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Chapter 1: Theory of endo-1, 4-Beta-D-Xylanase Assay Procedure
Chapter 2: Buffers & Reagents
Chapter 3: Assay Procedure
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|Content:||200 Tablets or 1,000 Tablets|
|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||Protease|
|Reproducibility (%):||~ 5%|
High purity dyed and crosslinked Protazyme AK tablets for the measurement of enzyme activity, for research, biochemical enzyme assays and in vitro diagnostic analysis.
endo-Protease test tablets. High sensitivity. Containing AZCL-Casein.
Please note the video above shows the protocol for assay of endo-xylanase using xylazyme tablets. The procedure for the assay of protease using Protazyme AK Tablets is equivalent to this.
Enzyme purity and activity in fibre determinations.
McCleary, B. V. (1999). Cereal Foods World, 44(8), 590-596.
Dietary fiber is mainly composed of plant cell wall polysaccharides such as cellulose, hemicellulose, and pectic substances, but it also includes lignin and other minor components (1). Basically, it covers the polysaccharides that are not hydrolyzed by the endogenous secretions of the human digestive tract (2,3). This definition has served as the target for those developing analytical procedures for the measurement of dietary fiber for quality control and regulatory considerations (4). Most procedures for the measurement of total dietary fiber (TDF), or specific polysaccharide components, either involve some enzyme treatment steps or are mainly enzyme-based. In the development of TDF procedures such as the Prosky method (AOAC International 985.29, AACC 32—05) (5), the Uppsala method (AACC32-25) (6), and the Englyst method (7), the aim was to remove starch and protein through enzyme treatment, and to measure the residue as dietary fiber (after allowing for residual, undigested protein and ash). Dietary fiber was measured either gravimetrically or by chemical or instrumental procedures. Many of the enzyme treatment steps in each of the methods, particularly the prosky (5) and the Uppsala (6) methods are very similar. As a new range of carbohydrates is being introduced as potential dietary fiber components, the original assay procedures will need to be reexamined, and in some cases slightly modified, to ensure accurate and quantitative measurement of these components and of TDF. These “new” dietary fiber components include resistant nondigestible oligosaccharides; native and chemically modified polysaccharides of plant and algal origin (galactomannan, chemically modified celluloses, and agars and carrageenans); and resistant starch. To measure these components accurately, the purity, activity, and specificity of the enzymes employed will become much more important. A particular example of this is the mesurement of fructan. This carbohydrate consists of a fraction with a high degree of polymerization (DP) that is precipitated in the standard Prosky method (5,8) and a low DP fraction consequently is not measured (9). Resistant starch poses a particular problem. This component is only partially resistant to degradation by α-amylase, so the level of enzyme used and the incubation conditions (time and temperature) are critical.Hide Abstract
Effect of pre‐harvest sprouting on physicochemical changes of proteins in wheat.
Simsek, S., Ohm, J. B., Lu, H., Rugg, M., Berzonsky, W., Alamri, M. S. & Mergoum, M. (2014). Journal of the Science of Food and Agriculture, 94(2), 205-212.
Background: High moisture before harvest can cause sprouting of the wheat kernel, which is termed pre-harvest sprouting (PHS). The aim of this study was to examine the variation in physicochemical properties of proteins in PHS-damaged (sprouted) hard red and white spring wheat genotypes. Specifically, protein content, enzyme activity and degradation of proteins were evaluated in sound and PHS-damaged wheat. Results: Protein contents of sprouted wheat samples were lower than that of non-sprouted samples; however, their differences were not significantly (P > 0.05) correlated with sprouting score. Sodium dodecyl sulfate (SDS) buffer extractable proteins (EXP) and unextractable proteins (UNP) were analyzed by high-performance size exclusion chromatography. PHS damage elevated endoprotease activity and consequently increased the degradation of polymeric UNP and free asparagine concentration in wheat samples. Free asparagine is known to be a precursor for formation of carcinogenic acrylamide during high heat treatment, such as baking bread. Free asparagine content had significant correlations (P < 0.01) with sprouting score, endoprotease activity and protein degradation. Conclusions: Genotypes with higher endoprotease activity tend to exhibit a larger degree of degradation of UNP and higher free asparagine concentration in sprouted wheat samples.Hide Abstract
Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering.
Waegeman, H., De Lausnay, S., Beauprez, J., Maertens, J., De Mey, M. & Soetaert, W. (2013). New Biotechnology, 30(2), 255-261.
