Analysis of feed enzymes.
McCleary, B. V. (2001). “Enzymes in Farm Animal Nutrition”, (M. Bedford and G. Partridge, Eds.), CAB International, pp. 85-107.
Enzymes are added to animal feed to increase its digestibility, to remove anti-nutritional factors, to improve the availability of components, and for environment reasons (Campbell and Bedford, 1992; Walsh et al., 1993). A wide-variety of carbohydrase, protease, phytase and lipase enzymes find use in animal feeds. In monogastric diets, enzyme activity must be sufficiently high to allow for the relatively short transit time. Also, the enzyme employed must be able to resist unfavourable conditions that may be experienced in feed preparation (e.g. extrusion and pelleting) and that exist in the gastrointestinal tract. Measurement of trace levels of enzymes in animal feed mixtures is difficult. Independent of the enzyme studied, many of the problems experienced are similar; namely, low levels of activity, extraction problems inactivation during feed preparation, non-specific binding to other feed components and inhibition by specific feed-derived inhibitors, e.g. specific xylanase inhibitors in wheat flour (Debyser et al., 1999).
Measurement of cereal α-Amylase: A new assay procedure.
McCleary, B. V. & Sheehan, H. (1987). Journal of Cereal Science, 6(3), 237-251.
A new procedure for the assay of cereal α-amylase has been developed. The substrate is a defined maltosaccharide with an α-linked nitrophenyl group at the reducing end of the chain, and a chemical blocking group at the non-reducing end. The substrate is completely resistant to attack by β-amylase, glucoamylase and α-glucosidase and thus forms the basis of a highly specific assay for α-amylase. The reaction mixture is composed of the substrate plus excess quantities of α-glucosidase and glucoamylase. Nitrophenyl-maltosaccharides released on action of α-amylase are instantaneously cleaved to glucose plus free p-nitrophenol by the glucoamylase and α-glucosidase, such that the rate of release of p-nitrophenol directly correlates with α-amylase activity. The assay procedure shows an excellent correlation with the Farrand, the Falling Number and the Phadebas α-amylase assay procedures.
A new procedure for the measurement of fungal and bacterial α-amylase.
Sheehan, H. & McCleary, B. V. (1988). Biotechnology Technniques, 2(4), 289-292.
A procedure for the measurement of fungal and bacterial α-amylase in crude culture filtrates and commercial enzyme preparations is described. The procedure employs end-blocked (non-reducing end) p-nitrophenyl maltoheptaoside in the presence of amyloglucosidase and α-glucosidase, and is absolutely specific for α-amylase. The assay procedure is simple, reliable and accurate.
Measurement of α-Amylase in Cereal, Food and Fermentation Products.
McCleary, B. V. & Sturgeon, R. (2002). Cereal Foods World, 47, 299-310.
In General, the development of methods for measuring α-amylase is pioneered in the clinical chemistry field and then translated to other industries, such as the cereals and fermentation industries. In many instances, this transfer of technology has been difficult or impossible to achieve due to the presence of interfering enzymes or sugars and to differences in the properties of the enzymes being analysed. This article describes many of the commonly used methods for measuring α-amylase in the cereals, food, and fermentation industries and discusses some of the advantages and limitations of each.
Measurement of α-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the ceralpha assay: Collaborative study.
McCleary, B. V., McNally, M., Monaghan, D. & Mugford, D. C. (2002). Journal of AOAC International, 85(5), 1096-1102.
This study was conducted to evaluate the method performance of a rapid procedure for the measurement of α-amylase activity in flours and microbial enzyme preparations. Samples were milled (if necessary) to pass a 0.5 mm sieve and then extracted with a buffer/salt solution, and the extracts were clarified and diluted. Aliquots of diluted extract (containing α-amylase) were incubated with substrate mixture under defined conditions of pH, temperature, and time. The substrate used was nonreducing end-blocked p-nitrophenyl maltoheptaoside (BPNPG7) in the presence of excess quantities of thermostable α-glucosidase. The blocking group in BPNPG7 prevents hydrolysis of this substrate by exo-acting enzymes such as amyloglucosidase, α-glucosidase, and β-amylase. When the substrate is cleaved by endo-acting α-amylase, the nitrophenyl oligosaccharide is immediately and completely hydrolyzed to p-nitrophenol and free glucose by the excess quantities of α-glucosidase present in the substrate mixture. The reaction is terminated, and the phenolate color developed by the addition of an alkaline solution is measured at 400 nm. Amylase activity is expressed in terms of Ceralpha units; 1 unit is defined as the amount of enzyme required to release 1 µmol p-nitrophenyl (in the presence of excess quantities of α-glucosidase) in 1 min at 40°C. In the present study, 15 laboratories analyzed 16 samples as blind duplicates. The analyzed samples were white wheat flour, white wheat flour to which fungal α-amylase had been added, milled malt, and fungal and bacterial enzyme preparations. Repeatability relative standard deviations ranged from 1.4 to 14.4%, and reproducibility relative standard deviations ranged from 5.0 to 16.7%.
