Initial Stage Of Hydrolysis Research Articles

Experimental study on cement-based materials for grouting/replacement of broken rock mass in coal mine

In deep high-pressure mines, the surrounding rock of soft broken roadways often faces issues such as poor anchorability, support difficulties, high maintenance costs, and the inadequacy of traditional materials for site requirements. To tackle these challenges, this study focuses on grouting reinforcement in coal mine sites and prefabricated bearing arch form (grouting or replacement) under specific conditions, aiming to meet the material requirements. Through an orthogonal test, various properties of cement-based materials were investigated, including fluidity, initial setting time, water separation rate, early (≤1d) compressive strength, fracture strength, and later (28d as a reference) strength. The objective was to develop a cost-effective cement-based material with high strength and toughness, suitable for underground coal mines. The experimental results demonstrate that the self-developed cement-based materials exhibit rapid setting, excellent fluidity, high early strength, and strong durability compared to traditional alternatives. Analyses of the comprehensive properties reveal that the influencing factors, in order of significance, include the mass ratio of cement and sand, water cement ratio, rubber content, and bonding mortar master material content. By employing extreme treatment and efficiency coefficient methods, the comprehensive performance of materials under different ratios was evaluated. Consistent findings were obtained, indicating that the optimal mass ratio of cement-based materials is 500:500:20:15:10:5:8:10 for cement, sand, fly ash, silica fume, mineral powder, steel slag powder, bonding mortar master material, and rubber, respectively. Additionally, the best comprehensive performance was achieved with a water cement ratio of 0.5, which confirms the results of the orthogonal test. Macroscopic failure characteristics and SEM microscopic analysis further validate the superior performance of the materials under the aforementioned ratio. The reaction process of the material can be roughly divided into four stages: initial hydrolysis stage, induction stage, acceleration stage, decay and stability stage, and finally the formation of stone body. A comparison with ordinary materials highlights the advantages of the developed cement-based materials, and suggestions are proposed for the development of field-applicable materials.

Comparative analysis of granular starch hydrolysis and multi-structural changes by diverse α-amylases sources: Insights from waxy rice starch

Three cultivars of waxy rice starch with different multi-scale structures were subjected to α-amylase hydrolysis to determine amylopectin fine structure, production of oligosaccharides, morphology, and crystallinity of the partially hydrolyzed starch granules. α-amylases hydrolyzed the amylopectin B2 chain during the initial stage of hydrolysis, suggesting that it is primarily located in the outer shell of the granules. For waxy rice starch with loose structure, α-amylases attacked the crystalline and amorphous regions simultaneously in the initial stage, while for starch granules with compact structure, the outer shell blocklet (crystalline structure) can be a hurdle for α-amylases to proceed to hydrolysis of the internal granule structure. The ability of α-amylases from porcine pancreatic α-amylases to attack the outer shell crystalline structure was lower than that of α-amylases from Bacillus amyloliquefaciens and Aspergillus oryzae. These results show that α-amylase source and rice cultivar combinations can be used to generate diverse structures in degraded waxy rice starch.

Hydrolysis hydrogen generation behavior of Mg10Ni10Ce alloy catalytic modified by isomeric carbon materials

Hydrolysis hydrogen generation from Mg-based alloys is considered as a potential energy carrier for global low-carbon and growing energy challenges. At present, it is a key problem to explore an efficient modification method for Mg-based alloys to obtain rapid initial hydrolysis kinetics and high H2 yield in short time. In this work, carbon-based isomeric catalysts were creatively employed to activate Mg10Ni10Ce alloy. Based on the rapid initial hydrolysis kinetics, the effects of carbon nanostructured catalysts on the hydrogen production of Mg10Ni10Ce were studied. Herein, the catalytically modified Mg10Ni10Ce samples with point-like carbon black (SP), one-dimensional tubular carbon nanotubes (CNTs) and two-dimensional layered graphite (GP) are simply prepared by short-time high-energy ball milling process. Due to the significant structural characteristics, the catalytically modified Mg10Ni10Ce samples have higher H2 yields at the initial hydrolysis stage, Mg10Ni10Ce-SP, Mg10Ni10Ce-CNTs and Mg10Ni10Ce-GP samples are 17.3%, 25.2% and 36.2% at 313 K for 30 s, which are higher than that of Mg10Ni10Ce (10.4%). The above work provides an effective strategy for hydrogen production by modifying Mg-based alloys with nanostructured catalysts, and lays a foundation for the application of portable hydrogen production devices.

