Pretreatment remove most of the soluble lignin by disturbing the complex lignin structure resulting in complete access for enzymes. We produced Peracetic acid (PAA) using recombinant acetyl xylan esterase (rAXE) from Aspergillus ficcum and it was determined by HPLC.10 µg of rAXE generates 1.33 mM of Peracetic acid in-situ.
Key words: Acetyl xylan esterase, Aspergillus ficuum, Peracetic acid (PAA), High performance liquid chromatography (HPLC).
Biomass is composed of carbohydrate a polymer which is the primary source for sugar molecules that can be converted into ethanol or alkanes for energy usage. The sugar polymers are embedded in plant cell walls, which naturally resist chemical, physical and enzymatic degradation. In particular, lignin, an aromatic polymer that is a major component of lignocellulosic biomass, blocks access of enzymes to sugar polymers. However, enzymatic hydrolysis is a promising way to obtain sugars from lignocellulosic materials as it requires less energy input. But the low enzymatic accessibility of the native cellulose is a key problem for biomass processing. Therefore, pretreatment is an essential element in the bioconversion of lignocellulosic substrates. The objective of biomass pretreatment is to alter the structure of the lignocellulosic matrix to increase cellulose digestibility using cellulose breaking enzymes, which can be done by removing lignin, hemicelluloses, or a combination of the two. Several processes have been developed for pretreatment of lignocellulosic biomass, including steam explosion, liquid hot water process, alkali pretreatment, wet oxidation and acid hydrolysis (1). All of these processes enhance the enzymatic digestibility of biomass to some extent. Most of them should be operated at high temperature resulting in high pressure, which increases the energy consumption, costs of equipment and usage of chemicals during the process creates pollution to the environment in all manners. Furthermore, these processes still leave most of the lignin in the material and limit the complete bioconversion of cellulose to sugar. Peracetic acid (PAA) is recognized as a powerful oxidizing agent and is a selective reagent towards the lignin structure. It oxidizes the aromatics in lignin, generating dicarboxylic acid and their lactones (2). The aliphatic propane side-chain is displaced to some extent during the oxidative process (3). The enzymatic digestibility of PAA-pretreated or PAA pre pretreated biomass was effectively enhanced. Peracetic acid-treated biomass can be efficiently fermented to make ethanol or other high energy density liquids like alkanes (4, 5). Peracetic acid, however, can be explosive in concentrated form and it is expensive. This safety hazard increases the cost of storage and transport across countries of peracetic acid. The transportation and storage can be eliminated by in-situ generation of peracetic acid using enzymes. Enzymes are potential catalysts for the synthesis of peracetic acid from hydrogen peroxide and acetate esters such as ethyl acetate. Perhydrolysis is the cleavage of an ester by hydrogen peroxide, and is mechanistically similar to hydrolysis. Many esterases catalyze perhydrolysis, but perhydrolases are a subgroup of esterases that are especially efficient at perhydrolysis. (6) Acetylxylan esterase (EC 126.96.36.199) is an enzyme that catalyzes a chemical reaction, the deacetylation of xylans and xylo-oligosaccharides. This enzyme belongs to the family of hydrolases, specifically those acting on carboxylic ester bonds. Several cellulolytic and hemicellulolytic microbes contain enzymes specific for deacetylation. In addition to degrading esterified acetyl groups, some fungal acetyl esterases have substrate specificity for highly acetylated hardwood xylans. In addition to the complete degradation of acetylated xylan, AXEs, with combinations of other xylolytic enzymes, can cooperatively hydrolyze acetyl xylan and partially deacetylate acetyl xylan. Acetyl xylan esterase can also be able to catalyze the perhydrolysis of acetate ester to peracetic acid. We used the acetyl xylan esterase from Aspergillus ficuum expressed in Pichia pastoris for the perhydrolysis of ethyl acetate thus generating peracetic acid. This enzymatically generated peracetic acid would be used for biomass pretreatment.
