Amylase Isoforms Research Articles

Differential biochemical and kinetic properties of α-amylases from Rhyzopertha dominica (F.) progenies reared on wheat varieties differing in α-amylase inhibitory activity

The present study was accomplished to gain insights into the biochemical and kinetic properties of Rhyzopertha dominica’s α-amylase isoforms (named RdA70, RdA79, and RdA90) expressed in progenies reared in wheat varieties differing in α-amylase inhibitory activity. An inverse relationship was observed between the progenies’ amylase activity and the wheat inhibitory activity. Wheat samples with a high and low infestation (named HI-Borlaug and LI-Villa Juarez, respectively) were chosen to simplify the study. The progenies amylases were isolated by hydrophobic interaction chromatography, while the wheat samples were analyzed in α-amylase inhibitory activity by size exclusion chromatography. The isoforms RdA70 and RdA90 from LI-Villa Juarez progeny showed higher enzyme activities (73.8 and 43.4%, respectively) than those from HI-Borlaug. When the amylase isoforms were tested in susceptibility to inhibition by the inhibitory albumins, those from LI-Villa Juarez were more susceptible than those of the HI-Borlaug. Determination of the kinetic parameters revealed that RdA70 from the HI-Borlaug progeny showed 3.0-fold less starch affinity than that from the LI-Villa Juarez (Km of 12.3 ± 1.8 versus 4.0 ± 0.3). The rest of the α-amylases did not show the same pattern as RdA70 in the HI-Bourlag as that their starch affinity was further reduced (RdA79 Km = 23.1 ± 4.3, RdA90 Km = 17.1 ± 2.9). Estimation of IC50 values confirmed the high sensitivity of the three α-amylases of the LI-Villa Juarez progeny to wheat α-amylase inhibitors. The inhibitor constant Ki was the lowest for RdA70 in the LI-Villa Juarez progeny indicating the inhibitor’ tight binding to that isoenzyme. These results suggest that R. dominica uses RdA70 to bind large amounts of wheat α-amylase inhibitors than RdA79 and RdA90 as a physiological defense mechanism.

Leaf carbohydrates influence transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in the facultative CAM plant, Mesembryanthemum crystallinum

Nocturnal degradation of transitory starch is a limiting factor for the optimal function of crassulacean acid metabolism and must be coordinated with phosphoenolypyruvate carboxylase (PEPC)-mediated CO2 uptake to optimise carbon gain over the diel cycle. The aim of this study was to test the hypothesis that nocturnal carboxylation is coordinated with starch degradation in CAM via a mechanism whereby the products of these pathways regulate diel transcript abundance and enzyme activities for both processes. To test this hypothesis, a starch and CAM-deficient mutant of Mesembryanthemum crystallinum was compared with wild type plants under well-watered and saline (CAM-inducing) conditions. Exposure to salinity increased the transcript abundance of genes required for nocturnal carboxylation, starch and sucrose degradation in both wild type and mutant, but the transcript abundance of several of these genes was not sustained over the dark period in the low-carbohydrate, CAM-deficient mutant. The diel pattern of transcript abundance for PEPC mirrored that of PEPC protein, as did the transcripts, protein, and activity of chloroplastic starch phosphorylase in both wild type and mutant, suggesting robust diel coordination of these metabolic processes. Activities of several amylase isoforms were low or lacking in the mutant, whilst the activity of a cytosolic isoform of starch phosphorylase was significantly elevated, indicating contrasting modes of metabolic regulation for the hydrolytic and phosphorylytic routes of starch degradation. Externally supplied sucrose resulted in an increase in nocturnal transcript abundance of genes required for nocturnal carboxylation and starch degradation. These results demonstrate that carbohydrates impact on transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in CAM.

Effect of glucose as a carbon repressor on the extracellular proteome of Aspergillus niger during the production of amylases by solid state cultivation

Abstract In comparison to traditional submerged cultivations, solid state cultivation (SSC) has been shown to reduce the effect of carbon catabolite repression during the production of important industrial enzymes, such as amylases. Nevertheless, the effect of a carbon repressor on the entire secretome during the production of enzymes of industrial interest has not been described. Therefore, the secretome composition and amylase production from Aspergillus niger grown in SSC using media containing starch and different concentrations of glucose (60 and 120 g/L) were evaluated. The results showed that proteins without an amino-terminal signal peptide, which is necessary for secretion through the classical secretory pathway, were detected at high glucose concentrations; some of these proteins were moonlighting proteins associated with pathogenesis. High concentrations of glucose also produced better amylase volumetric activities. α-Amylase activity was eight times higher in media containing glucose as compared to the medium containing only starch. Glucoamylase activity was 29 and 20 times higher at glucose concentrations of 60 and 120 g/L, respectively, compared to the medium containing only starch. Moreover, the presence of glucose had a variable effect on the abundance of the amylase isoforms produced by A. niger.