Sugarcane molasses-based biorefinery: Organic acids and ethanol production

Sugarcane molasses are the largest produced waste in sugar mills; in the last harvesting cycle 2,178,131 tons were obtained and only 2.46% were used for transformation processes. Molasses has great potential to be the main feedstock in a biorefinery concept. Its composition rich in fermentable sugars and its availability are interesting features considered in this study. Through the Aspen Plus © software a multi-product biorefinery scenario was design and analyzed, technically and economically. The three main products considered were ethanol, lactic acid and succinic acid. The overall process consists of an initial stage of hydrolysis or inversion of the sucrose present in the molasses to reducing sugars followed by a specific dilution for fermentation of each of the products considered, as well as subsequent separation and purification operations. Plant efficiencies conversions were 3.24 kg of molasses/L of ethanol, 3.08 kg of molasses/kg of lactic acid and 9.25 kg of molasses/kg of succinic acid. The economic assessment was positive for organic acids production. Ethanol production had a slightly worst economic performance compared to the other processes, but the proposed scenario managed to obtain a profitability index of 1.02. The expense recovery ratio of the whole biorefinery was 1.35 which means a surplus of 35% after the project investment has paid for itself. The biorefinery’s robustness in the economic aspect comes from organic acids production; meanwhile, the social and environmental impacts are from ethanol production.

Glucan Conversion and Membrane Recovery of Biomimetic Cellulosomes During Lignocellulosic Biomass Hydrolysis.

Enzyme immobilization has been identified as one way to recycle enzymes and reduce processing costs during enzymatic hydrolysis of lignocellulosic materials. However, most immobilization methods have not been attractive to lignocellulosic processing plants. In this study, cellulase enzymes were attached to a copolymer of glycidyl methacrylate (GMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) to make polymer-enzyme conjugates (PECs) and facilitate recovery using a 50-kDa molecular weight cutoff membrane. Glucan conversion during biomass hydrolysis was investigated using new PECs and PECs recovered after an initial hydrolysis stage. Enzyme immobilization on PECs did not reduce effectiveness during the initial hydrolysis. Temperature and pH showed similar effects on free enzymes and PECs. PECs facilitated higher conversion rates than free enzymes at high biomass loadings. Recovered PECs were used to achieve approximately 100% glucan conversion in a subsequent hydrolysis step when supplemented with 40% of the free enzyme used in the first stage. The combination of PECs and membrane recovery has the potential to reduce hydrolysis cost during cellulosic bioprocessing.

The inhibitory effect of xylan on enzymatic hydrolysis of cellulose is dependent on cellulose ultrastructure

The inhibitory effect of xylan on enzymatic hydrolysis of cellulose with different ultrastructure is not clear. In this work, cellulose II, cellulose III, and amorphous cellulose were prepared from cellulose I by NaOH, ethylenediamine, and ball-milling treatment, respectively. Their crystalline structure and enzymatic hydrolyzability were systematically characterized and compared with cellulose I. Furthermore, the inhibitory effect of xylan on cellulose with different ultrastructure was investigated in detail. Results suggested that the enzymatic hydrolyzability of different cellulose increased in sequence from cellulose I (25.4%), cellulose III (41.7%), cellulose II (56.2%) to amorphous cellulose (77.4%) at enzyme loadings of 5.56 mg protein/g substrate. The enzymatic hydrolyzability of allomorphic cellulose was strongly inhibited by xylan, while a little inhibition was observed for amorphous cellulose. It was found that cellobiose released from different cellulose was decreased after the addition of xylan. At the initial hydrolysis stage, the inhibition degree was obviously increased for all of different cellulose, and then, the inhibition degree of cellulose I and cellulose II gradually decreased as enzymatic hydrolysis proceeded, while the inhibition degree of cellulose III and amorphous cellulose had changed a little. In addition, at a longer time of hydrolysis, the inhibition degree was decreased with the increase of cellulase dosage except for cellulose III, which remained approximately unchanged. Overall, this study demonstrated that the inhibitory effect of xylan on cellulose hydrolysis was substantially affected by the ultrastructure of cellulose, and thus it provides new insights for relieving the inhibitory effect of xylan by altering the ultrastructure of cellulose.