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Pichia pastoris GS115 (his4) harboring the gene encoding AXE (Afaxe) from A. ficuum was maintained on YPD plates (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar). P. pastoris was cultured 100 mL in a buffered complex medium with glycerol (BMGY) for 24 h (7). After the culture reached a density of OD600 = 6.0, the cells were harvested, resuspended and cultured in 100 mL of 0.5% methanol medium (BMMY) to induce expression. To purify the recombinant Afaxe, the cell-free supernatants were collected on the third day of cultivation by centrifugation at 8000 g for 5 min, and filtered through 0.2µm filters. One hundred milliliters of filtered supernatant was mixed with 10 ml 10- binding buffer (20 mM sodium phosphate, 0.5 M NaCl, and 20 mM imidazole; pH 7.4). The mixture was applied to a Ni2+ his-tag column (Histrap, GE Healthcare) using the AKTA FPLC purification system and protein was eluted as per manufacturer instructions. All fractions containing purified enzyme were dialyzed in 1 mM phosphate buffer (pH 6.0) to remove salt and imidazole. Protein concentration was measured by the Bradford method, using Thermo Scientific (USA) protein assay kit with serum albumin as the standard protein. SDS-PAGE was performed as described in Sambrook (8) to confirm the presence of protein (Fig 1). Whether AXE could able to catalyze perhydrolysis of ethyl acetate under hydrogen peroxide presenting condition, a reaction of AXE mediated perhydrolysis was done by Karst assay (9). A solution of 1 mL containing 100 mM potassium phosphate buffer of pH 7.0, ethyl acetate 500 mM, hydrogen peroxide 1.0 M and AXE (0.1 mg/mL) was incubated for 30 min at 37â„ƒ. After reaction, 0.1 mL of this sample solution was mixed with 0.1 mL of a 20 mM solution of methyl p-tolyl sulfide (MTS) with acetonitrile and 0.8 mL of 60% acetonitrile. 10 mg of manganese dioxide was added into each 1 mL sample 30 minutes after addition of the MTS solution. Prior to HPLC analysis, centrifugation at 15,000 rpm was performed for 5 minutes. MTS oxidation was carried out at different time periods. The amount of MTS and its oxidizing derivative, such as methyl p-tolyl sulfoxide (MTSO) and methyl p-tolyl sulfone (MTSON), were analyzed by HPLC (Shimadzu, Japan) consisted of the following components; pump LC-20A, auto-sampler model SIL-20A, column ACE C18 (250 X 4.6 mm, ACE) in column oven CTO-20A, detector model SPD-M20A, and software LC solution. Elution was done with a gradient of water and acetonitrile as followed; 0-4 min, isocratic 40% acetonitrile; 4-5 min, linear gradient to 100% acetonitrile; 5-22 min, isocratic 100% acetonitrile; 22-24 min return to isocratic start conditions; 24-28 min, re-equilibration time. The flow rate was 0.8 mL/min. The retention time of MTS and methyl p-tolyl sulfone was determined by the monitoring the effluent at 230 nm and the concentration of these compounds were calculated by a calibration curve made with these reference compounds. MTS, MTSO, and methyl p-tolyl sulfone was also confirmed with HPLC/MS. HPLC/MS method for analyzing these compounds was briefly described below. The HPLC system was a 1200 series (Agilent Technologies, Wilmington, USA). The analytical HPLC column was a reverse phase column (Shiseido CAPCELL, C18, 5um, 2.0 mm X 100 mm). The flow rate was 0.23 mL/min and the elution was with a gradient of water and acetonitrile containing 0.1% formic acid. Mass spectrometric detection was done by Agilent 6410B instrument (Agilent Technologies, Wilmington, USA). Ions were generated in positive ionization mode using electrospray ionization interface. The fragmenter potential was set to 110 V and the interface heater was set to 300°C (Data not shown).
