Acetylesterase Activity Research Articles

Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose.

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ~70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1-expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production.

P. aeruginosa SGNH hydrolase-like proteins AlgJ and AlgX have similar topology but separate and distinct roles in alginate acetylation.

The O-acetylation of polysaccharides is a common modification used by pathogenic organisms to protect against external forces. Pseudomonas aeruginosa secretes the anionic, O-acetylated exopolysaccharide alginate during chronic infection in the lungs of cystic fibrosis patients to form the major constituent of a protective biofilm matrix. Four proteins have been implicated in the O-acetylation of alginate, AlgIJF and AlgX. To probe the biological function of AlgJ, we determined its structure to 1.83 Å resolution. AlgJ is a SGNH hydrolase-like protein, which while structurally similar to the N-terminal domain of AlgX exhibits a distinctly different electrostatic surface potential. Consistent with other SGNH hydrolases, we identified a conserved catalytic triad composed of D190, H192 and S288 and demonstrated that AlgJ exhibits acetylesterase activity in vitro. Residues in the AlgJ signature motifs were found to form an extensive network of interactions that are critical for O-acetylation of alginate in vivo. Using two different electrospray ionization mass spectrometry (ESI-MS) assays we compared the abilities of AlgJ and AlgX to bind and acetylate alginate. Binding studies using defined length polymannuronic acid revealed that AlgJ exhibits either weak or no detectable polymer binding while AlgX binds polymannuronic acid specifically in a length-dependent manner. Additionally, AlgX was capable of utilizing the surrogate acetyl-donor 4-nitrophenyl acetate to catalyze the O-acetylation of polymannuronic acid. Our results, combined with previously published in vivo data, suggest that the annotated O-acetyltransferases AlgJ and AlgX have separate and distinct roles in O-acetylation. Our refined model for alginate acetylation places AlgX as the terminal acetlytransferase and provides a rationale for the variability in the number of proteins required for polysaccharide O-acetylation.

Identification and functional characterization of the distinct plant pectin esterases PAE8 and PAE9 and their deletion mutants.

Main conclusion PAE8 and PAE9 have pectin acetylesterase activity and together remove one-third of the cell wall acetate associated with pectin formation in Arabidopsis leaves. In pae8 and pae9 mutants, substantial amounts of acetate accumulate in cell walls. In addition, the inflorescence stem height is decreased. Pectic polysaccharides constitute a significant part of the primary cell walls in dicotyledonous angiosperms. This diverse group of polysaccharides has been implicated in several physiological processes including cell-to-cell adhesion and pathogenesis. Several pectic polysaccharides contain acetyl-moieties directly affecting their physical properties such as gelling capacity, an important trait for the food industry. In order to gain further insight into the biological role of pectin acetylation, a reverse genetics approach was used to investigate the function of genes that are members of the Pectin AcetylEsterase gene family (PAE) in Arabidopsis. Mutations in two members of the PAE family (PAE8 and PAE9) lead to cell walls with an approximately 20 % increase in acetate content. High-molecular-weight fractions enriched in pectic rhamnogalacturonan I (RGI) extracted from the mutants had increased acetate content. In addition, the pae8 mutant displayed increased acetate content also in low-molecular-weight pectic fractions. The pae8/pae9-2 double mutant exhibited an additive effect by increasing wall acetate content by up to 37 %, suggesting that the two genes are not redundant and act on acetyl-substituents of different pectic domains. The pae8 and pae8/pae9-2 mutants exhibit reduced inflorescence growth underscoring the role of pectic acetylation in plant development. When heterologously expressed and purified, both gene products were shown to release acetate from the corresponding mutant pectic fractions in vitro. PAEs play a significant role in modulating the acetylation state of pectic polymers in the wall, highlighting the importance of apoplastic metabolism for the plant cell and plant growth.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2139-6) contains supplementary material, which is available to authorized users.