Escherichia coli strains are widely used as host for the production of recombinant proteins. Compared to E. coli K12, E. coli BL21 (DE3) has several biotechnological advantages, such as a lower acetate yield and a higher biomass yield, which have a beneficial effect on protein production. In a previous study (BMC Microbiol. 2011, 11:70) we have altered the metabolic fluxes of a K12 strain (i.e. E. coli MG1655) by deleting the regulators ArcA and IclR in such a way that the biomass yield is remarkably increased, while the acetate production is decreased to a similar value as for BL21 (DE3). In this study we show that the increased biomass yield beneficially influences recombinant protein production as a higher GFP yield was observed for the double knockout strain compared to its wild type. However, at higher cell densities (>2 g L-1 CDW), the GFP concentration decreases again, due to the activity of proteases which obstructs the application of the strain in high cell density cultivations. By further deleting the genes lon and ompT, which encode for proteases, this degradation could be reduced. Consequently, higher GFP yields were observed in the quadruple knockout strain as opposed to the double knockout strain and the MG1655 wild type and its yield approximates the GFP yield of E. coli BL21 (DE3), that is, 27±5mg g-1 CDW vs. 30±5mg g-1 CDW, respectively.Hide Abstract
Treatments with xylanase at high (90%) and low (40%) water content have different impacts on physicochemical properties of wheat bran.
Santala, O. K., Nordlund, E. A. & Poutanen, K. S. (2013). Food and Bioprocess Technology, 6(11), 3102-3112.
The aim of the work was to elucidate the impacts of treatment with xylanase at high (90%) and low (40%) water contents on the structural and physicochemical properties of wheat bran. The bran treatments at 40% water content, both with and without added xylanase, resulted in a smaller average bran particle size, more changes in bran microstructure, and higher solubilization of polysaccharides than the corresponding treatments at 90%. Also, the water holding capacity of bran (3.6 ± 0.1 g water/g bran dm), determined by Baumann method, decreased more already after 4-h xylanase treatments at 40% (2.4 ± 0.1) than at 90% (2.9 ± 0.2). The solubility of salt-extractable bran proteins decreased during the treatments, especially at 40%, also without added xylanase. Protein aggregation was detected in the SDS + DTT-extractable bran fraction, which also contained small proteins of 10–20 kDa not detectable in the untreated bran. The use of xylanase had only minor effect on bran proteins as compared to the treatments without added xylanase. The results indicate the large role of mechanical shear on the bran properties at 40% water content. The low arabinose/xylose ratio (0.32) in the bran water extract after 24-h xylanase treatment at 40%, however, suggests that the solubilization of arabinoxylan was caused by enzymatic action, and not by mechanical degradation. Arabinose/xylose ratio of the bran water extract decreased similarly during all the treatments, suggesting similar solubilization pattern of arabinoxylan at both water contents. The study showed that bran properties can be significantly modified by adjusting the water content and mechanical energy used in processing.Hide Abstract
Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin.
Yu, S., Cowieson, A., Gilbert, C., Plumstead, P. & Dalsgaard, S. (2012). Journal of Animal Science, 90(6), 1824-1832.
Phytic acid (IP(6)) and myo-inositol phosphate esters (IP(1-5)), including IP(5) isomers prepared chemically and enzymatically with bacterial and fungal phytases, were examined for their effects on protein aggregation of soy protein and β-casein, interaction with Fe(3+), and pepsin activity. The results indicated that the aggregating capabilities of IP esters (IP(1-6)) on the 2 proteins decreased dramatically from IP(6) to IP(5) and became negligible with IP(1-4). Among the IP(5) isomers tested, InsP(5)(1,2,3,4,5) produced by 6-phytase was slightly less powerful in aggregating protein than InsP(5)(1,2,4,5,6) produced by 3-phytase (P = 0.001). For protein hydrolysis, IP esters of IP(3-4) still showed inhibition of pepsin though to a lesser extent than IP(5-6). The in vitro data with IP(1-5) generated with microbial 3- and 6-phytases indicate that, for complete alleviation of pepsin inhibition, IP(6) needs to be broken down to IP(1-2.) In contrast to the aggregation with protein, the reactivity of IP(1-6) toward Fe(3+) decreased proportionally from IP(6) to IP(3.) Based on the radical decrease in turbidity of IP(6) -protein complex observed, as a result of IP(6) dephosphorylation to IP(5), a novel qualitative and semi-quantitative phytase plate assay was established using IP(6)-protein complex incorporated into an agarose petri-dish as substrate. Phytase activity was shown as the development of clear halos on the agarose plate with time. This simple phytase plate assay method can be used at animal farms, control laboratories, and even for the screening of engineered phytase variants. The current study, thus, stresses the importance of the efficient hydrolysis of IP(6) at lower pH range to alleviate the negative effect of phytic acid and its degradation products on protein and Fe(3+) digestion.Hide Abstract
Effects of tyrosinase and laccase on oat proteins and quality parameters of gluten-free oat breads.