Measurement of Starch: Critical evaluation of current methodology.
B. V. McCleary, L. M. J. Charmier & V. A. McKie. (2018). Starch‐Stärke, In Press.
Most commonly used methods for the measurement of starch in food, feeds and ingredients employ the combined action of α‐amylase and amyloglucosidase to hydrolyse the starch to glucose, followed by glucose determination with a glucose oxidase/peroxidase reagent. Recently, a number of questions have been raised concerning possible complications in starch analytical methods. In this paper, each of these concerns, including starch hydrolysis, isomerisation of maltose to maltulose, effective hydrolysis of maltodextrins by amyloglucosidase, enzyme purity and hydrolysis of sucrose and β‐glucans have been studied in detailed. Results obtained for a range of starch containing samples using AOAC Methods 996.11 and 2014 .10 are compared and a new simpler format for starch measurement is introduced. With this method that employs a thermostable α‐amylase (as distinct from a heat stable α‐amylase) which is both stable and active at 100°C and pH 5.0, 10 samples can be analysed within 2 h, as compared to the 6 h required with AOAC Method 2014.10.
Relationship between levels of diastatic power enzymes and wort sugar production from different barley cultivars during the commercial mashing process of brewing.
Hu, S., Yu, J., Dong, J., Evans, D. E., Liu, J., Huang, S., Huang, S., Fan W., Yin, H. & Li, M. (2014). Starch‐Stärke, 66(7-8), 615-623.
The fermentable carbohydrate composition of wort has a direct influence on yeast fermentation efficiency and resultant beer quality. In this study, the relationship between diastatic power enzymes (DPE) and their wort sugars products during the course of small-scale, emulated commercial mashing was investigated. Malts derived from 13 barley cultivars were mashed and assayed at five time points during mashing for the levels of DPE and fermentable sugars. Comparisons of the patterns of DPE activity and wort sugar production showed that the activity levels of β-amylase and limit dextrinase (LD) during mashing were variable between the 13 cultivars, in comparison to the level of α-amylase and resultant composition of wort sugars. Moreover, comparison of peak DPE activities indicated that α-amylase correlated positively and significantly with LD, while no obvious correlation was found between β-amylase and either α-amylase or LD, indicating that activity pattern of α-amylase and LD was closely related during mashing. Multiple linear regression models, based on levels of the DPE as various time points during mashing, thermostability of β-amylase and malt Kolbach index, were able to explain 42.9%, 91.9%, 94%, and 73.2% of wort maltotriose, maltose, glucose, and fermentable sugar composition, respectively. A combination of these insights into the dynamics of starch hydrolysis during mashing will assist brewers in malt cultivar selection and the adjustment of mashing conditions so as optimize the sugar content for the efficient production of high quality beer.
Influence of germination time and temperature on the properties of rye malt and rye malt based worts.
Hübner, F., Schehl, B. D., Gebruers, K., Courtin, C. M., Delcour, J. A. & Arendt, E. K. (2010). Journal of Cereal Science, 52(1), 72-79.
The effects of germination time and temperature on the quality of rye malt and worts derived thereof were investigated using Response Surface Methodology. Amylolytic and proteolytic enzyme activities were increased by long germination periods, while β-glucanase activity was not influenced. Total and Soluble Nitrogen content were also not significantly affected by the variations in germination conditions. Free Amino Nitrogen (FAN) was found in higher amounts in worts prepared from rye malts with long germination times. Extract contents were higher in rye malt than in the control barley malt and could be increased by a favourable germination regime, while no such impact on wort fermentability was found. High wort viscosities could be significantly reduced by a long germination period at low temperatures, but were still unacceptably high. The same conditions favoured the development of endoxylanase activity. Arabinoxylan (AX) accumulated during the germination process and their extractability increased. The results suggest that longer germination periods resulted in an increased number of AX molecules with lower molecular mass. Optimal rye malt qualities within the limits of this study were found for a germination time of 144 h at 10°C, which resulted in an acceptable FAN content and the lowest measured viscosity.
Starch degradation in buttercup squash (Cucurbita maxima).
Irving, D. E., Shingleton, G. J. & Hurst, P. L. (1999). Journal of the American Society for Horticultural Science, 124(6), 587-590.