Production of raw-starch-digesting α-amylase isoform from Bacillus sp. under solid-state fermentation and biochemical characterization

α-Amylase production by solid-state fermentation of different Bacillus sp. was studied previously on different fermentation media. However, no study has been reported on the influence of selected media on expression of desired amylase isoforms such as raw-starch-digesting amylase (RSDA). In this paper, the influence of different inexpensive and available agro-resources as solid media (corn, wheat and triticale) on α-amylase isoform induction from three wild-type Bacillus sp., selected among one hundred strains tested, namely 9B, 12B and 24A was investigated. For all three strains, tested amylases were detected in the multiple forms; however, number and intensity of each form differed depending on the solid media used for growth. To determine which isoform from Bacillus sp. 12B was RSDA, the suspected isoform was purified. The optimum pH for the purified α-amylase isoform was 6.0-8.0, while the optimum temperature was 60-90°C. Isoform was considerably thermostable and Ca(2+)-independent, and actually the only α-amylase active towards raw starch. Purification and characterization of RSDA showed that not all of the solid media tested induced RSDA. From an economic point of view, it might be significant to obtain pure isoenzyme for potential use in the raw-starch hydrolysis, since it was 5 times more efficient in raw corn starch hydrolysis than the crude amylase preparation.

Expression and distribution of cellulase, amylase and peptidase isoforms along the midgut of Morimus funereus L. (Coleoptera: Cerambycidae) larvae is dependent on nutrient substrate composition

The influence of diet composition--two substrates, wheat bran and sawdust--on isoform expression of digestive enzymes (cellulase, amylase and peptidase) in the midgut of Morimus funereus larvae was examined. Their impact on larval development was demonstrated by measuring the increase of larval weight during development and by analysis of digestive enzymes zymographic profiles, where the expression of cellulase isoforms from M. funereus larvae midgut has been examined for the first time in this study. Larvae reared on wheat bran had higher body weight between day 60 and day 100 than larvae reared on sawdust; however, both groups achieved similar body weight after day 110. Wheat bran as substrate induced different cellulase and amylase isoforms. Oak sawdust in substrate acted as inducer of peptidases. The highest cellulase activity and the greatest isoform variability were detected in the midgut extracts of larvae reared on wheat bran. From our results it can be assumed that M. funereus endocellulase, amylase and peptidase are secreted in the anterior midgut, and their concentration gradually decreases towards the hindgut.

Biochemical characterisation of α-amylase inhibitors fromAchyranthes asperaand their interactions with digestive amylases of coleopteran and lepidopteran insects

Starchy seeds are an important food and a source of dietary ingredients in many countries. However, they suffer from extensive predation by bruchids (weevils) and other pests. α-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. A proteinaceous α-amylase inhibitor from the seeds of Achyranthes aspera was identified, purified and characterised. In electrophoretic analysis, two prominent amylase inhibitor activity bands (AI1 and AI2) were detected. The inhibitor was purified 9.99-fold with 1206.95 total amylase inhibitor units mg⁻¹ protein. The molecular weight of the purified inhibitor was around 6 kDa. The isolated α-amylase inhibitor was found to be resistant to heat and proteolysis. Feeding analysis of Callosobruchus maculatus larvae on a diet containing seed powder of A. aspera revealed that survival of the larvae was severely affected, with the highest mortality rate occurring on the fifth day of feeding. The isolated inhibitor inhibited the majority of amylase isoforms of C. maculatus, Tribolium confusum and Helicoverpa armigera in electrophoretic analysis and solution assays. The information obtained in the present investigation could be useful for a genetic engineering approach that would make seeds resistant to storage pest infestations.

Host‐mediated induction of α‐amylases by larvae of the Mexican bean weevil Zabrotes subfasciatus (Coleoptera: Chrysomelidae: Bruchinae) is irreversible and observed from the initiation of the feeding period

Larvae of Zabrotes subfasciatus secrete alpha-amylases that are insensitive to the alpha-amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non alpha-amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible alpha-amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible alpha-amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days (age when the larvae stopped feeding), we could detect higher activity of the inducible alpha-amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible alpha-amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible alpha-amylase isoforms. Incubations of brush border membrane vesicles with the alpha-amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes.

Comparison of α-amylase isoforms from the midgut of Cerambyx cerdo L. (Coleoptera: Cerambycidae) larvae developed in the wild and on an artificial diet

?-Amylase isoforms of Cerambyx cerdo larvae from the wild (ML and SL) and reared in the laboratory (ADL) were compared. Three amylase isoforms were presented in the SL and ML extracts while two isoforms were presented in the ADL according to zymogram after isoelectric focusing (IEF). All C. cerdo amylase isoforms were acidic proteins (pI < 3.5). Seven amylase isoforms (ACC 1-7) from the midgut of C. cerdo larvae were found in the ML midgut extract, six in the SL extract, and four in the ADL extract according to native PAGE zymogram. The ADL amylase had the highest activity. All crude midgut extracts of C. cerdo larvae were fractionated on a Superose 12 HR column. The molecular mass of the ACC was estimated to be 34 kDa. .