Flander, L., Holopainen, U., Kruus, K. & Buchert, J. (2011). Journal of Agricultural and Food Chemistry, 59(15), 8385-8390.
Effects of a Trichoderma reesei tyrosinase (TYR) and a Trametes hirsuta laccase (LAC) on breadmaking performance of gluten-free oat flour were investigated by SDS-PAGE analysis of oat protein fractions, large deformation rheology, and microscopy of the doughs, as well as on the basis of specific volume and firmness of the gluten-free breads. TYR induced the formation of higher molecular weight proteins in the SDS-PAGE assay. Microscopical analysis showed more intensive protein aggregation in the TYR-treated dough than in the dough without TYR. TYR also increased the firmness of the dough, which was assumed to be because of the cross-linking of oat globulins. LAC did not affect the oat globulins. TYR alone, or together with a commercial Thermomyces lanuginosus xylanase (XYL), increased significantly the specific volume of the gluten-free oat bread. A combination of TYR and XYL also increased the softness of the bread, whereas a combination of LAC and XYL improved the specific volume but did not affect the softness of oat bread. The results thus indicate that cross-linking of oat globulins by TYR, especially with the addition of XYL, was beneficial for improving the texture of gluten-free oat bread.Hide Abstract
Effects of laccase and xylanase on the chemical and rheological properties of oat and wheat doughs.
Flander, L., Rouau, X., Morel, M. H., Autio, K., Seppänen-Laakso, T., Kruus, K. & Buchert, J. (2008). Journal of Agricultural and Food Chemistry, 56(14), 5732-5742.
The effects of Trametes hirsuta laccase and Pentopan Mono BG xylanase and their combination on oat, wheat, and mixed oat−wheat doughs and the corresponding breads were investigated. Laccase treatment decreased the content of water-extractable arabinoxylan (WEAX) in oat dough due to oxidative cross-linking of feruloylated arabinoxylans. Laccase treatment also increased the proportion of water-soluble polysaccharides (WSNSP) apparently due to the β-glucanase side activity present in the laccase preparation. As a result of the laccase treatment, the firmness of fresh oat bread was increased. Xylanase treatment doubled the content of WEAX in oat dough and slightly increased the amount of WSNSP. Increased stiffness of the dough and firmness of the fresh bread were detected, probably because of the increased WEAX content, which decreased the amount of water available for β-glucan. The combination of laccase and xylanase produced slight hydrolysis of β-glucan by the β-glucanase side activity of laccase and enhanced the availability of AX for xylanase with concomitant reduction of the amount and molar mass of WSNSP. Subsequently, the volume of oat bread was increased. Laccase treatment tightened wheat dough, probably due to cross-linking of WEAX to higher molecular weight. In oat−wheat dough, laccase slightly increased the proportion of WSNSP between medium to low molecular weight and increased the specific volume of the bread. Xylanase increased the contents of WEAX and WSNSP between medium to low molecular weight in oat−wheat dough, which increased the softness of the dough, as well as the specific volume and softness of the bread. The results thus indicate that a combination of laccase and xylanase was beneficial for the textures of both oat and oat−wheat breads.Hide Abstract
Effects of laccase, xylanase and their combination on the rheological properties of wheat doughs.
Selinheimo, E., Kruus, K., Buchert, J., Hopia, A. & Autio, K. (2006). Journal of Cereal Science, 43(2), 152-159.
The effects of Trametes hirsuta laccase alone and in combination with Aspergillus oryzae and Bacillus subtilis xylanases on dough extensibility were studied using the Kieffer test to determine the dough extensibility (Ex) and the resistance to stretching (Rmax). Laccase treatment resulted in dough hardening: the Rmax of dough increased and the Ex at Rmax decreased as a function of dosage (5–50 nkat/g flour). Xylanases softened flour and gluten doughs. Hardening by laccases and softening by xylanases was weaker in gluten doughs. Dough hardening, observed in the laccase treatments, decreased as a function of dough resting time. The softening effect occurred especially at higher laccase dosages (≈50 nkat/g flour). The softening phenomenon was related to the laccase-mediated depolymerization of the cross-linked AX network. In combined laccase and xylanase treatments, the effect of laccase was predominant, especially at low xylanase dosage, but when xylanase was added to flour dough at high concentrations, the hardening effect of laccase on dough was decreased. In combined laccase and xylanase treatments in gluten doughs, similar decreases in laccase-mediated hardening were not seen.Hide Abstract