Enzymatic degradation of Elephant grass (Pennisetum purpureum) stems: Influence of the pith and bark in the total hydrolysis

The internal pith of a high energy plant, Elephant grass (EG), was more extensively degraded (>50% dry matter) compared to the outer cortex (31%) or the whole stem (35%) by an enzyme preparation from Humicola insolens, Ultraflo. Reducing sugars and acetic acid release from the pith was also higher compared to the cortex. Supplementation of Ultraflo with a type-C feruloyl esterase increased the level of deacetylation but also led to reduced solubilisation. The addition of 20% dimethyl sulfoxide (DMSO) as a co-solvent also reduced the solubility of EG by Ultraflo, although acetic acid release was increased, complimenting previous results found on model substrates. The presence of DMSO was also shown to have a protective effect on xylanase activity but not acetyl esterase activity in Ultraflo. Xylan in the biomass was preferentially solubilised by DMSO, while Ultraflo removed more glucose than xylose.

Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates

The acetylesterases are hydrolytic enzymes which in plants cleave acetyl groups from acetylated cell wall components, primarily polysaccharides. To estimate acetylesterase activity in plant apoplast, two assays can be used. First assay is a direct measurement of the acetylesterase activity in protein extract using synthetic substrate, pNP-acetyl. In this assay, amount of pNP released after hydrolysis of pNP-acetyl is determined by measuring the intensity of developed yellow color using spectrophotometer. The absorbance of reaction mixture is directly proportional to the activity of acetylesterases in the reaction mixture. Second assay is a determination of acetylesterase activity and its specificity towards natural polysaccharides and based on interaction between ferric perchlorate and acetyl residues resulting in ferric acetohydroxamic complex that can be quantified using spectrophotometer. In this assay, commercially available acetylated polysaccharides (xylan from Birchwood for acetylxylan esterase; pectin from citrus fruit for rhamnogalacturonan acetylesterase; or any other available polysaccharide of interest) incubated with apoplastic extract and amount of acetyl residues released from this polysaccharide is estimated using ferric perchlorate reagent (protocol was modified from McComb and McCready, 1957). The absorbance of produced colored complex is directly proportional to the amount of acetyls released from acetylated polysaccharide.

Structural and Functional Characterization of Pseudomonas aeruginosa AlgX: ROLE OF AlgX IN ALGINATE ACETYLATION

The exopolysaccharide alginate, produced by mucoid Pseudomonas aeruginosa in the lungs of cystic fibrosis patients, undergoes two different chemical modifications as it is synthesized that alter the properties of the polymer and hence the biofilm. One modification, acetylation, causes the cells in the biofilm to adhere better to lung epithelium, form microcolonies, and resist the effects of the host immune system and/or antibiotics. Alginate biosynthesis requires 12 proteins encoded by the algD operon, including AlgX, and although this protein is essential for polymer production, its exact role is unknown. In this study, we present the X-ray crystal structure of AlgX at 2.15 Å resolution. The structure reveals that AlgX is a two-domain protein, with an N-terminal domain with structural homology to members of the SGNH hydrolase superfamily and a C-terminal carbohydrate-binding module. A number of residues in the carbohydrate-binding module form a substrate recognition "pinch point" that we propose aids in alginate binding and orientation. Although the topology of the N-terminal domain deviates from canonical SGNH hydrolases, the residues that constitute the Ser-His-Asp catalytic triad characteristic of this family are structurally conserved. In vivo studies reveal that site-specific mutation of these residues results in non-acetylated alginate. This catalytic triad is also required for acetylesterase activity in vitro. Our data suggest that not only does AlgX protect the polymer as it passages through the periplasm but that it also plays a role in alginate acetylation. Our results provide the first structural insight for a wide group of closely related bacterial polysaccharide acetyltransferases.