An aggregated understanding of cellulase adsorption and hydrolysis for ball-milled cellulose

This study evaluated the effects of physicochemical properties of a series of ball-milled cellulose on cellulase adsorption and glucose yield. The relationship between cellulase adsorption and initial hydrolysis rate was also discussed. We found that hydrophobicity and surface charge are the key factors affecting cellulase adsorption on ball-milled cellulose. The results demonstrated that glucose yield had a positive correlation with specific surface area, while showed a negative correlation with particle size, degree of polymerization and crystallinity. Among these properties, specific surface area and crystallinity are the key factors affecting glucose yield. As ball milling progressed, cellulose showed lower enzyme adsorption capacity/amount of bound enzyme during initial stage of hydrolysis, but had higher initial hydrolysis rate. The enhanced rate is attributed to the fact that the amorphous region produced by ball milling reduces the free energy required for decrystallization thus increases the catalytic efficiency of the bound enzyme.

The inhibition of anaerobic digestion by model phenolic compounds representative of those from Sargassum muticum

Practical yields of biogas from the anaerobic digestion of macroalgae and, Sargassum muticum in particular, are substantially below the theoretical maximum. There is considerable conjecture about the reasons for the relatively low practical methane yields from seaweed, and polyphenols are suggested as one of the elements in the low yield of methane from brown seaweeds. However, there appears to be little information on the effect of specific phenolics on defined substrates. This paper examines the effect of some simple phenolic compounds, representative of those reported in S. muticum on methane production from a range of model substrates. Three simple phenolics were selected, gallic acid, epicatechin and phloroglucinol; at four addition levels, 0, 0.5, 3.5 and 7.5% w/w of substrate; for four substrates, a readily digested simple organic substance, glycerol, and three polymers found in seaweed, cellulose, alginic acid and the sodium salt of alginic acid. Alginic acid and its sodium salt were found to be recalcitrant with average methane yields of equivalent to only 23–28% of their theoretical methane potential. Methane yield was further reduced by the presence of high concentrations (7% of substrate equivalent to 17.5 mg L−1) of phloroglucinol and epicatechin. None of the phenolic compounds studied appeared to inhibit the breakdown of the simple and readily digested compound, glycerol. Low methane yield in seaweed may be due to the recalcitrance of complex hydrocolloids and phenolic inhibition of the breakdown of more complex molecules in the initial hydrolysis stage of anaerobic digestion, but further research is required.

Soy and Rapeseed Protein Hydrolysis by the Enzyme Preparation Protosubtilin

A comparative study of soybean and rapeseed protein hydrolysis by protosubtilin, an original Russian enzyme preparation widely used in animal feed production, has been performed. SDS-PAG electrophoresis, HPLC, and mass spectrometry have been employed to analyze the obtained products. The soybean protein isolate used for hydrolysate production was obtained from a commercial supplier, and rapeseed proteins were prepared from the meal by alkali extraction. Low molecular weight impurities were removed by ultrafiltration. The degree of protein hydrolysis has been shown to depend on the substrate-to-enzyme preparation ratio, hydrolysis time, and protein concentration. Rapeseed protein hydrolysis by protosubtilin at an enzyme/protein ratio of 1: 20 and hydrolysis time of 20 h resulted in complete cleavage of the proteins present in the raw material and the accumulation of oligopeptides (molecular weight less than 14 kDa) and free amino acids, which accounted for 53 and 8% of the initial protein weight, respectively. In contrast to rapeseed proteins, soybean proteins showed considerable gelling at the initial stages of hydrolysis, and the formation of insoluble hydrolysis-resistant fragments was observed. The soluble part of the hydrolysate contained short oligopeptides and free amino acids, which accounted for 13% of the initial protein weight only.

