Alder substrate to give the spirocyclic system of the antibiotic abyssomicin C. Using synthetic substrate analogues, it is shown that AbyA5 catalyzes stereospecific acetate elimination, establishing the (R)-tetronate acetate as a biosynthetic intermediate. The X-ray crystal structure of AbyA5, the first of an acetate-eliminating enzyme, reveals a deviant acetyl esterase fold. Molecular dynamics simulations and enzyme assays show the use of a His-Ser dyad to catalyze either elimination or hydrolysis, via disparate mechanisms, under substrate control.

An Esterase‐like Lyase Catalyzes Acetate Elimination in Spirotetronate/Spirotetramate Biosynthesis

AbstractSpirotetronate and spirotetramate natural products include a multitude of compounds with potent antimicrobial and antitumor activities. Their biosynthesis incorporates many unusual biocatalytic steps, including regio‐ and stereo‐specific modifications, cyclizations promoted by Diels–Alderases, and acetylation‐elimination reactions. Here we focus on the acetate elimination catalyzed by AbyA5, implicated in the formation of the key Diels–Alder substrate to give the spirocyclic system of the antibiotic abyssomicin C. Using synthetic substrate analogues, it is shown that AbyA5 catalyzes stereospecific acetate elimination, establishing the (R)‐tetronate acetate as a biosynthetic intermediate. The X‐ray crystal structure of AbyA5, the first of an acetate‐eliminating enzyme, reveals a deviant acetyl esterase fold. Molecular dynamics simulations and enzyme assays show the use of a His‐Ser dyad to catalyze either elimination or hydrolysis, via disparate mechanisms, under substrate control.

Lactic Acid Fermentation of Arabinoxylan From Nejayote by Streptococcus infantarius ssp. infantarius 25124 Isolated From Pozol.

Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) is a lactic acid bacterium (LAB) isolated from pozol, a refreshing beverage prepared by suspending fermented nixtamal (a thermal and alkali-treated maize dough) in water. Although Lactobacillus are the predominant strains in fermented doughs, such as sourdoughs, and non-nixtamalized fermented maize foods, the pozol microbiota is markedly different. This may be the result of the nixtamalization process, which could act as a selective force of some strains. Sii-25124 has been reported as the main amylolytic LAB in pozol; starch is the primary carbon source on nixtamal since monosaccharides and disaccharides are lost during nixtamalization; however, non-amylolytic LAB counts are higher than amylolytic LAB in pozol after 24-h fermentation suggesting that another carbon source is being used by the former bacteria. Hemicellulose (arabinoxylan in maize) becomes available via nixtamalization and is subsequently metabolized by LAB. The aim of this work was to determine whether this bacterium is able to use arabinoxylan as the only carbon source in a defined medium containing arabinoxylan extracted from either nejayote (wash water produced during nixtamal preparation), or beechwood xylan. Xylanase activity in the presence of nejayote arabinoxylan (135.8 ± 48.7 IU/mg protein) was higher than that of beechwood (62.5 ± 19.8 IU/mg protein). Other enzymatic activities, such as arabinofuranosidase and acetyl esterase, were also detected, suggesting the adaptation of the bacterium studied to nixtamal dough. It was concluded that Streptococcus infantarius 25124 isolated from pozol was able to use arabinoxylans, which are present in nixtamal dough, so fermentation does not depend exclusively on free sugars and starch.

Brettanomyces anomalus, a double drawback for cider aroma

Sixty-six French ciders were collected and characterized to build up a data base of eighty-eight variables, including sensory descriptors, aroma description and physicochemical variables. A classification in four main sensory categories was made on the basis of sensorial data. Fruity-Floral ciders were rich in acetate esters and low in volatile phenols, in opposition with phenol-like or off-flavour ciders having an inverse ratio. In order to explain this observation, an essential result was the demonstration of Brettanomyces anomalus implication, not only in volatile phenol synthesis, but also in acetate ester degradation. Brettanomyces anomalus exhibited an intracellular acetyl esterase activity. At last, both mechanisms, acetate ester degradation and phenol synthesis, contribute to the decrease of cider fruitiness.

Bacterial Fermentation of Water-Soluble Cellulose Acetate Raises Large-Bowel Acetate and Propionate and Decreases Plasma Cholesterol Concentrations in Rats.

We hypothesized that water-soluble cellulose acetate (WSCA) could be useful tool for the delivery of short-chain fatty acids to the large intestine. Rats were fed a control diet or a diet containing graded levels of WSCA for up to 21 days. Consuming WSCA dose-dependently increased large-bowel acetate and propionate concentrations through the bacterial fermentation. When WSCA was used as substrate, acetyl esterase activity in the cecal bacteria was detected solely in rats fed WSCA, in which the activity increased over time accompanied by an increased number of Bacteroides xylanisolvens. Consuming WSCA at a 4% level increased the goblet cell numbers and mucin contents in the cecum and lowered plasma cholesterol concentrations, which tended to correlate with the portal plasma concentrations of propionate. The results suggest that bacterial fermentation of WSCA is characterized by the greater production of acetate and propionate, which may contribute to the physiologic alterations.

Role of an N-terminal extension in stability and catalytic activity of a hyperthermostable α/β hydrolase fold esterase.

The carbohydrate esterase family 7 (CE7) enzymes catalyze the deacetylation of acetyl esters of a broad range of alcohols and is unique in its activity towards cephalosporin C. The CE7 fold contains a conserved N-terminal extension that distinguishes it from the canonical α/β hydrolase fold. The hexameric quaternary structure indicates that the N-terminus may affect activity and specificity by controlling access of substrates to the buried active sites via an entrance tunnel. In this context, we characterized the catalytic parameters, conformation and thermal stability of two truncation variants lacking four and ten residues of the N-terminal region of the hyperthermostable Thermotoga maritima CE7 acetyl esterase (TmAcE). The truncations did not affect the secondary structure or the fold but modulated the oligomerization dynamics. A modest increase was observed in substrate specificity for acetylated xylose compared with acetylated glucose. A drastic reduction of ~30-40°C in the optimum temperature for activity of the variants indicated lower thermal stability. The loss of hyperthermostability appears to be an indirect effect associated with an increase in the conformational flexibility of an otherwise rigid neighboring loop containing a catalytic triad residue. The results suggest that the N-terminal extension was evolutionarily selected to preserve the stability of the enzyme.