The Acetyl-Esterase Activity of the Hemagglutinin-Esterase Protein of Human Coronavirus OC43 Strongly Enhances the Production of Infectious Virus

Most betacoronaviruses possess an hemagglutinin-esterase (HE) protein, which appears to play a role in binding to or release from the target cell. Since this HE protein possesses an acetyl-esterase activity that removes acetyl groups from O-acetylated sialic acid, a role as a receptor-destroying enzyme has been postulated. However, the precise function of HE and of its enzymatic activity remains poorly understood. Making use of neutralizing antibody and of molecular clones of recombinant human coronavirus OC43 (HCoV-OC43), our results suggest that the HE protein of this HCoV could be associated with infection of target cells and, most notably, is important in the production of infectious viral particles. Indeed, after transfecting BHK-21 cells with various cDNA infectious clones of HCoV-OC43, either lacking the HE protein or bearing an HE protein with a nonfunctional acetyl-esterase enzymatic activity, we were reproducibly unable to detect recombinant infectious viruses compared to the reference infectious HCoV-OC43 clone pBAC-OC43(FL). Complementation experiments, using BHK-21 cells expressing wild-type HE, either transiently or in a stable ectopic expression, demonstrate that this protein plays a very significant role in the production of infectious recombinant coronaviral particles that can subsequently more efficiently infect susceptible epithelial and neuronal cells. Even though the S protein is the main viral factor influencing coronavirus infection of susceptible cells, our results taken together indicate that a functionally active HE protein enhances the infectious properties of HCoV-OC43 and contributes to efficient virus dissemination in cell culture.

Development of “Field Level” Chromogenic Assay for Aflatoxin M1 Detection in Milk

Out of eighteen Bacillus spp. screened for aflatoxin M1 (AFM1) sensitivity Bacillus brevis MTCC 3136 was found most sensitive followed by B. megaterium ATCC 9885 at 0.5 ppb (Codex limit). Bacillus brevis 3136 was found weakly positive for lipase activity while B. megaterium 9885 showed significant acetyl esterase activity. Maximum sporulation was achieved in Arret and Krishbaum sporulation agar (90.63%) followed by tryptone yeast extract broth, tryptone glucose yeast extract broth, minimal media and sucrose salt medium. A field level chromogenic assay for AFM1 in milk has been developed using spores of B. megaterium 9885 grown in Arret and Krishbaum sporulation agar. Spores were added with milk containing germinants (dextrose, sucrose, tryptone) and chromogenic substrate followed by incubation at 37EšC for 75 ± 5 min. The colour change from colourless to sky blue colour within 75 ± 5 min is indication of spore germination followed by release of enzyme and its action on chromogenic substrate. However, no colour change indicates that milk is positive for AFM1 at 0.5 ppb. The developed assay was validated with raw, pasteurized and dried milks with a 96% correlation by comparing with radioimmunoassay i.e., Charm 6602 assay. The reliability of assay has also been checked by spiking AFM1 in milk. The developed assay was also validated by certified reference materials of heavy metals and pesticides for checking the cross reactivity of these with our developed assay. The developed chromogenic assay is cost effective, portable, having no interference of antibiotics, detergents, sanitizers, heavy metals and pesticides. The developed assay can be used in dairy farm and laboratories with fewer resources for AFM1 detection in milk.

Heterologous expression and biochemical characterization of acetyl xylan esterase from Coprinopsis cinerea

Acetyl xylan esterase (AXE) from basidiomycete Coprinopsis cinerea Okayama 7 (#130) was functionally expressed in Pichia pastoris with a C-terminal tag under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 1.5 mg l(-1). Its molecular mass was estimated to be 65.5 kDa based on the SDS-PAGE analysis, which is higher than the calculated molecular mass of 40 kDa based on amino acid composition. In-silico analysis of the amino acid sequence predicted two potential N-glycosylation sites. Results from PNGase F deglycosylation and mass spectrum confirmed the presence of N-glycosylation on the recombinant AXE with predominant N-glycans HexNAc2Hex9-16. The recombinant AXE showed best activity at 40 °C and pH 8. It showed not only acetyl esterase activity with a Km of 4.3 mM and a Vmax of 2.15 U mg(-1) for hydrolysis of 4-nitrophenyl acetate but also a butyl esterase activity for hydrolysis of 4-nitrophenyl butyrate with a Km of 0.11 mM and Vmax of 0.78 U mg(-1). The presence of two additional amino acid residues at its native N-terminus was found to help stabilize the enzyme against the protease cleavages without affecting its activity.