Responses of midgut amylases of Helicoverpa armigera to feeding on various host plants

Midgut digestive amylases and proteinases of Helicoverpa armigera, a polyphagous and devastating insect pest of economic importance have been studied. We also identified the potential of a sorghum amylase inhibitor against H. armigera midgut amylase. Amylase activities were detected in all the larval instars, pupae, moths and eggs; early instars had lower amylase levels which steadily increased up to the sixth larval instar. Qualitative and quantitative differences in midgut amylases of H. armigera upon feeding on natural and artificial diets were evident. Natural diets were categorized as one or more members of legumes, vegetables, flowers and cereals belonging to different plant families. Amylase activity and isoform patterns varied depending on host plant and/or artificial diet. Artificial diet-fed H. armigera larvae had comparatively high amylase activity and several unique amylase isoforms. Correlation of amylase and proteinase activities of H. armigera with the protein and carbohydrate content of various diets suggested that H. armigera regulates the levels of these digestive enzymes in response to macromolecular composition of the diet. These adjustments in the digestive enzymes of H. armigera may be to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. H. armigera, a generalist feeder experiences a great degree of nutritional heterogeneity in its diet. An investigation of the differences in enzyme levels in response to macronutrient balance and imbalance highlight their importance in insect nutrition.

Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: Bruchidae) and the induction of α–amylases in response to different diets

Zabrotes subfasciatus larvae possess three α–amylase isoforms as determined by in gel assays following SDS–PAGE. The two minor isoforms present lower electrophoretic mobility than the major form, and seem to occur as a heterodimer. When developed inside Vigna unguiculata (cowpea) seeds, fourth instar larvae have minor quantities of the slow-migrating forms, but when reared on seeds of Phaseolus vulgaris (common bean) or Phaseolus lunatus, the two slow-migrating forms are expressed in higher amounts, while activity of the major form was independent of the host seed. Larvae developing inside cowpea seeds at the beginning of the fourth instar were fed on flour from cotyledons of cowpea or common bean. Larvae fed on the common bean flour started to express the dimer in higher amounts when compared with the control larvae fed on cowpea flour. In an attempt to correlate differences between starch granules and the induction of α–amylases, a detailed study on the digestive process of the granules was conducted. Incorporation of purified starch granules into artificial diets did not induce the two minor α–amylases. The in vitro hydrolysis rates of purified granules and the pattern of dextrins liberated by the different α–amylases were similar for the two legume species. The starch granules enter the midgut extensively damaged, which may facilitate the access to the more susceptible parts of the granules to enzymatic attack.

Enzymes of Starch Metabolism in Poplar Wood During Fall and Winter

In poplar wood starch is converted to soluble sugars, mostly sucrose, raffinose, stachyose, and maltose, at the beginning of the dormant season. In a first attempt to understand the mechanism of starch degradation during this period the following enzymes were extracted and quantified in weekly intervals: total amylase, a-amylase (EC 3.2.1.1), starch phosphorylase (EC 2.4.1.1), debranching enzyme (EC 3.2.1.41), D-enzyme (EC 2.4.1.25), maltase (EC 3.2.1.20), maltose phosphorylase (EC 2.4.1.8), starch synthase (EC 2.4.1.21), and ADPGlcPPase (EC 2.7.7.27). Maximum extractable activity of the starch-degrading enzymes was observed during the phase of rapid starch decline in October. Later in the year, only D-enzyme was closely correlated with changes of the starch content. Native PAGE and activity staining revealed that only four of 13 amylolytic bands were associated with periods of starch decline. Two of these bands were detectable also at low temperature and at a slightly alkaline pH. They were not inhibited by maltose. It is suggested that these amylase isoforms are located in amyloplasts and take part in starch degradation. The activities of enzymes involved in starch synthesis, ADPGlcPPase and starch synthase also increased in fall. This observation is consistent with the occurrence of high temperature induced starch resynthesis during the dormant season.

Three methods compared for isoamylase separation in tissue homogenates.

Using homogenates of autopsy tissue, we compared three widely available techniques for separating amylase isoenzymes: wheat-germ inhibition (WI), and electrophoresis on cellulose acetate (CA) or agarose (AG). WI separated amylase into two isoforms, CA into seven (three pancreatic and four salivary), and AG into nine (five pancreatic and four salivary). CA and WI had similar isoamylase detection limits (8-10 U/L) and similar imprecision in measuring percent S-type vs P-type isoamylase (within-run SD 1-2%), and they demonstrated a linear response to added S or P isoamylase. In contrast, the AG method had higher detection limits (10-15 U/L), greater imprecision (within-run SD 3%), and showed a nonlinear response to added S or P isomylase. We conclude that CA and WI have essentially equivalent assay attributes, superior to AG, but that CA resolves more amylase isoforms than WI.