Comparison of the influence of pH on the selectivity of free and immobilized trypsin for β-lactoglobulin hydrolysis

Although immobilized trypsin is a viable alternative to the free one in solution for producing protein hydrolysates, the change of selectivity introduced by immobilization is unclear. In this study, we compared the selectivity of free and immobilized trypsin towards different cleavage sites of β-lactoglobulin (β-Lg) with a focus on the impact of environmental pH. Both free and immobilized trypsin exhibited greater accessibility to native β-Lg at elevated pH (from pH 7.2 to 8.7). Additionally, free trypsin preferred to attack cleavage sites located at the C-terminus at pH 7.8, whereas an opposite preference for the N-terminus was observed at pH 8.7. Regarding the immobilized trypsin, the pH did not significantly influence its preference for the C- or N-terminus. Generally, immobilization of trypsin resulted in more focused cleavage within its specificity during the initial stage of hydrolysis, and some peptides were formed more rapidly by the immobilized trypsin.

Genotype and environment effects on the susceptibility of wheat starch to amylolysis

The present study addresses an aspect of genotype by environmental influences on cereal grain quality that has not been researched extensively. The susceptibility of raw and cooked starch to enzymic hydrolysis has been examined using a set of well-characterized wheat endosperm starch samples obtained from five wheat varieties grown in four different locations in 2 years. Native starch granules were hydrolyzed slowly, with genotype contributing most of variance between the samples during the initial stages of hydrolysis. Surface properties of the granules influenced the initial stages of hydrolysis. Environmental factors became increasingly influential as hydrolysis progressed, accounting for almost 50% of variance at 24 h, when 70–80% of the starch had been hydrolyzed. Of the environmental conditions associated with growing locations, temperatures before flowering and during grain filling were identified as contributing to variability in the susceptibility of native starch to enzyme hydrolysis. Cooked starch was hydrolyzed much more rapidly than native granules, with 80% of starch breakdown occurring within 90 min. For cooked starch, genotype contributed about 60–70% of the variance in the amount of starch hydrolyzed within 30 min and in the amount of starch remaining after 2 h. The strong genetic influence on the susceptibility of both native and cooked wheat starch to enzymic hydrolysis indicates the heritability of these characters.

Hydrolytic mechanism of α-maltotriohydrolase on waxy maize starch and retrogradation properties of the hydrolysates

In this study, we explored the hydrolytic mechanism of glucan 1,4-α-maltotiohydrolase (AMTS). Kinetic parameters analysis of AMTS were performed using the amylopectin with different external chain lengths (ECL). Then, the waxy maize starch was gelatinized and hydrolyzed by AMTS to various degrees; retrogradation of starch hydrolysates were determined. Michaelis constant (Km) of the β-dextrin-3 (ECL 2.3) was eight folds than the amylopectin (ECL 17.8), but its catalytic efficiency (kcat/Km) was only 6.5% of amylopectin. AMTS preferred the external chains to the internal chains for hydrolysis, and amylopectin was hydrolyzed by AMTS through combined exo- and endo-action. In the initial hydrolysis stage, the exo-action of AMTS was predominant with slow reduction in Mw and little changes in the internal chains of amylopection. The percentage of maltotriose in the soluble oligosaccharides (PM) was 90.5% at 2.1% degree of hydrolysis (DH), and gradually decreased from 90.5% to 71.2% at 35.4% DH. The PM decreased could be partially attributed to the increased transglycosylation reactions of AMTS. Complete inhibition of amylopectin retrogradation was observed with >15.9% hydrolysis for the increased shorter outer chains and low molecular weight dextrins. These results indicate that AMTS may be effective in retarding starch retrogradation in baked products.

Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii

The novel chitinase (PbChi67) from the marine bacterium Paenicibacillus barengoltzii CAU904 was purified and biochemically characterized. PbChi67 was purified to apparent homogeneity with 10.2 fold purification and 8.0% recovery yield. The molecular mass of the enzyme was 67.0kDa by SDS-PAGE and 67.9kDa by gel filtration, respectively. PbChi67 was most active at pH 3.5 and was stable within pH 3.0–9.0. The optimal temperature of PbChi67 was 60°C and it was stable up to 55°C with a thermal denaturing half-life of 43min at 65°C. The enzyme exhibited strict substrate specificity towards colloidal chitin and glycol chitin but showed no or trace activities towards other tested substrates. The Km and Vmax values of PbChi67 for colloidal chitin and glycol chitin were 3.35mg/mL and 17.1μmol/min/mg, and 2.66mg/mL and 15.0μmol/min/mg, respectively. PbChi67 hydrolyzed colloidal chitin to yield N-acetyl chitooligosaccharides (COSs) with degree of polymerization (DP) of 2–4 at the initial hydrolysis stage, indicating that it is an endo-type chitinase. These properties make the enzyme as a good candidate for recycling of chitin materials.