Extractable activities of α-amylase, β-amylase, and starch phosphorylase were investigated in order to understand the mechanism of starch degradation in buttercup squash (Cucurbita maxima Duchesne ex Lam. `Delica') with the ultimate goal of improving the conversion of starch into sweet sugars. During rapid starch synthesis (0 to 30 days after flowering), extractable activities of α-amylase and β-amylase were low, but those of starch phosphorylase increased. After harvest, during ripening at 12°C, or in fruit left in the field, activities of α-amylase and β-amylase increased. Starch contained 20% to 25% amylose soon after starch synthesis was initiated and until 49 days after harvest irrespective of whether the crop remained in the field or in storage at 12°C. Maltose concentrations were low prior to harvest, but levels increased during fruit ripening. Data suggest starch breakdown is hydrolytic in buttercup squash, with α-amylase being the primary enzyme responsible for initiating starch breakdown.
Optimizing red sorghum malt quality when Bacillus subtilis is used during steeping to control mould growth.
Tawaba, J. C. B., Béra, F. & Thonart, P. (2012). Journal of the Institute of Brewing, 118(3), 295-304.
Previous work has shown that Bacillus subtilis-S499-based biocontrol treatments applied without aeration at the steeping stage of red sorghum malting offer good mould reduction, but yield malts with low levels of key hydrolytic enzymes. Thus we attempted to raise these levels by aerating the steeping liquor, varying the steeping time (from 8 to 40 h) and temperature (from 25 to 35°C), and combining a biocontrol treatment with prior steeping in 0.2% NaOH. Aeration proved particularly important whenever B. subtilis cells were present in the steep liquor. The optimal temperatures for α- and β-amylase were 30 and 25°C, respectively. By increasing the steeping time, it was possible to improve the α-amylase activity, but the β-amylase activity peaked sharply between 16 and 20 h, depending on the steeping medium. A good compromise was steeping in a biocontrol medium for 14–16 h at 30°C. Combination steeping treatments (0.2% NaOH for 8 h followed by biocontrol for 8 h) yielded malts of a quality approaching that afforded by dilute alkaline treatment.
Effect of drying temperature and time on alpha-amylase, beta-amylase, limit dextrinase activities and dimethyl sulphide level of teff (Eragrostis tef) malt.
Gebremariam, M. M., Zarnkow, M. & Becker, T. (2013). Food and Bioprocess Technology, 6(12), 3462-3472.
Teff is a gluten-free cereal with attractive nutritional profile. This research was aimed to study the influence of kilning on the enzyme activities and dimethyl sulphide (DMS) level of DZ-Cr-387 teff variety and suggest a kilning condition that yields teff malt with low DMS with no or little damage on its enzyme activities. The malts were dried using isothermal conditions at 30, 40, 50, 60 and 70°C for 40 h with sampling in certain time interval. To set up kilning program, two temperature regimens 18 h at 30°C + 1h at 60°C + 3 or 5 h at 65°C (R1) and 18 h at 30°C + 1 h at 60°C + 3 or 5 h at 80°C (R2) were selected. Results from isothermal kilning indicated that enzyme activities, DMS and moisture contents were affected (P < 0.05) by time and temperature. The values of α-amylase, β-amylase, limit dextrinase activities and DMS content while using the first regimen (R1) with 3 h curing at 65°C were 68 U/g, 440 U/g, 1,072 U/kg and 3.3 mg/kg, respectively. Whereas in the second regimen with 3 h curing at 80°C, the values were 42 U/g, 406 U/g, 736 U/kg and 2.15 mg/kg, respectively. Prolonged curing in both kilning regimens caused an adverse effect on the amylolytic enzyme activities. R1 with shorter curing time is considered to be the best condition in preserving enzymes. The enzyme activities and DMS level show that teff can be an alternative raw material for production of gluten-free malt.
Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae.
Hou, J., Tyo, K., Liu, Z., Petranovic, D. & Nielsen, J. (2012). Metabolic Engineering, 14(2), 120-127.
The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often restricted due to the limitations of the host strain. In the protein secretory pathway, the protein trafficking between different organelles is catalyzed by the soluble NSF (N-ethylmaleimide-sensitive factor) receptor (SNARE) complex and regulated by the Sec1/Munc18 (SM) proteins. In this study, we report that over-expression of the SM protein encoding genes SEC1 and SLY1, improves the protein secretion in S. cerevisiae. Engineering Sec1p, the SM protein that is involved in vesicle trafficking from Golgi to cell membrane, improves the secretion of heterologous proteins human insulin precursor and α-amylase, and also the secretion of an endogenous protein invertase. Enhancing Sly1p, the SM protein regulating the vesicle fusion from endoplasmic reticulum (ER) to Golgi, increases α-amylase production only. Our study demonstrates that strengthening the protein trafficking in ER-to-Golgi and Golgi-to-plasma membrane process is a novel secretory engineering strategy for improving heterologous protein production in S. cerevisiae.
Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress.
Tyo, K. E., Liu, Z., Petranovic, D. & Nielsen, J. (2012). BMC Biology, 10(1), 16.
Background: The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).
Results: HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.
Conclusions: Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.
Evaluation of exopolysaccharide producing Weissella cibaria MG1 strain for the production of sourdough from various flours.
Wolter, A., Hager, A. S., Zannini, E., Galle, S., Gänzle, M. G., Waters, D. M. & Arendt, E. K. (2014). Food Microbiology, 37, 44-50.
This study determined exopolysaccharide (EPS) production by Weissella cibaria MG1 in sourdoughs prepared from gluten-free flours (buckwheat, oat, quinoa and teff), as well as wheat flour. Sourdoughs (SD) were fermented without sucrose, or by replacing 10% flour with sucrose to support EPS production. The amount of EPS depended on the substrate: high amounts of EPS corresponding to low amounts of oligosaccharides were found in buckwheat (4.2 g EPS/kg SD) and quinoa sourdoughs (3.2 g EPS/kg SD); in contrast, no EPS but panose-series oligosaccharides (PSO) were detected in wheat sourdoughs. Organic acid production, carbohydrates and rheological changes during fermentation were compared to the EPS negative control without added sucrose. Corresponding to the higher mineral content of the flours, sourdoughs from quinoa, teff and buckwheat had higher buffering capacity than wheat. Fermentable carbohydrates in buckwheat, teff and quinoa flours promoted W. cibaria growth; indicating why W. cibaria failed to grow in oat sourdoughs. Endogenous proteolytic activity was highest in quinoa flour; α-amylase activity was highest in wheat and teff flours. Protein degradation during fermentation was most extensive in quinoa and teff SD reducing protein peaks 18-29, 30-41 and 43–55 kDa extensively. Rheological analyses revealed decreased dough strength (AF) after fermentation, especially in sucrose-supplemented buckwheat sourdoughs correlating with amounts of EPS. High EPS production correlated with high protein, fermentable sugars (glucose, maltose, fructose), and mineral contents in quinoa flour. In conclusion, W. cibaria MG1 is a suitable starter culture for sourdough fermentation of buckwheat, quinoa and teff flour.
An evaluation of exogenous enzymes with amylolytic activity for dairy cows.
Klingerman, C. M., Hu, W., McDonell, E. E., DerBedrosian, M. C. & Kung Jr, L. (2009). Journal of dairy science, 92(3), 1050-1059.
An experimental (7B) and a commercial (AMA) formulation of enzymes, both primarily with α-amylase activity, were evaluated for activity at various pH values, stability in ruminal fluid, the potential to improve in vitro ruminal fermentations, and the potential to improve production performance of lactating cows. When incubated (40°C) in buffer with a pH between 5.4 and 6.0, 7B had about 10 to 25 times greater amylase activity than AMA, and enzyme activity in this range increased by 100% for 7B, whereas activity decreased by about 26% for AMA. Both formulations maintained enzyme activity when they were incubated in in vitro ruminal fermentations for 24 h. After 6 h of ruminal in vitro fermentation, additions of 7B resulted in linear increases in apparent total volatile fatty acid production for flint and dent corn but had no effect on floury corn. In a lactation trial, 28 Holstein cows (68 ± 31 d in milk, 46.9 ± 9.1 kg of milk/d) were fed a total mixed ration (TMR) supplemented with nothing (CON), a low dose of 7B [7BL, 0.88 mL/kg of TMR dry matter (DM)], a high dose of 7B (7BH, 4.4 mL/kg of TMR DM), or AMA (0.4 g/kg of TMR DM). The experiment was conducted as a 4 x 4 Latin square design with 21-d periods. Cows fed 7BL, 7BH, and AMA ate similar amounts of DM, and cows fed the latter 2 diets consumed more DM than did cows fed CON. Cows fed 7BL produced more milk than cows fed CON and 7BH, but produced similar amounts to cows fed AMA. The production of 3.5% fat-corrected milk was greater from cows fed 7BL and AMA compared with cows fed CON. The percentages of milk fat and milk protein were unaffected by treatment. Total-tract digestion of DM and organic matter were greater for cows fed 7BL compared with those fed CON. The addition of exogenous amylase enzymes to the diets of lactating dairy cows has the potential to improve animal productivity.