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AXE has ability to perhydrolyse ethyl acetate to peracetic acid with H2O2. The amount of peracetic acid produced was indirectly determined by MTS oxidization. MTS was oxidized with AXE enzymatic solution and products from MTS oxidation were determined using HPLC with UV/visible detection. The HPLC separation of an excess of MTS and its reaction products with peracetic acid (PAA) was presented in Fig 2. After MTS oxidation four major peaks were detected. Among these peaks, the absorption spectra of Peak A and peak B corresponds to MTSO and MTSON respectively. It exhibited maxima near 224 nm, respectively (data not shown). The identity these products as MTSO and methyl p-tolyl sulfone was verified by comparison of the retention time with the MTSO and methyl p-tolyl sulfone standards and collected samples were analyzed with HPLC/MS. In HPLC/MS, the retention time of these substances were identical with the retention time of these standards. The corresponding mass spectra of samples and standards were coincided. To verify MTSO and methyl p-tolyl sulfone production owing to the PAA produced from AXE mediated reaction and to determine the amounts of MTS and methyl p-tolyl sulfone produced, MTS oxidation was done and analyzed by HPLC/UV. According to increase of the incubation times of PAA production, MTS oxidation increased and the production of MTSO and methyl p-tolyl sulfone increased (Fig 3). MTSO appeared and it oxidized to form methyl p-tolyl sulfone in solution subsequently. At the end of incubation, the amounts of MTSO and methyl p-tolyl sulfone were 0.72 mM and 0.31 mM. These results indicated that 1.33 mM of PAA produced by 10 µg of AXE. In previous reports (9, 10) acidification of solution containing ethyl acetate and H2O2 might oxidize MTS to MTSO in excess H2O2 conditions. To reduce this, all the reactions were incubated in 100 mM Phosphate Buffer at pH 7.0. 10 mg of Manganese dioxide (MnO2) was also used prior to HPLC detection to degrade the excess H2O2 to avoid the oxidation of MTS to MTSO by H2O2. In control no MTS oxidation happened after this treatment with MnO2 represents the absence of peracetic acid completely.
The biodegradability of lignocellulosic biomass is limited by several factors like crystallinity of cellulose, available surface area and lignin content. Pretreatments with concentrated acids, wet oxidation, solvents and metal complexes are effective, but too expensive compared Steam pretreatment, lime pretreatment, and ammonia based cause environmental problems when they are used and released. Whereas pretreatment with concentrated acids solubilize some amount cellulose present in the biomass along with the lignin, thus reducing the production of ethanol. But peracetic acid is a strong oxidizing agent which solubilizes lignin alone. Enzymatic generation of peracetic acid is a good way to avoid the cost and hazardous issue when compared to chemical generation. We attempt to produce to peracetic acid using acetyl xylan esterase form Aspergillus ficuum. The acetyl xylan esterase from A.ficuum is similar to acetyl xylan esterase from Phanerochaete chrysoporium (11) as it secretes naturally for the de-acetylation of xylan. P.chrysosporium may produce peracetic acid during de-acetylation process for the solubilization of lignin thereby resulting in the accessibility of enzymes like lignin peroxidases and manganese peroxidase for complete lignin removal. Thus acetyl xylan esterase would be a better enzyme to generate peracetic acid in situ. In our experiments 1.33 mM peracetic acid was generated using 10 µg of AXE. The presence of peracetic acid (PAA) was detected by using HPLC, where the presence of MTSON (MTS oxidized by PAA) clearly shows the amount of PAA. The amount of peracetic acid produced was little higher when compared to previous experiments (6). But it may not enough to use in large scale pretreatment plant. Immobilization technologies as well as improvements in the catalytic properties of the enzyme will make much larger improvements. It is easy to recycle this enzyme for continuous cycles during pretreatment process. This enzyme utilizes acetyl xylan esters present in the biomass as the acetyl source for the peracetic acid generation. This would reduce the amount of acetate ester needed and also remove the acetyl groups, which enhances the sugar release from the biomass. Enzymatic generation of peracetic acid as a pretreatment for biomass offers advantages by generating peracetic acid as needed, thereby eliminating issues related to storage and transportation (explosion and stability). It also will sterilize the biomass and prevent microbial contamination problems in biomass storage and fermentation. The amount of enzyme needed is not economical, but improvements in enzyme stability and activity could overcome this barrier. Thus generation of peracetic acid form acetyl xylan esterase would be a good source for the pretreatment of biomass to produce cost effective ethanol and also for the biobleaching of biomass in pulp and paper industry.
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