Investigating the mechanisms of amylolysis of starch granules by solution-state NMR.

Starch is a prominent component of the human diet and is hydrolyzed by α-amylase post-ingestion. Probing the mechanism of this process has proven challenging, due to the intrinsic heterogeneity of individual starch granules. By means of solution-state NMR, we demonstrate that flexible polysaccharide chains protruding from the solvent-exposed surfaces of waxy rice starch granules are highly mobile and that during hydrothermal treatment, when the granules swell, the number of flexible residues on the exposed surfaces increases by a factor of 15. Moreover, we show that these flexible chains are the primary substrates for α-amylase, being cleaved in the initial stages of hydrolysis. These findings allow us to conclude that the quantity of flexible α-glucan chains protruding from the granule surface will greatly influence the rate of energy acquisition from digestion of starch.

An s‐triazine briged silylalkyl sol–gel precursor—synthesis, structure and coating formation for electrical insulation of copper wire and corrosion protection of construction steel

An aminoalkylalkoxysilane modified s‐triazine was prepared and used as single‐source precursor for hybrid coatings. These coating materials were applied and tested for the electrical insulation of fine copper wires and for corrosion protection of construction steel. The molecular precursor was comprehensively analyzed using FT‐IR, 1H, 13C and 29Si NMR spectroscopy. Besides, the initial stages of hydrolysis and condensation reactions under acidic conditions were examined with solution 29Si NMR spectroscopy. Structures of gelled and heat‐treated samples were analyzed with CP/MAS 29Si NMR and FT‐IR spectroscopy. Sols were applied on fine copper wires using industrial coating and curing techniques. The performance of the hybrid coatings was characterized by electrical breakdown tests, pinhole tests and mechanical strength measurements. The anti‐corrosion behavior of the s‐triazine precursor derived coatings on steel substrates was studied by electrical impedance spectroscopy. The spectra were interpreted with respect to different hydrolysis and condensation rates, curing temperatures and coating thicknesses. Copyright © 2014 John Wiley & Sons, Ltd.

Expression and Characterization of Recombinant GH11 Xylanase from Thermotolerant Streptomyces sp. SWU10

Xylans are major hemicellulose components of plant cell wall which can be hydrolyzed by xylanolytic enzymes. Three forms of endo-β-1,4-xylanases (XynSW1, XynSW2A, and XynSW2B) produced by thermotolerant Streptomyces sp. SWU10 have been reported. In the present study, we described the expression and characterization of the fourth xylanase enzyme from this bacteria, termed XynSW3. The gene containing 726 bp was cloned and expressed in Escherichia coli. The recombinant enzyme (rXynSW3) was purified from cell-free extract to homogeneity using Ni-affinity column chromatography. The apparent molecular mass of rXynSW3 was 48 kDa. Amino acid sequence analysis revealed that it belonged to a xylanase of glycoside hydrolase family 11. The optimum pH and temperature for enzyme activity were 5.5-6.5 and 50 °C, respectively. The enzyme was stable up to 40 °C and in wide pH ranges (pH 0.6-10.3). Xylan without arabinosyl side chain is the most preferable substrate for the enzyme. By using birch wood xylan as substrate, rXynSW3 produced several oligosaccharides in the initial stage of hydrolysis, and their levels increased with time, demonstrating that the enzyme is an endo-acting enzyme. The major products were xylobiose, triose, and tetraose. The rXynSW3 can be applied in several industries such as food, textile, and biofuel industries, and waste treatment.

Assessment of the influence of amylose-LPC complexation on the extent of wheat starch digestibility by size-exclusion chromatography

Amylose forms inclusion complexes with lysophosphatidylcholine (LPC), that decrease the susceptibility of amylose to amylase degradation. This study on the influence of complexation on starch susceptibility to amylase explains the nature of this protective effect. Wheat starch suspensions (9% w/w) containing 0.5–5% LPC were subjected to hydrolysis by porcine pancreatic α-amylase at 37°C for several digestion times. The digesta were analysed by size-exclusion chromatography (SEC). The molar mass distribution was closely dependent on the digestion time and amount of LPC. This study precisely demonstrates the alteration of the digestion profile of starch on a molecular level, influenced by amylose-LPC complexation; however the effect depends on the digestion time. During 15 and 30min digestion, inclusion complexes not only protect amylopectin in the initial hydrolysis stage, but also demonstrate lower susceptibility of the molecular amylose complexes to amylase hydrolysis. Digestion for 240min resulted in a lower oligosaccharide peak concentration, in the presence of a high LPC concentration, which is related to less degradation of complexed amylose fraction.