Enzymatic characteristics of crude feruloyl and acetyl esterases of rumen fungus Neocallimastix sp. YAK11 isolated from yak (Bos grunniens)

Rumen fungus Neocallimastix sp. YAK11 was isolated from yak (Bos grunniens), and three consecutive 10-day pure cultures were anaerobically performed at 39 °C in 20-ml Hungate's tubes to explore ferulic acid esterase (FAE) and acetyl esterase (AE) activity profiles of the fungus grown on whole hay fraction of Chinese wildrye grass (Leymus chinensis) (WHOcw , n = 4) and its neutral detergent fibre fraction (NDFcw , n = 4), respectively. An aliquot of 0.7-ml culture was sampled daily using a sterile syringe, and 0.7-ml fresh medium was immediately added to the tubes to compensate for the withdrawn samples. Peak esterase activity occurred for FAE on day 5 (p < 0.001) and for AE on day 6 (p < 0.001). The mean activities of FAE and AE in WHOcw were 2.07 and 1.29 times of those in NDFcw (p < 0.001). Both FAE and AE activities were positively correlated with xylanase (r > 0.65, p < 0.001) and carboxymethyl cellulase (r > 0.57, p < 0.001) activities. Total volatile fatty acid concentration was positively correlated with enzyme activities of AE (r > 0.87, p < 0.001), FAE (r > 0.82, p < 0.001) and xylanase (r > 0.56, p < 0.001). Crude enzyme solution was harvested for the fungus grown on WHOcw , and the pH optimum of FAE activity was 8.0 while the optimum for AE was 9.0. Both FAE and AE had a broad pH stability range. The optimal temperatures for FAE and AE activity were 40 and 50 °C. The Michaelis constant (Km ) and maximum velocity (Vmax ) for FAE against methyl ferulate at pH 6.0 and 39 °C were 0.078 mm and 2.93 mU, respectively. The Km and Vmax for AE against p-nitrophenyl acetate at pH 7.0 and 39 °C were 2.73 mm and 666.67 mU, respectively. Both FAE and AE may have prospective advantages for the enzymatic degradation of roughages in ruminant animals.

Effect of roughage fibre content on fibrolytic activities and volatile fatty acid profiles of Neocallimastix sp. YAK11 isolated from rumen fluids of yak ( Bos grunniens)

A 10-day pure culture of Neocallimastix sp. YAK11, isolated from rumen fluids of yak ( Bos grunniens) was evaluated for fibrolytic activity, apparent dry matter disappearance (ADMD), ferulic acid, reducing sugars and volatile fatty acids (VFA). The fungus was grown on oat straw (OS), corn stalks (CS), rice straw (RS) and wheat straw (WS). Recovered activity of ferulic acid esterase peaked at day 5 in all roughages, and the lowest activity was found with low fibre OS throughout the incubation (P<0.0001). The recovered activity of acetyl esterase peaked at day 6 with WS and RS and at day 7 with CS and OS (P<0.0001). The highest acetyl esterase activity was found with high fibre WS throughout the incubation (P<0.0001). Xylanase activity increased with all roughages and was maintained at a high level throughout the incubation (P=0.0102). The lowest xylanase activity was found in WS (P<0.0001), but cultures grown on OS, CS and RS showed no differences. The highest ADMD and reducing sugar values occurred in OS and the lowest in WS (P<0.0001). Ferulic acid release did not differ between four roughages. ADMD was positively correlated with xylanase activity ( r = 0.51, P<0.01). Total VFA concentration was greatest in OS (P<0.0001) and highly correlated with fibrolytic enzyme activities ( r > 0.42, P<0.01). Molar proportions of acetate and valerate did not differ in the roughages. The lowest propionate and branch chained VFA levels occurred in WS (P=0.0115), and the highest butyrate levels occurred in OS throughout the 10-d incubation (P=0.0464). The highest ratio of non-glucogenic to glucogenic acids (NGR) occurred in WS (P=0.0481). NGR peaked for all roughages around day 3 and then slightly declined for the rest of the experiment. In general, the isolate was capable of a pronounced degradation that would depend on the fibre content of the roughages. Screening of fungi from rumen fluids of yak would appear to be a useful strategy to obtain highly active esterases and polysaccharide hydrolases.

Effect of glucose addition and N sources in defined media on fibrolytic activity profiles of Neocallimastix sp. YQ1 grown on corn stover

Cleavage of plant cell wall arabinoxylans occurs by the action of ferulic acid esterase (FAE) and acetyl esterase (AE), which cleave feruloyl groups substituted at the 5'-OH group of arabinosyl residues and acetyl groups substituted at O-2/O-3 of the xylan backbone, respectively. In this study, we examined the enzyme profiles of the anaerobic rumen fungus Neocallimastix sp. YQ1 for FAE, AE and polysaccharide hydrolases when grown on corn stover, a lignin-rich waste biomaterial. A 2 × 4 factorial experiment in 10-days pure cultures was used to test glucose addition (G(+) : glucose at 1.0 g/l, G(-) : no glucose) and four N sources (N1: 1.0 g/l yeast extract, 1.0 g/l tryptone and 0.5 g/l (NH(4))(2) SO(4); N2: 2.8 g/l yeast extract and 0.5 g/l (NH(4))(2) SO(4) ; N3: 1.6 g/l tryptone and 0.5 g/l (NH(4))(2) SO(4); N4: 1.4 g/l tryptone and 1.7 g/l yeast extract) in defined media. The optimal combinations of glucose and N sources to promote FAE and AE activity were G(+) N2 and G(+) N4, respectively. The peak activities of FAE and AE occurred on days 9 and 10, respectively. Addition of glucose and an increase in yeast extract and/or tryptone to a Hungate's medium favoured fungal production of volatile fatty acids, which could be just a consequence of more organic matter available to digest. This suggests that enzymatic release of ferulic acid by a synergistic action of lignin hydrolytic esterase and polysaccharide hydrolases may be essential for plant cell wall biodegradation in the rumen.

Fermentative characteristics and fibrolytic activities of anaerobic gut fungi isolated from wild and domestic ruminants

Fermentative characteristics and fibrolytic enzyme activities of anaerobic gut fungi from wild (17 isolates) and domestic ruminants (15 isolates) were examined. In a medium containing 0.5% wheat straw and 0.02% cellobiose as energy source, activities of carboxymethyl cellulase (CMCase), avicelase, xylanase, acetyl esterase and protease produced by the fungal isolates were investigated. Average activity of CMCase (17.4 vs. 8.25 mIU ml−1), acetyl esterase (134 vs. 57 mIU ml−1) and protease (4400 vs. 1683 mIU ml−1) were significantly higher in isolates from wild ruminants than those from domestic ruminants. Xylanase and avicelase activities were comparable. When compared irrespective of source, fungal isolates having monocentric growth pattern produced more fibrolytic enzymes than isolates having polycentric growth pattern. CMCase, xylanase, avicelase activities were highest in Neocallimastix isolates. Acetyl esterase activity was highest in Piromyces and Neocallimastix isolates. Protease activity was highest in Piromyces isolates followed closely by Neocallimastix isolates. Between isolates from wild and domestic ruminants few differences were observed in pattern of carbohydrate utilisation and end products of fermentation. Inter-strain differences in the end product formation were apparent. All of the isolates produced acetate, lactate and formate; only a few isolates produced succinate. For isolation of superior fibrolytic isolates of anaerobic fungi, greater emphasis should be given to the screening of enzyme activities of isolates of genera Neocallimastix and Piromyces.