The susceptibility of large and small granules of waxy, normal and high-amylose genotypes of barley and corn starches toward amylolysis at sub-gelatinization temperatures

Starches from waxy (CDC Candle), normal (CDC McGwire) and high-amylose (SH 99250) genotypes of hull-less barley were isolated and fractionated into large and small granules. The unfractionated and fractionated barley starches were then characterized toward their composition, morphology and structure, and compared with commercial corn starches of similar genotypes. The effect of starch properties on degree of hydrolysis (DH) at 55°C for 1h followed by at 30°C for 72h using granular starch hydrolyzing enzymes (mixture of α-amylase and glucoamylase) was also evaluated. Morphological changes of starches before and after hydrolysis were visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The proportion (% weight) of small granules in high-amylose (HA) genotypes of barley (46.5%) and corn (32.4%) starches were higher than in the waxy (WX) and normal (NM) genotypes. Apparent amylose (AM) content of corn genotypes ranged from 1 to 1.1% (WX), 21 to 26% (NM), and 66 to 70% (HA). Whereas, the corresponding ranges for barley genotypes were 4–6% (WX), 17–24% (NM), and 35–40% (HA). HA corn starch exhibited a mixed ‘A+V’-type X-ray pattern, whereas the other genotypes of corn and barley starches exhibited an ‘A’-type X-ray pattern. The relative crystallinity (RC) of the genotypes of corn and barley starches followed the order: WX>NM>HA. The AM content and RC of small granules in corn and barley starches were lower than in large granules. Regardless of genotypes and starch sources, smaller granules had a higher gelatinization temperature range (15.5–42.8°C) and a lower enthalpy of gelatinization (9.7–21.2J/g) than those of larger granules (11.1–41.9°C and 13.4–23.9J/g, respectively). Compared to large granules, the small granules were initially hydrolyzed to a greater extent (18% higher). However after 72h, hydrolysis was 10% higher in the large granules. SEM and CLSM images revealed that the distribution of surface pores and internal channels varied between genotypes of corn and barley starches. Although small variations exist between the NM corn and NM barley starches with respect to their proximate composition and amylose content, the observed large variations in the extent of amylolysis at the initial stages of hydrolysis is indicative that the molecular architecture and granule porosity influence amylolysis.

Morphology and structural characterization of high-amylose rice starch residues hydrolyzed by porcine pancreatic α-amylase

High-amylose starches are attracting considerable attention because of their potential health benefits and industrial uses. Enzyme hydrolysis of starch is involved in many biological and industrial processes. In this paper, starches were isolated from mature grains of high-amylose transgenic rice line (TRS) and its wild type rice cultivar Te-qing (TQ). The morphological and structural changes of starch residues following porcine pancreatic α-amylase (PPA) hydrolysis were characterized by SEM, DSC, XRD, 13C CP/MAS NMR, and ATR-FTIR. TQ starch was hydrolyzed faster than TRS starch. PPA pitted the starch granule surface first, then penetrated into the interior and hydrolyzed the granule from the inside out. The peripheral region of subgranule and the surrounding band of TRS starch were more resistant to PPA hydrolysis than the external region of TQ starch. Both amorphous and crystalline (the long- and short-range ordered) structures were simultaneously hydrolyzed in TQ starch. A-type polymorph of TRS C-type starch was hydrolyzed faster than B-type polymorph. The long-range ordered structure was hydrolyzed faster than the amorphous structure, whereas the short-range ordered structure was hydrolyzed slower than the amorphous structure in TRS starch in the initial hydrolysis stage. The above results indicated that B-type crystallinity and short-range ordered structure in the peripheral region of subgranule and the surrounding band of TRS starch increased the resistance of TRS starch to PPA hydrolysis.

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPAinsol) should be measured between 10 and 20min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.