Effects of extracts of spices on rumen methanogenesis, enzyme activities and fermentation of feeds in vitro

An experiment was conducted to study the effects of boiling water, methanol and ethanol extracts (0, 0.25 and 0.50 mL) of seeds of Foeniculum vulgare (fennel), flower buds of Syzygium aromaticum (clove), bulbs of Allium sativum (garlic), bulbs of Allium cepa (onion) and roots of Zingiber officinalis (ginger) on rumen methanogenesis, fibrolytic enzyme activities and fermentation characteristics in vitro. Ethanol and methanol extracts of fennel, clove and garlic at 0.50 mL and clove at 0.25 mL inhibited (P < 0.05) methane production. Carboxymethylcellulase activity was reduced (P < 0.05) by ethanol and methanol extracts (0.50 mL) of fennel and clove (0.25 and 0.50 mL). The extracts of clove reduced (0.25 and 0.50 mL) xylanase and acetylesterase activities, and the fennel extract (0.50 mL) reduced (P < 0.05) xylanase activity. However, the extracts of garlic (0.50 mL) increased (P < 0.05) acetylesterase activity. Concentrations of volatile fatty acids were reduced (P < 0.05) by the extracts of garlic and onion. The extracts of garlic caused a decrease (P < 0.05) in acetate:propionate ratio (A:P) at 0.50 mL, whereas A:P was increased (P < 0.05) by the inclusion of 0.50 mL extracts of clove. Methanol and ethanol extracts of clove decreased (P < 0.05) in vitro organic matter degradability. Extracts (0.50 mL) of clove decreased (P < 0.05) the numbers of total protozoa, small entodiniomorphs and holotrichs, whereas extracts of onion, ginger and garlic enhanced (P < 0.05) protozoal numbers (both entodiniomorphs and holotrichs). Ethanol and methanol extracts of fennel and garlic have potential to inhibit rumen methanogenesis without adversely affecting rumen fermentation.

Functionalization of cellulose acetate fibers with engineered cutinases

In the present work, we describe for the first time the specific role of cutinase on surface modification of cellulose acetate fibers. Cutinase exhibits acetyl esterase activity on diacetate and triacetate of 0.010 U and 0.007 U, respectively. An increase on the hydroxyl groups at the fiber surface of 25% for diacetate and 317% for triacetate, after a 24 h treatment, is estimated by an indirect assay. Aiming at further improvement of cutinase affinity toward cellulose acetate, chimeric cutinases are genetically engineered by fusing the 3'-end coding sequence with a bacterial or a fungal carbohydrate-binding module and varying the linker DNA sequence. A comparative analysis of these genetic constructions is presented showing that, the superficial regeneration of cellulose hydrophilicity and reactivity on highly substituted cellulose acetates is achieved by chimeric cutinases.

Bioactive xylo-oligosaccharides from wheat bran soluble polysaccharides

Soluble polysaccharides (SP, 35 g) isolated from wheat bran (100 g) consisted mainly of arabinose and xylose with minor quantities of rhamnose, mannose, galactose and glucose. Wheat bran SP was subjected to purified endoxylanase (from 96 h ragi malt) treatment to obtain xylo-oligosaccharides (0.3 g/1 g wheat bran). The oligosaccharide mixture was purified on Biogel P-2 column into four major peaks designated as WO-1, WO-2, WO-3 and WO-4. Individual oligosaccharide purity was ascertained by HPLC and their composition was determined by GLC. The purified oligosaccharides were characterized by ESI-MS and 1H NMR analysis. WO-1 and WO-2 were identified as arabinose containing xylotetraose and xylotriose, respectively, whereas WO-3 and WO-4 were identified as unsubstituted xylotriose and xylobiose, respectively. In vitro studies carried out using Bifidobacterium spp and Lactobacillus spp suggested the prebiotic nature of the crude as well as purified xylo-oligosaccharides as revealed by growth characteristics such as high O.D. of the culture broth, decrease in its pH, increase in cell mass and the resultant fermentation products. β-xylanase, β-xylosidase, α-arabinofuranosidase, α/β-galactosidases and acetyl esterase activities were determined in 24 h old culture broth and xylanase activity (440–830 μU/ml) was found to be the most preponderant among all of them.

A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate

A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K M values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.

Production of Xylooligosaccharides from Xylans by Extracellular Xylanases fromThermobifida fusca

Xylooligosaccharides are produced for use as a valuable food sweetener or additive. They have many beneficial biomedical and health effects. In this study, a process for producing xylooligosaccharides from lignocellulolytic agricultural waste was developed. Bagasse, corncob, wheat bran, and peanut shell were used as carbon sources for production of xylanolytic enzymes from Thermobifida fusca NTU22. When using bagasse as the carbon source, the xylanolytic enzymes that simultaneously accumulated in the broth in a 500 mL Hinton flask after 72 h of cultivation at 50 degrees C were measured as xylanase (14.0 U/mL), beta-xylosidase (74.1 mU/mL), and acetyl esterase (29.1 mU/mL). The optimum pH and temperature for xylanases were 6.0-8.0 and 70 degrees C, respectively. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gel. This was followed by heat treatment at 70 degrees C for 30 min that eliminated 90% of the beta-xylosidase activity. The xylanase and acetyl esterase activities were still 100%. Two percent of xylan extracted from the bagasse was then hydrolyzed by heat-treated crude xylanase preparation at 60 degrees C, pH 7.0, for 10 h. The xylooligosaccharides that accumulated in the broth were about 23.7%. After the purification process by activated charcoal chromatography, the purity of xylooligosaccharides was 71.4%.

Determination of enzymes from Colletotrichum sp. AHU9748 essential for lepidimoide production from okra polysaccharide

The allelopathic substance lepidimoide (Lp), which exhibits multiple functions in the growth and development of plants, was produced by Colletotrichum sp. AHU9748 from okra polysaccharide. Okra polysaccharide has the repeating structure (1-->4)-O-alpha-(d-galactopyranosyluronic acid)-(1-->2)-O-alpha-l-rhamnopyranose in its hexasaccharide repeating unit of its main chain. To determine the enzymes essential for Lp production, the supernatant of a culture broth was fractionated by repeated column chromatographies to identify two serial fractions responsible for Lp production and non-Lp production by measuring Lp production together with beta-galactosidase (beta-gal), rhamnogalacturonan lyase (RG-lyase) and acetylesterase (AE) activities, which we hypothesized to be necessary for Lp production from the structure of Lp. We confirmed the presence of these three enzymatic activities in the highest-Lp-producing fraction. The addition of purified RG-lyase to fractions producing no or a small amount of Lp demonstrated that beta-gal and RG-lyase activities are necessary for Lp production. The N-terminal amino acid sequences of the three separated proteins on SDS-PAGE confirmed the presence of enzymes identical to beta-gal, RG-lyase and AE in the Lp-producing fractions.

The cell-fusion activity of influenza C viral glycoprotein

The hemagglutinin-esterase (HE) glycoprotein of the influenza C virus causes cell fusion under acidic conditions. Because the influenza C virus grows more efficiently at 34 °C than at 37 °C, we examined whether the cell fusion activity of HE synthesized at 34 °C was higher than that at 37 °C. COS-1 cells, transfected with HE cDNA of C/Ann Arbor/1/50 virus, were incubated at 34 or 37 °C, and the cell fusion activity was analyzed after incubation for 24 h at each temperature. The results showed that cell fusion was observed with HE synthesized at 34 °C but not at 37 °C, although both HEs were transported to the cell surface. The fusion pore formation process did not proceed sufficiently when HE was synthesized at 37 °C. Comparison of the acetylesterase activities and antigenic structures between the HE synthesized at 34 °C and that at 37 °C showed that there were little or no differences in their properties. In contrast, the hemadsorption test with human erythrocytes suggested that the cell surface expression of HE synthesized at 37 °C was less efficient than that at 34 °C.